The most detailed study of Yugoslavian landraces was conducted by Pavlicic and Trifunovic (1966), when all the collected populations were aligned into 18 agro-ecological groups using the method of "natural classification" proposed by Anderson & Cutler (1942). The 18 agroecological groups are: Montenegrin flints, Bosnian early dents, Kosmet flinty dents, Macedonian flints, Eight rowed corn type of North eastern America, Derived flints, Mediterranean flint, Small-kernelled flints, Eight rowed soft dents, Romanian flints, Largeeared flints, White flinty dents Moravac, Dent type of USA corn belt dents, Derived dents, Dent type of southern east USA, Serbian dents, Flinty dents and Denty flints (Figure 1).

components content as phytate or a large grains content of proteins, oil or starch.

diversity using morphological and molecular markers.

et al., 2003).

genotypes.

its neighbouring continental area.

flints and denty flints were collected.

**2. Establishment of genetic diversity of maize landraces**

The utilization of genetic diversity of landraces in breeding programs started with development of the first inbred lines in Yugoslavia form Ruma Golden Dent, Vukovar Dent, Šidski, Beljski and Novi Sad Golden Dent populations. Crosses of self pollinated landraces with American inbred lines, which expressed a high level of heterosis, were the base for development of line-hybrids. First Yugoslav maize hybrids were created from inbred lines of local origin and foreign inbred lines genetically divergent from local germplasm. In modern maize breeding program landraces are used for creation of broad base synthetic populations, development of core collections and as potential sources of favorable traits.

Fig. 1. Agroecological groups of maize landraces from ex-Yugoslav territories

A core collection consists of a limited set of genotypes derived from the existing collection and has to represent the genetic diversity of that collection. The Maize Genebank adopted two approaches to develop core collections. Representatives of each eco-geographical group (20%) were chosen based on morphological and biological traits for development of eco-core collection. Core subsets are used as donors for traits typical for each agoecological group. On the other hand, the main criterion for selecting landraces for the elite-core was the combining ability. Combining abilities of 900 medium and late maturing landraces, from regions with temperate growing conditions, were tested with four commercial inbred lines, Mo17, B73, V395/31 and LD230 (Radovic et al., 2000). Four core subsets of high combining abilities with specific inbred lines were obtained for further use in breeding programs.

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 93

a check since they are expected to be genetically distant from local populations. These

All populations were sown at Zemun Polje in 2008, in two different sowing densities – 44640 and 64935 plants per ha. The experimental design was RCBD (*Randomized Complete Block Design*) with two replicas, four rows per replica and 20 plants per row. For morphological trait measurements two medium rows per population were used. We measured for each plot 15 morphological traits (plant height, ear height, leaf number, husk leaf length, tassel length, central spike/tassel length, number of tassel primary branches, branched part/tassel length, ear length, ear row number, ear kernel/row number, ear diameter, kernel length, kernel width and kernel thickness) taken from 20 competitive plants (40 plants per

Based on the average values of 15 morphological traits, as well as ASI (anthesis - silking interval) and their standard deviations cluster analysis was performed using square Euclidean distance and Ward complete linkage method. All the statistical analyses were

Cluster analysis based on phenotypic traits highlights distance between populations originating from ex-Yugoslav territories including two populations from France (clusters 1, 2 and cluster 3) and introduced populations from Georgia and China (cluster 4). Dendrogram can be divided into four clusters. The first cluster includes only two populations, one from Montenegrin flints and the other from Mediterranean flint agro-ecological groups. Cluster II encompasses only local flint populations. The third cluster can further be divided in two subclusters (Figure 2). Sub-cluster IIIA includes mostly local flint and intermediary (flinty-dents and denty-flints) populations, while sub-cluster IIIB includes mostly local dent and intermediary populations. Both French populations, with flint type of kernels, fall into cluster III. Introduced populations from Georgia and China form a distinctive cluster IV, which also

Phenotypic analysis revealed large heterogeneity among local populations indicating that populations are highly adapted to specific environmental conditions and uses. As such, they could be a valuable source of genetic variability. These results are in agreement with the findings that maize is the most diverse crop known, containing extensive diversity at both the phenotypic and molecular levels (Buckler et al., 2006). More ever, studies on European maize germplasm have already pointed out the presence of a great variability in morphological traits

Development of molecular marker techniques, which enable estimation of genetic diversity directly at the DNA sequence level, are expected to supplement and refine the morphologicalbased classification. Molecular markers provide a direct measure of genetic diversity and go beyond indirect diversity measures, based on morphological traits or geographical origin.

RFLP (restriction fragment length polymorphisms), as well as PCR (polymerase chain reaction) based markers like RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphisms) and SSR (simple sequence repeats) have been successfully used in maize genetic diversity studies. RFLP have been extensively used in assessing genetic variability in maize (Dubreil & Charchosset, 1998; Gauthier et al., 2002; Rebourg et al., 2001; Rebourg et al., 2003) due to their high discriminative power. However,

and large molecular diversity in traditional populations (Gauthier et al., 2002).

populations originate from France, Georgia and China.

population) and from two kernels per ear.

performed using program NTSYSpc 2.1.

**3.2 Molecular marker analysis**

includes two local populations (one dent and one flinty dent).

#### **3. Genetic diversity assessment**

Significant maize genetic diversity decrease in the last few decades is a consequence of development of modern hybrids and agricultural systems. The use of a limited number of elite lines and synthetics heightens the risk of genetic uniformity in commercial maize production fields (Hallauer et al., 1988). Thus, maize breeders became more aware of the need for both maintaining genetic diversity among hybrid varieties and improving the management of genetic resources through the conservation of landraces (Goodman, 1994).

Landraces are the cultivated maize material with the highest genetic variation as well as with the best adaptation to the natural and anthropological environment were they have evolved (Maxted et al., 1997). They contain locally adapted alleles and represent an irreplaceable bank of highly co-adapted genotypes (Quals et al., 1997). In this sense, genetic variability is a valuable natural source of beneficial traits providing plant adaptation to different stresses and undergoing climatic changes.

Different methods can be used to assess genetic variability in plant species, such as pedigree data, morphological and molecular markers. Data obtained by landrace description are further statistically processed. Multivariate analyses (e.g., cluster analysis, principal component analysis and discriminate analysis) for measuring the degree of divergence among populations have been useful in different fields of research, allowing obtaining a summary of the most relevant characteristics. Populations (landraces) can be grouped together based on informative data and be used directly in a breeding program.

A broad approach using phenotypic and molecular markers is required to analyze diversity and to support conservation, management, and development of plant genetic resources (Hammer et al., 1999). Both kinds of markers have their advantages and drawbacks and their combined utilization is recommended to increase the resolving power of genetic diversity analyses (Singh et al., 1991).

Considering the origins of the populations and the fact that trade and communications have always been very intensive on a relatively small ex-Yugoslav territory it can be assumed that genetic relationships between the 2217 landraces from Maize Research Institute gene bank could be inconsistent with the existing classifications. Moreover, accessions that are maintained in the collection are samples of the original populations which, through propagation, have lost a part of variability due to genetic drift and inbreeding. A new phenotypic analysis, as well as molecular analysis, is being performed on these landraces in order to assess their genetic variability and verify their relationships.

#### **3.1 Phenotypic analysis**

Phenotypic markers have been of great value in studies of maize landraces (Galarreta & Alvarez, 2001; Lucchin et al., 2003; Ortiz et al., 2008). The most commonly used descriptors are related to plant architecture traits, tassel traits, ear and kernel characteristics. Earliness is also frequently a part of the landrace description. Relationships between traits are statistically analyzed using graphs, correlation coefficient estimations and principal component analysis (PCA). Genetic similarity can be calculated from the morphologic data by unweighted pairgroup method (UPGMA) cluster analysis based on Euclidian distance coefficients. Cluster analysis is used to reveal the association between landraces.

A sample of 54 maize landraces was analyzed with three populations per agro-ecological group. Besides ex-Yugoslavian landraces, six introduced populations were included, used as

Significant maize genetic diversity decrease in the last few decades is a consequence of development of modern hybrids and agricultural systems. The use of a limited number of elite lines and synthetics heightens the risk of genetic uniformity in commercial maize production fields (Hallauer et al., 1988). Thus, maize breeders became more aware of the need for both maintaining genetic diversity among hybrid varieties and improving the management of genetic resources through the conservation of landraces (Goodman, 1994). Landraces are the cultivated maize material with the highest genetic variation as well as with the best adaptation to the natural and anthropological environment were they have evolved (Maxted et al., 1997). They contain locally adapted alleles and represent an irreplaceable bank of highly co-adapted genotypes (Quals et al., 1997). In this sense, genetic variability is a valuable natural source of beneficial traits providing plant adaptation to

Different methods can be used to assess genetic variability in plant species, such as pedigree data, morphological and molecular markers. Data obtained by landrace description are further statistically processed. Multivariate analyses (e.g., cluster analysis, principal component analysis and discriminate analysis) for measuring the degree of divergence among populations have been useful in different fields of research, allowing obtaining a summary of the most relevant characteristics. Populations (landraces) can be grouped

A broad approach using phenotypic and molecular markers is required to analyze diversity and to support conservation, management, and development of plant genetic resources (Hammer et al., 1999). Both kinds of markers have their advantages and drawbacks and their combined utilization is recommended to increase the resolving power of genetic

Considering the origins of the populations and the fact that trade and communications have always been very intensive on a relatively small ex-Yugoslav territory it can be assumed that genetic relationships between the 2217 landraces from Maize Research Institute gene bank could be inconsistent with the existing classifications. Moreover, accessions that are maintained in the collection are samples of the original populations which, through propagation, have lost a part of variability due to genetic drift and inbreeding. A new phenotypic analysis, as well as molecular analysis, is being performed on these landraces in

Phenotypic markers have been of great value in studies of maize landraces (Galarreta & Alvarez, 2001; Lucchin et al., 2003; Ortiz et al., 2008). The most commonly used descriptors are related to plant architecture traits, tassel traits, ear and kernel characteristics. Earliness is also frequently a part of the landrace description. Relationships between traits are statistically analyzed using graphs, correlation coefficient estimations and principal component analysis (PCA). Genetic similarity can be calculated from the morphologic data by unweighted pairgroup method (UPGMA) cluster analysis based on Euclidian distance

A sample of 54 maize landraces was analyzed with three populations per agro-ecological group. Besides ex-Yugoslavian landraces, six introduced populations were included, used as

coefficients. Cluster analysis is used to reveal the association between landraces.

together based on informative data and be used directly in a breeding program.

order to assess their genetic variability and verify their relationships.

**3. Genetic diversity assessment**

different stresses and undergoing climatic changes.

diversity analyses (Singh et al., 1991).

**3.1 Phenotypic analysis**

a check since they are expected to be genetically distant from local populations. These populations originate from France, Georgia and China.

All populations were sown at Zemun Polje in 2008, in two different sowing densities – 44640 and 64935 plants per ha. The experimental design was RCBD (*Randomized Complete Block Design*) with two replicas, four rows per replica and 20 plants per row. For morphological trait measurements two medium rows per population were used. We measured for each plot 15 morphological traits (plant height, ear height, leaf number, husk leaf length, tassel length, central spike/tassel length, number of tassel primary branches, branched part/tassel length, ear length, ear row number, ear kernel/row number, ear diameter, kernel length, kernel width and kernel thickness) taken from 20 competitive plants (40 plants per population) and from two kernels per ear.

Based on the average values of 15 morphological traits, as well as ASI (anthesis - silking interval) and their standard deviations cluster analysis was performed using square Euclidean distance and Ward complete linkage method. All the statistical analyses were performed using program NTSYSpc 2.1.

Cluster analysis based on phenotypic traits highlights distance between populations originating from ex-Yugoslav territories including two populations from France (clusters 1, 2 and cluster 3) and introduced populations from Georgia and China (cluster 4). Dendrogram can be divided into four clusters. The first cluster includes only two populations, one from Montenegrin flints and the other from Mediterranean flint agro-ecological groups. Cluster II encompasses only local flint populations. The third cluster can further be divided in two subclusters (Figure 2). Sub-cluster IIIA includes mostly local flint and intermediary (flinty-dents and denty-flints) populations, while sub-cluster IIIB includes mostly local dent and intermediary populations. Both French populations, with flint type of kernels, fall into cluster III. Introduced populations from Georgia and China form a distinctive cluster IV, which also includes two local populations (one dent and one flinty dent).

Phenotypic analysis revealed large heterogeneity among local populations indicating that populations are highly adapted to specific environmental conditions and uses. As such, they could be a valuable source of genetic variability. These results are in agreement with the findings that maize is the most diverse crop known, containing extensive diversity at both the phenotypic and molecular levels (Buckler et al., 2006). More ever, studies on European maize germplasm have already pointed out the presence of a great variability in morphological traits and large molecular diversity in traditional populations (Gauthier et al., 2002).

#### **3.2 Molecular marker analysis**

Development of molecular marker techniques, which enable estimation of genetic diversity directly at the DNA sequence level, are expected to supplement and refine the morphologicalbased classification. Molecular markers provide a direct measure of genetic diversity and go beyond indirect diversity measures, based on morphological traits or geographical origin.

RFLP (restriction fragment length polymorphisms), as well as PCR (polymerase chain reaction) based markers like RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphisms) and SSR (simple sequence repeats) have been successfully used in maize genetic diversity studies. RFLP have been extensively used in assessing genetic variability in maize (Dubreil & Charchosset, 1998; Gauthier et al., 2002; Rebourg et al., 2001; Rebourg et al., 2003) due to their high discriminative power. However,

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 95

dominant and multiallelic. Pejic et al. (1998) suggested that SSR appear to be suited for the analysis of outcrossing heterozygous individuals and AFLP are advantageous when genome coverage is a major issue due to the presence of linkage disequilibrium, such as in inbred lines and breeding material. The choice among the markers to use depends on the material

Two studies with SSR and AFLP markers were conducted at MRI on the landraces from different agro-ecological groups. In the first study 21 landraces belonging to seven different agro ecological groups have been subjected to SSR analysis with aim to develop genetic fingerprint for characterization, identification and classification of the landraces, as well as for estimation of their genetic diversity (Ignjatovic-Micic et al., 2008). In the second study eighteen landraces from six different agro-ecological groups were assayed with AFLP and SSR markers (Ignjatovic-Micic et al., 2007). The main objectives of the work were to compare the level of information provided by SSR and AFLP and the genetic similarities (GS) obtained with the two marker systems. In both studies a DNA-pooling strategy that proved to be effective in RFLP analysis of maize populations (Dubreuil et al., 1999) was employed in order to assess if it could be applied with the same success for population analysis with SSR

SSR analysis was performed with 25 primer pairs covering all 10 maize chromosomes. The amplification reaction was carried out in 25μl reaction volume containing 1x enzyme buffer, 2.4 mM MgCl2, 200 μM dNTPs, 0.5 μM primers, 1 x BSA, 1U *Taq* polymerase and 200 ng of DNA. The amplification profiles followed were: an initial denaturation at 95°C/5min, followed by 15 cycles each of denaturation at 95°C/30sec, annealing at 63.5°C/1min (-0.5°C/cycle) and extension at 72°C/1min; another 22 cycles of 95°C/30sec, 56°C/1min and

AFLP analysis was conducted using three primer combinations: EACA/M-CAG, E-AGA/M-CAG, E-ACA/M-CAT and E-AAA/CAG. *EcoRI* was used as a rare cutter and *MseI*  as a frequent cutter. Pre-amplification was done by the following program: 1. 94°C/60sec, 2. 94°C/30sec, 3. 55°/30sec, 4. 72°C/60sec (30 cycles of steps 2-4). Amplification program consisted of: 1. 94°C/60sec, 2. 94°C/30sec, 3. 62°C/30sec, 4. 72°C/60sec (40 cycles of steps 2-4). Both SSR and AFLP amplified fragments were separated on 6% denaturated polyacrilamide gels and visualized by silver staining (Bassam et al., 1991). The gels were

The SSR probes used detected single loci and each detected band was assumed to be an allele. Allele frequency was scored as percentage of individual bands within the sample using peak height estimated by scanning. Genetic distances (GD) between populations were evaluated by the *Rogers' Distance* (Rogers, 1972). For the AFLP primer combinations bands were binary scored (1 and 0) with each band being considered a locus and the genetic similarities among tested populations were calculated by the *Jaccard coefficient* (Jaccard, 1908). Cluster analysis was performed with unweighted pair-group method (UPGMA).

Twenty five SSR markers generated a total of 224 polymorphic bands, while three AFLP primers combinations generated 188 bands with 79.25 being polymorphic. The average polymorphism content was higher for SSR and higher genetic similarity values were estimated for AFLPs. In our study ranking of the agro ecological groups (order from the

scanned and band profiles for each primer were scored visually.

Statistical analyses were done by NTSYSpc2.1 program package.

highest to the lowest average GS) was congruent for both marker systems.

to be analyzed, the aim of the experiment and the facilities available.

and AFLP markers.

72°C/1min were performed.

RFLP technique is labor intensive and time consuming, needs large quantities of DNA and is difficult to automate. On the other hand, RAPD which overcome some of the RFLP drawbacks have shown some problems with reproducibility of amplification and scoring of error data (Demeke et al., 1997; Karp et al., 1997).

Abbreviations for the 18 agroecological groups are: MF - Montenegrin flints, BED -Bosnian early dents, KFD - Kosmet flinty dents, McF - Macedonian flints, ERCT - Eight rowed corn type of North eastern America, DerF - Derived flints, MdF - Mediterranean flint, SKF - Small-kernelled flints, ERSD - Eight rowed soft dents, RF - Romanian flints, LEF - Large-eared flints, WFDM - White flinty dents Moravac, DTCB - Dent type of USA corn belt dents, DD - Derived dents, DTSE - Dent type of southern east USA, SD - Serbian dents, FD - Flinty dents and DenF - Denty flints.

Fig. 2. Cluster analysis of the analyzed populations based on phenotypic traits using square Euclidean distance and Ward method

SSR and AFLP, the most informative among DNA-based markers, seem to be a better choice for genetic diversity studies (Smith et al., 1997; Pejic et al., 1998; Lubberstedt et al., 2000), since they overcome the problem of reproducibility and error data of RAPD markers, are generally much simpler to apply than RFLP, can be automated and yet provide results correlated with those from RFLP analysis (Pejic et al., 1998). They have been successfully utilized in maize genetic diversity studies (Lubberstedt et al., 2000; Barcaccia et al., 2003; Dubreil et al., 2006; van Eten et al., 2008; Eschholz et al., 2010; Hartings et al., 2010).

Although often used for the same scientific research AFLP and SSR present major distinctions. AFLP markers are genomic fragments detected after selective PCR amplification, while SSR markers consist of tandemly repeated units of short (1-6 bp) nucleotide motifs that show extensive length variation between individuals (Jarne & Lagoda, 1996). AFLP markers are dominant and biallelic, while SSR markers are co-

RFLP technique is labor intensive and time consuming, needs large quantities of DNA and is difficult to automate. On the other hand, RAPD which overcome some of the RFLP drawbacks have shown some problems with reproducibility of amplification and scoring of

Abbreviations for the 18 agroecological groups are: MF - Montenegrin flints, BED -Bosnian early dents, KFD - Kosmet flinty dents, McF - Macedonian flints, ERCT - Eight rowed corn type of North eastern America, DerF - Derived flints, MdF - Mediterranean flint, SKF - Small-kernelled flints, ERSD - Eight rowed soft dents, RF - Romanian flints, LEF - Large-eared flints, WFDM - White flinty dents Moravac, DTCB - Dent type of USA corn belt dents, DD - Derived dents, DTSE - Dent type of southern east USA,

Fig. 2. Cluster analysis of the analyzed populations based on phenotypic traits using square

SSR and AFLP, the most informative among DNA-based markers, seem to be a better choice for genetic diversity studies (Smith et al., 1997; Pejic et al., 1998; Lubberstedt et al., 2000), since they overcome the problem of reproducibility and error data of RAPD markers, are generally much simpler to apply than RFLP, can be automated and yet provide results correlated with those from RFLP analysis (Pejic et al., 1998). They have been successfully utilized in maize genetic diversity studies (Lubberstedt et al., 2000; Barcaccia et al., 2003;

Although often used for the same scientific research AFLP and SSR present major distinctions. AFLP markers are genomic fragments detected after selective PCR amplification, while SSR markers consist of tandemly repeated units of short (1-6 bp) nucleotide motifs that show extensive length variation between individuals (Jarne & Lagoda, 1996). AFLP markers are dominant and biallelic, while SSR markers are co-

Dubreil et al., 2006; van Eten et al., 2008; Eschholz et al., 2010; Hartings et al., 2010).

error data (Demeke et al., 1997; Karp et al., 1997).

SD - Serbian dents, FD - Flinty dents and DenF - Denty flints.

Euclidean distance and Ward method

dominant and multiallelic. Pejic et al. (1998) suggested that SSR appear to be suited for the analysis of outcrossing heterozygous individuals and AFLP are advantageous when genome coverage is a major issue due to the presence of linkage disequilibrium, such as in inbred lines and breeding material. The choice among the markers to use depends on the material to be analyzed, the aim of the experiment and the facilities available.

Two studies with SSR and AFLP markers were conducted at MRI on the landraces from different agro-ecological groups. In the first study 21 landraces belonging to seven different agro ecological groups have been subjected to SSR analysis with aim to develop genetic fingerprint for characterization, identification and classification of the landraces, as well as for estimation of their genetic diversity (Ignjatovic-Micic et al., 2008). In the second study eighteen landraces from six different agro-ecological groups were assayed with AFLP and SSR markers (Ignjatovic-Micic et al., 2007). The main objectives of the work were to compare the level of information provided by SSR and AFLP and the genetic similarities (GS) obtained with the two marker systems. In both studies a DNA-pooling strategy that proved to be effective in RFLP analysis of maize populations (Dubreuil et al., 1999) was employed in order to assess if it could be applied with the same success for population analysis with SSR and AFLP markers.

SSR analysis was performed with 25 primer pairs covering all 10 maize chromosomes. The amplification reaction was carried out in 25μl reaction volume containing 1x enzyme buffer, 2.4 mM MgCl2, 200 μM dNTPs, 0.5 μM primers, 1 x BSA, 1U *Taq* polymerase and 200 ng of DNA. The amplification profiles followed were: an initial denaturation at 95°C/5min, followed by 15 cycles each of denaturation at 95°C/30sec, annealing at 63.5°C/1min (-0.5°C/cycle) and extension at 72°C/1min; another 22 cycles of 95°C/30sec, 56°C/1min and 72°C/1min were performed.

AFLP analysis was conducted using three primer combinations: EACA/M-CAG, E-AGA/M-CAG, E-ACA/M-CAT and E-AAA/CAG. *EcoRI* was used as a rare cutter and *MseI*  as a frequent cutter. Pre-amplification was done by the following program: 1. 94°C/60sec, 2. 94°C/30sec, 3. 55°/30sec, 4. 72°C/60sec (30 cycles of steps 2-4). Amplification program consisted of: 1. 94°C/60sec, 2. 94°C/30sec, 3. 62°C/30sec, 4. 72°C/60sec (40 cycles of steps 2-4). Both SSR and AFLP amplified fragments were separated on 6% denaturated polyacrilamide gels and visualized by silver staining (Bassam et al., 1991). The gels were scanned and band profiles for each primer were scored visually.

The SSR probes used detected single loci and each detected band was assumed to be an allele. Allele frequency was scored as percentage of individual bands within the sample using peak height estimated by scanning. Genetic distances (GD) between populations were evaluated by the *Rogers' Distance* (Rogers, 1972). For the AFLP primer combinations bands were binary scored (1 and 0) with each band being considered a locus and the genetic similarities among tested populations were calculated by the *Jaccard coefficient* (Jaccard, 1908). Cluster analysis was performed with unweighted pair-group method (UPGMA). Statistical analyses were done by NTSYSpc2.1 program package.

Twenty five SSR markers generated a total of 224 polymorphic bands, while three AFLP primers combinations generated 188 bands with 79.25 being polymorphic. The average polymorphism content was higher for SSR and higher genetic similarity values were estimated for AFLPs. In our study ranking of the agro ecological groups (order from the highest to the lowest average GS) was congruent for both marker systems.

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 97

undetected. They also concluded that differences between populations are emphasized due to a concentration on alleles with high frequencies. However, in a simulated SSR analysis of bulks they showed that the correlation between genetic distance values of individuals and

Assessing molecular marker diversity among heterogeneous populations using a DNA bulking strategy was shown to be successful in different plant species (Yu & Pauls, 1993; Sweeny & Dannenberger, 1997; Dubreuil et al., 1999; Mian et al., 2002). The question is what marker system to use in order to obtain the most accurate results with maximum efficiency. In our work we compared SSRs and AFLPs, the markers suggested as the best choice in genetic diversity studies. Based on results of this comparison it could be concluded that AFLPs are more reliable than SSRs in assessing diversity of maize populations when using a

Absence of pollen and plant inability to produce functional pollen grains is known as male sterility. Male sterility can be determined by nuclear (genic male sterility) or cytoplasmic (cytoplasmic genetic male sterility – **CGMS**) genes. CGMS (onwards referred to as CMS) is successfully used in commercial production of hybrid seed, avoiding the drawbacks of hand or mechanical emasculation (Kaul, 1988). Identification of CMS types is important because commercial production of maize hybrid seed today stands upon utilization of C and S cytoplasmic types, as self-pollination in plants can severely jeopardize its production. Detasseling (removal of flowers from maize plants by hand) can be replaced by introducing male sterility in maize. This reduces possibility of self-pollination and also saves seed

Three main types of CMS were identified in maize: CMS-T, CMS-S and CMS-C. Male sterile cytoplasm is distinguished by specific nuclear genes (*Rf* genes) that restore pollen fertility. These genes, restorers of fertility, suppress the male-sterile effect of the cytoplasm, allowing

The use of uniform materials can be a threat in crop production and genetic vulnerability must be a constant concern in plant breeding for all species. An example is the extensive use of CMS-T in 1950-ies and 1960-ies, which showed to be extremely susceptible to *Helminthosporium maydis* race T. Severe yield losses occurred in the United States in 1970-ies and the next year in other regions of the world, due to the *Helminthosporium maydis*  pandemic. For new CMS sources landrace genotypes worldwide were searched and one

Tester lines containing nuclear *Rf* genes are traditionally used for identification and classification of CMS types. However, test-crossing procedure is time consuming, labor intensive and not precise. Development and appliance of molecular methods revealed that mutations responsible for CMS are located in mitochondrial DNA (mtDNA) in many plant species (Shnable & Wise, 1998). It was confirmed that chimeric genes – DNA parts with open reading frames (ORF) that comprise sequences derived from different genes, are responsible for cytoplasmic male sterility. Himeric T-*urf13* gene was detected in mtDNA CMS-T (Dewey et al., 1986) chimeric *atp6-C* gene in mtDNA CMS-C (Dewey et al.,

source was found within Maize Research Institute gene bank collection.

**4. Genetic diversity of landraces as source for cytoplasmic male**

bulks is high, but it is still to be checked for final conclusions.

producers millions of dollars per year in labor costs.

the production of viable pollen.

DNA bulking strategy.

**sterility (CMS)**

The cluster analysis did not group the populations precisely with expectations based upon their agro-ecological groups. Better agreement with expected results was accomplished with AFLP analysis (Figure 3). In the work of Reif et al. (2005) it was suggested that SSR analysis of bulked populations cannot detect alleles with a frequency below 0.2, so that a large number of alleles with low frequencies remains undetected. They also concluded that differences between populations are emphasized due to a concentration on alleles with high frequencies.

Fig. 3. Dendrograms of 18 maize populations (bulked population samples) constructed using UPGMA cluster analysis of A) Roger's distance values obtained by SSR analysis and B) Jaccard similarity values obtained by AFLP analysis. Abbreviations are the same as in Fig. 2.

The results in our work which indicate that SSRs are not a good choice for bulk analysis of populations could be explained by several reasons, besides the low sensitivity of bulk approach in detecting low frequency alleles. Use of di-nucleotide repeat SSR (excluded in the work of Reif et al.*,* 2005) which tend to be more polymorphic than SSR with longer repeat motifs (Smith et al*.*, 1997) could have caused higher scoring errors. The silver staining of the gels is probably not the right choice for bulk analysis due to its low sensitivity. It would be much better to use fluorescence as was done in the work of Reif et al. (2005), in which it was shown that the allele frequencies of a population could be estimated with high accuracy from the band intensities of SSR profiles of bulks. Populations cannot detect alleles with a frequency below 0.2, so that a large number of alleles with low frequencies remain

The cluster analysis did not group the populations precisely with expectations based upon their agro-ecological groups. Better agreement with expected results was accomplished with AFLP analysis (Figure 3). In the work of Reif et al. (2005) it was suggested that SSR analysis of bulked populations cannot detect alleles with a frequency below 0.2, so that a large number of alleles with low frequencies remains undetected. They also concluded that differences between populations are emphasized due to a concentration on alleles with high

Fig. 3. Dendrograms of 18 maize populations (bulked population samples) constructed using UPGMA cluster analysis of A) Roger's distance values obtained by SSR analysis and B) Jaccard

The results in our work which indicate that SSRs are not a good choice for bulk analysis of populations could be explained by several reasons, besides the low sensitivity of bulk approach in detecting low frequency alleles. Use of di-nucleotide repeat SSR (excluded in the work of Reif et al.*,* 2005) which tend to be more polymorphic than SSR with longer repeat motifs (Smith et al*.*, 1997) could have caused higher scoring errors. The silver staining of the gels is probably not the right choice for bulk analysis due to its low sensitivity. It would be much better to use fluorescence as was done in the work of Reif et al. (2005), in which it was shown that the allele frequencies of a population could be estimated with high accuracy from the band intensities of SSR profiles of bulks. Populations cannot detect alleles with a frequency below 0.2, so that a large number of alleles with low frequencies remain

similarity values obtained by AFLP analysis. Abbreviations are the same as in Fig. 2.

frequencies.

undetected. They also concluded that differences between populations are emphasized due to a concentration on alleles with high frequencies. However, in a simulated SSR analysis of bulks they showed that the correlation between genetic distance values of individuals and bulks is high, but it is still to be checked for final conclusions.

Assessing molecular marker diversity among heterogeneous populations using a DNA bulking strategy was shown to be successful in different plant species (Yu & Pauls, 1993; Sweeny & Dannenberger, 1997; Dubreuil et al., 1999; Mian et al., 2002). The question is what marker system to use in order to obtain the most accurate results with maximum efficiency. In our work we compared SSRs and AFLPs, the markers suggested as the best choice in genetic diversity studies. Based on results of this comparison it could be concluded that AFLPs are more reliable than SSRs in assessing diversity of maize populations when using a DNA bulking strategy.

#### **4. Genetic diversity of landraces as source for cytoplasmic male sterility (CMS)**

Absence of pollen and plant inability to produce functional pollen grains is known as male sterility. Male sterility can be determined by nuclear (genic male sterility) or cytoplasmic (cytoplasmic genetic male sterility – **CGMS**) genes. CGMS (onwards referred to as CMS) is successfully used in commercial production of hybrid seed, avoiding the drawbacks of hand or mechanical emasculation (Kaul, 1988). Identification of CMS types is important because commercial production of maize hybrid seed today stands upon utilization of C and S cytoplasmic types, as self-pollination in plants can severely jeopardize its production. Detasseling (removal of flowers from maize plants by hand) can be replaced by introducing male sterility in maize. This reduces possibility of self-pollination and also saves seed producers millions of dollars per year in labor costs.

Three main types of CMS were identified in maize: CMS-T, CMS-S and CMS-C. Male sterile cytoplasm is distinguished by specific nuclear genes (*Rf* genes) that restore pollen fertility. These genes, restorers of fertility, suppress the male-sterile effect of the cytoplasm, allowing the production of viable pollen.

The use of uniform materials can be a threat in crop production and genetic vulnerability must be a constant concern in plant breeding for all species. An example is the extensive use of CMS-T in 1950-ies and 1960-ies, which showed to be extremely susceptible to *Helminthosporium maydis* race T. Severe yield losses occurred in the United States in 1970-ies and the next year in other regions of the world, due to the *Helminthosporium maydis*  pandemic. For new CMS sources landrace genotypes worldwide were searched and one source was found within Maize Research Institute gene bank collection.

Tester lines containing nuclear *Rf* genes are traditionally used for identification and classification of CMS types. However, test-crossing procedure is time consuming, labor intensive and not precise. Development and appliance of molecular methods revealed that mutations responsible for CMS are located in mitochondrial DNA (mtDNA) in many plant species (Shnable & Wise, 1998). It was confirmed that chimeric genes – DNA parts with open reading frames (ORF) that comprise sequences derived from different genes, are responsible for cytoplasmic male sterility. Himeric T-*urf13* gene was detected in mtDNA CMS-T (Dewey et al., 1986) chimeric *atp6-C* gene in mtDNA CMS-C (Dewey et al.,

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 99

breeding procedures when presence of specific sterile cytoplasm is required, with the final

A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Lanes A – Cms-T (440bp), B – Cms-S (799bp), C – Cms-C (398bp), D – N type of cytoplasm; 1 – no band, 2 to 6 - Cms-S, 7 to 9 - Cms-C, 10 to 11 – no band, 12 to 13 - Cms-T, 14 to 20 - Cms-S.

Fig. 4. PCR amplification of CMS types with specific primers for C, T and S cytoplasm

Fig. 5. The structure of maize sterile cytoplasm types within a gene bank collection

Drought can be defined as the absence of adequate moisture necessary for normal plant growth and to complete the life cycle (Zhu, 2002). It is a "single most common cause of severe food shortage" (FAO) in developing countries and predicted global warming in XXI century will increase drought impact on crop production. Estimations of Intergovernmental Panel on Climate Change (IPCC, www.ipcc.org) are that between 1990 and 2100, globall increase of temperature will be 1.4 to 5.8 °C. Although the effect of this warming will be regionally distributed, it is assumed that increase in temperature, reduction in rainfalls, together with increase of incidents of insects and pests will reduce crops grain yield,

**5. Genetic diversity as a source of drought tolerance**

particularly in tropical and subtropical area (Ribaut, 2006).

goal of commercial production of maize hybrids.

1991) and a repeated DNA region "R" containing two ORFs in mtDNA CMS-S (Zabala et al., 1997).

The unique characteristics of himeric mtDNA regions were used for developing several molecular methods. RFLP analysis is time consuming to be applied routinely in breeding programs, while PCR-based markers are known to be rapid and definitive identifiers of the cytoplasm (Sato, 1998; Nakajima et al., 1999). Liu et al. (2002) developed a multiplex PCR assay combining three primer pairs specific for each type of CMS in a single reaction. This assay reveals 398, 440 and 799 bp specific DNA fragments identifying C, T and S cytoplasm, respectively. No DNA fragment is amplified from the N-type cytoplasm in this assay because an mtDNA fragment unique for the normal type does not exist. It was shown to be a quick and a reliable method, convenient for analysis of a huge number of samples.

The multiplex PCR assay was successfully used for CMS identification of maize accessions from Maize Research Institute (MRI) Genebank collection. Over 100 sources (2% of the total accession numbers) of cytoplasmic male sterility (CMS) were found in field trials, distinguishable by their overwhelming frequency of male sterile plants in segregating test progenies. As the types of CMS involved were unknown, a screening experiment was performed using a multiplex PCR with specific primers for S, C and T cytoplasm (Liu et al., 2002).

For PCR analysis *e*ach source of CMS was tested in triplicate, i.e. three separate samples per genotype were analyzed. DNA extraction was done from ten seeds per sample with CTAB buffer by the modified method of (Saghai-Maroof et al., 1984). The PCR reaction mix contained 1× PCR buffer, 1.5 mM MgCl2, 0.8 mM dNTP, 50 pmol of CMSSF, CMSSR, CMSTF, CMSTR, CMSCF and CMSCR primers, 1U of *Taq* polymerase and 50 ng of DNA. The PCR amplification consisted of 40 cycles (steps 2, 3 and 4), as follows: 1) initial denaturation 94°C/2 min, 2) denaturation 94°C/1 min, 3) annealing 55°C/1 min, 4) elongation 72°C/1.30 min and 5) final elongation 72°C/5 min. The amplified fragments were separated on 1.5% agarose gels, stained with ethidium bromide and photographed. An illustration of the multiplex PCR is given in Figure 4.

The multiplex PCR method used in our research, which is recommended for large scale screening, gave conclusive results for most of the analyzed accessions. A small number of accessions could not be identified for cytoplasmic male sterility type as, for example, different PCR products were obtained. The structure of maize sterile cytoplasm types within a gene bank collection is given in Figure 5. The predominant CMS type within the analyzed accessions was S cytoplasm. Clear presence of some type of CMS was found in 4.56% of all the former Yugoslav landraces, which is a significant percentage and S type makes 84% of all CMS found. S type was also predominant (44%) within introduced populations. This significantly lesser presence of CMS-S within introduced populations could be the consequence of a smaller sample size analyzed. On the other hand, introduced populations and lines are of different provenience, coming from different parts of the world and it was shown that, for example, among populations from Argentina the dominant type was CMS-C and from Italy CMS-S (Vancetovic et al., 2010).

The multiplex PCR approach enabled a simple, fast and reliable large scale screening of maize cytoplasm within MRI gene bank accessions, reducing time for cytoplasm characterizations from several years to a few weeks. Indirectly, this also helps in pre-

1991) and a repeated DNA region "R" containing two ORFs in mtDNA CMS-S (Zabala et

The unique characteristics of himeric mtDNA regions were used for developing several molecular methods. RFLP analysis is time consuming to be applied routinely in breeding programs, while PCR-based markers are known to be rapid and definitive identifiers of the cytoplasm (Sato, 1998; Nakajima et al., 1999). Liu et al. (2002) developed a multiplex PCR assay combining three primer pairs specific for each type of CMS in a single reaction. This assay reveals 398, 440 and 799 bp specific DNA fragments identifying C, T and S cytoplasm, respectively. No DNA fragment is amplified from the N-type cytoplasm in this assay because an mtDNA fragment unique for the normal type does not exist. It was shown to be

a quick and a reliable method, convenient for analysis of a huge number of samples.

The multiplex PCR assay was successfully used for CMS identification of maize accessions from Maize Research Institute (MRI) Genebank collection. Over 100 sources (2% of the total accession numbers) of cytoplasmic male sterility (CMS) were found in field trials, distinguishable by their overwhelming frequency of male sterile plants in segregating test progenies. As the types of CMS involved were unknown, a screening experiment was performed using a multiplex PCR with specific primers for S, C and T cytoplasm (Liu et

For PCR analysis *e*ach source of CMS was tested in triplicate, i.e. three separate samples per genotype were analyzed. DNA extraction was done from ten seeds per sample with CTAB buffer by the modified method of (Saghai-Maroof et al., 1984). The PCR reaction mix contained 1× PCR buffer, 1.5 mM MgCl2, 0.8 mM dNTP, 50 pmol of CMSSF, CMSSR, CMSTF, CMSTR, CMSCF and CMSCR primers, 1U of *Taq* polymerase and 50 ng of DNA. The PCR amplification consisted of 40 cycles (steps 2, 3 and 4), as follows: 1) initial denaturation 94°C/2 min, 2) denaturation 94°C/1 min, 3) annealing 55°C/1 min, 4) elongation 72°C/1.30 min and 5) final elongation 72°C/5 min. The amplified fragments were separated on 1.5% agarose gels, stained with ethidium bromide and photographed. An

The multiplex PCR method used in our research, which is recommended for large scale screening, gave conclusive results for most of the analyzed accessions. A small number of accessions could not be identified for cytoplasmic male sterility type as, for example, different PCR products were obtained. The structure of maize sterile cytoplasm types within a gene bank collection is given in Figure 5. The predominant CMS type within the analyzed accessions was S cytoplasm. Clear presence of some type of CMS was found in 4.56% of all the former Yugoslav landraces, which is a significant percentage and S type makes 84% of all CMS found. S type was also predominant (44%) within introduced populations. This significantly lesser presence of CMS-S within introduced populations could be the consequence of a smaller sample size analyzed. On the other hand, introduced populations and lines are of different provenience, coming from different parts of the world and it was shown that, for example, among populations from Argentina the dominant type was CMS-C

The multiplex PCR approach enabled a simple, fast and reliable large scale screening of maize cytoplasm within MRI gene bank accessions, reducing time for cytoplasm characterizations from several years to a few weeks. Indirectly, this also helps in pre-

illustration of the multiplex PCR is given in Figure 4.

and from Italy CMS-S (Vancetovic et al., 2010).

al., 1997).

al., 2002).

breeding procedures when presence of specific sterile cytoplasm is required, with the final goal of commercial production of maize hybrids.

Lanes A – Cms-T (440bp), B – Cms-S (799bp), C – Cms-C (398bp), D – N type of cytoplasm; 1 – no band, 2 to 6 - Cms-S, 7 to 9 - Cms-C, 10 to 11 – no band, 12 to 13 - Cms-T, 14 to 20 - Cms-S.

Fig. 5. The structure of maize sterile cytoplasm types within a gene bank collection

#### **5. Genetic diversity as a source of drought tolerance**

Drought can be defined as the absence of adequate moisture necessary for normal plant growth and to complete the life cycle (Zhu, 2002). It is a "single most common cause of severe food shortage" (FAO) in developing countries and predicted global warming in XXI century will increase drought impact on crop production. Estimations of Intergovernmental Panel on Climate Change (IPCC, www.ipcc.org) are that between 1990 and 2100, globall increase of temperature will be 1.4 to 5.8 °C. Although the effect of this warming will be regionally distributed, it is assumed that increase in temperature, reduction in rainfalls, together with increase of incidents of insects and pests will reduce crops grain yield, particularly in tropical and subtropical area (Ribaut, 2006).

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 101

Ideally, secondary traits should be genetically associated with grain yield under drought, carry no yield penalty under favorable conditions, highly heritable, cheap and rapid to measure, stable over time and be able to be observed at or before flowering so that

A characteristic of maize under drought stress is a delay in silking resulting in an increase in the anthesis-silking interval (ASI), incomplete fertilization and decreased kernel development (Hall et al., 1984). The recommended secondary traits, routinely used by CIMMYT, for use in maize breeding programs are: flowering date (anthesis-silking interval, ASI), ears per plant (bareness), leaf rolling, tassel size and stay green

Considering tremendous genetic diversity and importance of drought for maize production, long term project for improved drought tolerance has been started. All maize accessions, 6371, from Maize Research Institute Zemun Polje genebank were imposed to controlled water stress in Egypt, at the Sids Agricultural Research station (150 km south of Cairo). Among all accessions, 2217 landraces from former Yugoslavia were included into the

 Based on existing data, the material was divided into five groups, according to the duration of the vegetation season: extra early, early, medium, medium late and late. Groups were sown separately and have been irrigated until the appearance of the first tassels. After that, no irrigation was applied. The genotypes were estimated for ASI, staygreen (on the 1-5 scale, where 5 was the highest rate), total appearance of the plant (on the 1-8 scale, where 8 was the highest rate), barrenness, seed set and grain filing. A total of 167 landraces was selected according to reported criteria for further testing in Egypt and on two locations in temperate climate (Macedonia/Skopje and Serbia/Zemun Polje). According to the average of these traits for the three studied locations in two years, 14 most drought tolerant

Besides field performance *per se*, development of good maize hybrids requires establishment of the best combination among heterotic groups (Barata & Carena, 2006). Therefore, chosen landraces were crossed to inbred lines from three heterotic groups (BSSS, Lancaster and independent source) in order to classify them into heterotic groups. The testcrosses for combining ability were studied in field trials at three locations in two years. The criterion for the selection of good testcrosses was the yield not statistically different from the check or performance index (based on grain yield and moisture at harvest) that was over 100% in

Plant responses to environmental stresses include many adaptive mechanisms, changes in physiological and biochemical processes. Adaptation is associated with metabolic adjustments that lead to the accumulation of several organic solutes like sugars, betaines and proline (Yancey et al., 1982). It enables plants to maintain high rates of photosynthesis and can be advantageous in environments with large variation in water availability (Morgan, 1980). The experiments on maize genotypes (Moussa & Abdel-Aziz, 2008) pointed out that stress tolerance mechanism exists in seedlings stage and that early root and shoot development may provide useful information to enable the prediction of maize performance

undesirable parents are not crossed (Edmeades et al., 1999).

(Monneveux et al., 2008).

landraces were selected.

**5.1 Drought at seedling stage**

comparison to the check (Andjelkovic et al., 2010).

experiment (Vancetovic et al., 2008).

In Southern Europe, drought followed by heat wave, reduces maize production for 20% (EC, 2004). The similar effect was in Serbia in 2007, when July the most critical period for grain yield formation was the hottest in last 50 years.

Drought severely affects maize grain yields by restricting season length and through unpredictable stress than can occur at any time during the vegetation season. Drought occurring at flowering has the greatest impact on yield losses compared to all the other developmental stages (Grant et al., 1989). Water deficit lasting one or two days during pollination may cause more than 20% reduction in yield (Hall et al., 1981).

However, drought tolerance is the most difficult task for maize breeders to be solved. It is polygenic and complex trait with high genotype x environment interaction. Moreover, this stress occurs randomly in timing and severity, making identification of drought tolerant genotypes more difficult. In agriculture, drought tolerance is generally evaluated at the end of growing season using grain yield (Dhanda et al., 2002). Maize, like other crops, can achieve drought tolerance through different approaches, involving escape, avoidance and tolerance of stress that influenced growth, development and grain yield formation (Cooper et al., 2004). Possible choice could be selection for earliness (drought avoidance or escape) but in years with adequate water supply it could bring lower yields. On the other hand, by given adequate rainfall, yield is usually correlated with late maturity, in maize, sorghum and sunflower (Edmeades et al., 1989). The most widely used strategy is to select for yield under optimal conditions, and latter on field testing under different moisture conditions. Assumptions of the approach are that genes for drought tolerance are present in high yielding material and the selection under optimal conditions can increase performance under sub-optimal conditions (Bolaños & Edmeades, 1996). However, in the absence of stress, drought tolerance in genotypes provides yield losses (Carena et al., 2009). Thus, many different strategies and traits have been proposed for using in maize breeding programs for improved drought tolerance and best improvement is achieved in long term processes.

Environmental conditions that caused drought and expression of genetic variation for drought tolerance are very important for successful breeding. The use of appropriate locations where drought stress can be controlled and applied at designated time and intensity (MSE- manage stress environment) has made significant improvement in maize breeding (Barker et al., 2005).

Moreover, because of difficulties in selecting for yield *per se* under drought conditions, it is recommended to use secondary traits to complement phenotypic selection. Measurement of secondary traits as well as grain yield is necessary in objective identification of drought tolerant genotypes. Secondary traits are "plant characteristics other than grain yield that give additional information about how yield will change under drought" (Fisher et al., 2003). Useful secondary trait is the trait that used together with yield makes expected progress from selection, greater than the progress made using grain yield alone. The use of secondary traits with grain yield, rather than selection for grain yield alone, has been shown to increase selection efficiency by about 20% in maize grown under stress induced by low nitrogen (Bänziger & Lafitte, 1997). Many physiological and morphological secondary traits related to survival or increased production in water limited environments have been proposed (Richards, 1996). Some of them have never been applied in breeding programs, because of their complexity or impossibility of routine application (Ribaut et al., 2006).

In Southern Europe, drought followed by heat wave, reduces maize production for 20% (EC, 2004). The similar effect was in Serbia in 2007, when July the most critical period for grain

Drought severely affects maize grain yields by restricting season length and through unpredictable stress than can occur at any time during the vegetation season. Drought occurring at flowering has the greatest impact on yield losses compared to all the other developmental stages (Grant et al., 1989). Water deficit lasting one or two days during

However, drought tolerance is the most difficult task for maize breeders to be solved. It is polygenic and complex trait with high genotype x environment interaction. Moreover, this stress occurs randomly in timing and severity, making identification of drought tolerant genotypes more difficult. In agriculture, drought tolerance is generally evaluated at the end of growing season using grain yield (Dhanda et al., 2002). Maize, like other crops, can achieve drought tolerance through different approaches, involving escape, avoidance and tolerance of stress that influenced growth, development and grain yield formation (Cooper et al., 2004). Possible choice could be selection for earliness (drought avoidance or escape) but in years with adequate water supply it could bring lower yields. On the other hand, by given adequate rainfall, yield is usually correlated with late maturity, in maize, sorghum and sunflower (Edmeades et al., 1989). The most widely used strategy is to select for yield under optimal conditions, and latter on field testing under different moisture conditions. Assumptions of the approach are that genes for drought tolerance are present in high yielding material and the selection under optimal conditions can increase performance under sub-optimal conditions (Bolaños & Edmeades, 1996). However, in the absence of stress, drought tolerance in genotypes provides yield losses (Carena et al., 2009). Thus, many different strategies and traits have been proposed for using in maize breeding programs for improved drought tolerance and best improvement is achieved in

Environmental conditions that caused drought and expression of genetic variation for drought tolerance are very important for successful breeding. The use of appropriate locations where drought stress can be controlled and applied at designated time and intensity (MSE- manage stress environment) has made significant improvement in maize

Moreover, because of difficulties in selecting for yield *per se* under drought conditions, it is recommended to use secondary traits to complement phenotypic selection. Measurement of secondary traits as well as grain yield is necessary in objective identification of drought tolerant genotypes. Secondary traits are "plant characteristics other than grain yield that give additional information about how yield will change under drought" (Fisher et al., 2003). Useful secondary trait is the trait that used together with yield makes expected progress from selection, greater than the progress made using grain yield alone. The use of secondary traits with grain yield, rather than selection for grain yield alone, has been shown to increase selection efficiency by about 20% in maize grown under stress induced by low nitrogen (Bänziger & Lafitte, 1997). Many physiological and morphological secondary traits related to survival or increased production in water limited environments have been proposed (Richards, 1996). Some of them have never been applied in breeding programs, because of their complexity or impossibility of routine application (Ribaut et al., 2006).

pollination may cause more than 20% reduction in yield (Hall et al., 1981).

yield formation was the hottest in last 50 years.

long term processes.

breeding (Barker et al., 2005).

Ideally, secondary traits should be genetically associated with grain yield under drought, carry no yield penalty under favorable conditions, highly heritable, cheap and rapid to measure, stable over time and be able to be observed at or before flowering so that undesirable parents are not crossed (Edmeades et al., 1999).

A characteristic of maize under drought stress is a delay in silking resulting in an increase in the anthesis-silking interval (ASI), incomplete fertilization and decreased kernel development (Hall et al., 1984). The recommended secondary traits, routinely used by CIMMYT, for use in maize breeding programs are: flowering date (anthesis-silking interval, ASI), ears per plant (bareness), leaf rolling, tassel size and stay green (Monneveux et al., 2008).

Considering tremendous genetic diversity and importance of drought for maize production, long term project for improved drought tolerance has been started. All maize accessions, 6371, from Maize Research Institute Zemun Polje genebank were imposed to controlled water stress in Egypt, at the Sids Agricultural Research station (150 km south of Cairo). Among all accessions, 2217 landraces from former Yugoslavia were included into the experiment (Vancetovic et al., 2008).

 Based on existing data, the material was divided into five groups, according to the duration of the vegetation season: extra early, early, medium, medium late and late. Groups were sown separately and have been irrigated until the appearance of the first tassels. After that, no irrigation was applied. The genotypes were estimated for ASI, staygreen (on the 1-5 scale, where 5 was the highest rate), total appearance of the plant (on the 1-8 scale, where 8 was the highest rate), barrenness, seed set and grain filing. A total of 167 landraces was selected according to reported criteria for further testing in Egypt and on two locations in temperate climate (Macedonia/Skopje and Serbia/Zemun Polje). According to the average of these traits for the three studied locations in two years, 14 most drought tolerant landraces were selected.

Besides field performance *per se*, development of good maize hybrids requires establishment of the best combination among heterotic groups (Barata & Carena, 2006). Therefore, chosen landraces were crossed to inbred lines from three heterotic groups (BSSS, Lancaster and independent source) in order to classify them into heterotic groups. The testcrosses for combining ability were studied in field trials at three locations in two years. The criterion for the selection of good testcrosses was the yield not statistically different from the check or performance index (based on grain yield and moisture at harvest) that was over 100% in comparison to the check (Andjelkovic et al., 2010).

#### **5.1 Drought at seedling stage**

Plant responses to environmental stresses include many adaptive mechanisms, changes in physiological and biochemical processes. Adaptation is associated with metabolic adjustments that lead to the accumulation of several organic solutes like sugars, betaines and proline (Yancey et al., 1982). It enables plants to maintain high rates of photosynthesis and can be advantageous in environments with large variation in water availability (Morgan, 1980). The experiments on maize genotypes (Moussa & Abdel-Aziz, 2008) pointed out that stress tolerance mechanism exists in seedlings stage and that early root and shoot development may provide useful information to enable the prediction of maize performance

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 103

reduction

L1 18.4 20.8 11.5 7.8 8.8 11.4 L2 9.7 11.1 12.6 6.5 6.6 1.5 L3 18.2 22.3 18.4 8.2 9.4 12.8 L4 17.1 20.0 **14.5** 7.4 9.2 **19.5**  L5 22.5 23.7 **5.1** 10.2 11.9 **14.3**  L6 17.0 18.0 5.5 8.3 8.5 2.4 L7 17.3 19.5 11.2 7.7 8.3 7.2 L8 17.1 17.2 **0.6** 8.4 9.4 **10.6**  L9 17.1 18.0 **5.0** 8.0 8.8 **9.1**  L10 14.7 16.3 9.8 7.5 8.2 8.5 L11 13.8 16.6 **16.8** 5.7 7.0 **18.6**  L12 16.4 17.7 **7.3** 9.3 10.2 **8.8**  L13 18.8 20.3 **7.4** 8.1 9.9 **18.2**  L14 20.4 22.4 **8.9** 8.4 10.1 **16.8**

Table 1. Root and shoot length and percent (%) of reduction under control and stress

enable the prediction of maize performance under field conditions.

**6. Genetic diversity of landraces for grain quality**

2010, Mittelmann et al., 2011).

In the genetic study of quantitative trait loci (QTLs) in maize seedling some QTLs were specific to water stress, such as QTLs for root dry weight, shoot dry weight, and leaf area-toroot length ratio (Ruta, 2010). The QTLs for shoot-to-root ratio, were collocated and overlapped with QTLs for ear number in the field under intermediate and severe stresses (Ribaut et al., 1996) and for the anthesis-silking interval were collocated with QTLs for the numbers of crown roots and seminal roots. An early increase in growth of main roots seemed to be linked to a later decrease in ASI, while an early increase in lateral root length distribution on the main axile root may extend ASI. Considering that conventional breeding may take about ten years from crossing to the final release of new maize hybrids, it is important to find rapid screening technique under laboratory conditions. QTLs controlling the balance between early root and shoot development may provide useful information to

Characterization of genetic diversity of maize landraces aids efficient exploring of the allelic variation for genetic improvement of economically desirable traits, such as grain quality. Maize is a relevant food source, so the quantification of the nutritionally important grain constituents is important for the best exploitation of different genotypes. In this context, the landraces represent a good source of genetic variability and may help to identify the most suitable materials for the development of more nutritious foods. Many studies have documented genetic and phenotypic variability for grain composition traits in maize (Pollak & Scott, 2005; Reynolds et al., 2005; Berardo et al., 2009; Has et al.,

To determine genetic diversity for grain quality traits a set of 54 landraces was grown in a randomized complete block design (RCBD) with two replications at one location. Grain

tretment control tretment control reduction

Shoot lenght %

Landraces Root lenght %

condition (PEG)

under field conditions (Ruta, 2010). Usually, in laboratory conditions drought stress is investigated by withholding water. But, very often, different osmolytes (manitol, sorbitol, inorganic salts or polyethylene glycol-PEG) can be used to mimic water stress. Polymers of PEG with a molecular weight above 6000 Da do not penetrate cell wall and therefore mimic dry soil more closely (Verslues et al., 2006).

Maize landraces chosen within our project were analyzed in response to PEG treatment. Seeds were germinated for three days on moistened filter paper and then transferred into plastic pots containing Knopp solution with modified nitrogen content. For the next four days, plants were grown on ¼ strength nutrient solution supplemented with different forms of nitrogen. Nitrogen was supplied in the form of KNO3, Ca (NO3)2 and (NH4)2SO4. The initial pH of the solution was adjusted to 5.6. Plants were kept in a growth chamber under a 12-h photoperiod at 22/18oC, with the irradiance of 40Wm-2 and relative humidity of 70%. For the terminal 48 h of the growing period, one half of the plants (treatment) were grown on the fresh aerated nutrient solution supplemented with 4% polyethylene glycol (PEG, Mr 10 000), parallel to control plants grown on the nutrient solution without any addition. Root and shoot length, fresh and dry weight and prolin content were measured (Kravic et al., 2010).

Roots, responsible for water uptake, are the first plant organs sensing water stress. But roots and shoots usually respond differently to water deficit: in contrary to the shoot, relative or absolute size of the root might increase, depending on the root type and duration of stress (van der Weele et al., 2000). In our experiments drought stress at seedling stage affected shoot growth to a greater extent than root growth (Table 1).

In the experimental study of drought tolerant and sensitive maize seedlings (Ruta, 2010) the plant traits most severely affected by water shortage were shoot growth, (−75%) and leaf area (−68%), followed by root growth (−51%). It should be noted that at the same developmental stage, water stressed plants were two days older than the well-watered plants. This slower growth at water stress was much more pronounced at the shoot level than at the root level. Limited water availability retarded shoot development and promoted root development, consistent with results from plants stressed in solid substrates (Sharp & Davies, 1989; Weerathaworn et al., 1992a; 1992b). Genes for root development and proliferation during the seedling stage are also active at later growth stages, i.e. investigations of the genetic control of root traits at the seedling stage provide valuable information about its behavior at later stages of the plant development under the field conditions.

Although drought stress reduces plant water potential, it affects root and leaf growth differently (Frensch, 1997). It shifts the weight based shoot to root ratios in favor of the roots and it is a well known adaptive mechanism of preferring root over shoot development. The larger root-to-shoot ratio under severe stress is a further indicator that a moderate water stress had been achieved as it promotes a relatively better functioning of the roots (El Nadi et al., 1969; Sharp & Davies, 1979; Westgate & Boyer, 1985). Tendency of plants to increase the root to shoot ratio by allocating assimilates to the roots is typical response of plants in the field. In this experiment, and some others (Suharjo, 2007), plants were grown in solution, with mild (4% PEG) osmotic stress and root was not stimulated to grow deeper to extract more water (as in the soil). For this reason, root to shoot ratio does not differ too much compared to control conditions (Figure 6).

under field conditions (Ruta, 2010). Usually, in laboratory conditions drought stress is investigated by withholding water. But, very often, different osmolytes (manitol, sorbitol, inorganic salts or polyethylene glycol-PEG) can be used to mimic water stress. Polymers of PEG with a molecular weight above 6000 Da do not penetrate cell wall and therefore mimic

Maize landraces chosen within our project were analyzed in response to PEG treatment. Seeds were germinated for three days on moistened filter paper and then transferred into plastic pots containing Knopp solution with modified nitrogen content. For the next four days, plants were grown on ¼ strength nutrient solution supplemented with different forms of nitrogen. Nitrogen was supplied in the form of KNO3, Ca (NO3)2 and (NH4)2SO4. The initial pH of the solution was adjusted to 5.6. Plants were kept in a growth chamber under a 12-h photoperiod at 22/18oC, with the irradiance of 40Wm-2 and relative humidity of 70%. For the terminal 48 h of the growing period, one half of the plants (treatment) were grown on the fresh aerated nutrient solution supplemented with 4% polyethylene glycol (PEG, Mr 10 000), parallel to control plants grown on the nutrient solution without any addition. Root and shoot length, fresh and dry weight and prolin content were

Roots, responsible for water uptake, are the first plant organs sensing water stress. But roots and shoots usually respond differently to water deficit: in contrary to the shoot, relative or absolute size of the root might increase, depending on the root type and duration of stress (van der Weele et al., 2000). In our experiments drought stress at seedling stage affected

In the experimental study of drought tolerant and sensitive maize seedlings (Ruta, 2010) the plant traits most severely affected by water shortage were shoot growth, (−75%) and leaf area (−68%), followed by root growth (−51%). It should be noted that at the same developmental stage, water stressed plants were two days older than the well-watered plants. This slower growth at water stress was much more pronounced at the shoot level than at the root level. Limited water availability retarded shoot development and promoted root development, consistent with results from plants stressed in solid substrates (Sharp & Davies, 1989; Weerathaworn et al., 1992a; 1992b). Genes for root development and proliferation during the seedling stage are also active at later growth stages, i.e. investigations of the genetic control of root traits at the seedling stage provide valuable information about its behavior at later stages of the plant development under the field

Although drought stress reduces plant water potential, it affects root and leaf growth differently (Frensch, 1997). It shifts the weight based shoot to root ratios in favor of the roots and it is a well known adaptive mechanism of preferring root over shoot development. The larger root-to-shoot ratio under severe stress is a further indicator that a moderate water stress had been achieved as it promotes a relatively better functioning of the roots (El Nadi et al., 1969; Sharp & Davies, 1979; Westgate & Boyer, 1985). Tendency of plants to increase the root to shoot ratio by allocating assimilates to the roots is typical response of plants in the field. In this experiment, and some others (Suharjo, 2007), plants were grown in solution, with mild (4% PEG) osmotic stress and root was not stimulated to grow deeper to extract more water (as in the soil). For this reason, root to shoot ratio does not differ too much

dry soil more closely (Verslues et al., 2006).

measured (Kravic et al., 2010).

conditions.

compared to control conditions (Figure 6).

shoot growth to a greater extent than root growth (Table 1).

Table 1. Root and shoot length and percent (%) of reduction under control and stress condition (PEG)

In the genetic study of quantitative trait loci (QTLs) in maize seedling some QTLs were specific to water stress, such as QTLs for root dry weight, shoot dry weight, and leaf area-toroot length ratio (Ruta, 2010). The QTLs for shoot-to-root ratio, were collocated and overlapped with QTLs for ear number in the field under intermediate and severe stresses (Ribaut et al., 1996) and for the anthesis-silking interval were collocated with QTLs for the numbers of crown roots and seminal roots. An early increase in growth of main roots seemed to be linked to a later decrease in ASI, while an early increase in lateral root length distribution on the main axile root may extend ASI. Considering that conventional breeding may take about ten years from crossing to the final release of new maize hybrids, it is important to find rapid screening technique under laboratory conditions. QTLs controlling the balance between early root and shoot development may provide useful information to enable the prediction of maize performance under field conditions.

#### **6. Genetic diversity of landraces for grain quality**

Characterization of genetic diversity of maize landraces aids efficient exploring of the allelic variation for genetic improvement of economically desirable traits, such as grain quality. Maize is a relevant food source, so the quantification of the nutritionally important grain constituents is important for the best exploitation of different genotypes. In this context, the landraces represent a good source of genetic variability and may help to identify the most suitable materials for the development of more nutritious foods. Many studies have documented genetic and phenotypic variability for grain composition traits in maize (Pollak & Scott, 2005; Reynolds et al., 2005; Berardo et al., 2009; Has et al., 2010, Mittelmann et al., 2011).

To determine genetic diversity for grain quality traits a set of 54 landraces was grown in a randomized complete block design (RCBD) with two replications at one location. Grain

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 105

Abbreviations: MF-Montenegrin flints; BED-Bosnian early dent; KFD-Kosmet flinty dents;MaF-Macedonian flints; ERME-Eight rawed corn type of NE America; DF-Derived flints; MEF-

Fig. 7. Protein content of landraces of agro ecological groups

Denty flints.

Mediterranean flint, SKF-Small kernel flint; ERSD-Eight rowed soft dents; RF-Romanian flints; LEFlarge-earted flints, WFCM- White flinty dents Moravac; DUSA-Dent type of USA corn belt dents; DSEUSA-Dent type of southern east USA; DD-Derived dents; SD-Serbian dents; FD-Flinty dents; DF-

Fig. 8. Oil content of agroecological groups of landraces, abbreviations are same as on Fig. 7

Fig. 6. Root to shoot ratio (dry weight) in maize landraces (L1-L14) at seedling stage

protein, oil and starch content were measured using Infratec 1241 Grain analyzer, (Foss Tecator, Sweden) and expressed in percents (%). Phytate and Pi were determined by the method of Latta & Eskin (1980), modified by Sredojević & Dragicevic (2009).

The genetic variability for protein, oil and starch content was recorded in the analyzed landraces (Andjelkovic et al., 2011). The protein content ranged from a low level of 10.10% (a population from Derivated dents) to a high level of 14.85% (a population from Smallkernelled flints) and averaged 12.34%. The highest average protein content of 14.05% had population from Montenegrin flints and the lowest of 10.75%, one population from Derived dents (Figure 7). Landraces belonging to flint types have higher protein content than dent type landraces.

Populations from Montenegrin flints and Macedonian flints had the highest average and the second highest average oil content, 4.8% and 4.75%, respectively (Figure 8). The lowest average oil content had populations from Large-eared flints, 3.63%. Landraces belonging to flint types had higher oil content than landraces of dents type.

The starch content of maize landraces ranged from 66.35% to 71.50% and averaged 68.61% (Figure 9). The highest average starch content had populations from Derived flints (70.43%), and the lowest populations from Montenegrin flints (67.53%). Among landraces some interesting forms with high level of starch content were identified: Dent flints pop. 709 (71.10%), Derived flints pop. 190 (71.50%) and pop. 186 (70.95%), Dent type of USA Corn Belt Dents pop. 268 (70.85%).

It could be concluded that many tested landraces show an exceptional kernel quality based on the oil, protein and starch content, and this makes them suitable to be used for futher breeding improvment programs.

Fig. 6. Root to shoot ratio (dry weight) in maize landraces (L1-L14) at seedling stage

method of Latta & Eskin (1980), modified by Sredojević & Dragicevic (2009).

flint types had higher oil content than landraces of dents type.

type landraces.

Belt Dents pop. 268 (70.85%).

breeding improvment programs.

protein, oil and starch content were measured using Infratec 1241 Grain analyzer, (Foss Tecator, Sweden) and expressed in percents (%). Phytate and Pi were determined by the

The genetic variability for protein, oil and starch content was recorded in the analyzed landraces (Andjelkovic et al., 2011). The protein content ranged from a low level of 10.10% (a population from Derivated dents) to a high level of 14.85% (a population from Smallkernelled flints) and averaged 12.34%. The highest average protein content of 14.05% had population from Montenegrin flints and the lowest of 10.75%, one population from Derived dents (Figure 7). Landraces belonging to flint types have higher protein content than dent

Populations from Montenegrin flints and Macedonian flints had the highest average and the second highest average oil content, 4.8% and 4.75%, respectively (Figure 8). The lowest average oil content had populations from Large-eared flints, 3.63%. Landraces belonging to

The starch content of maize landraces ranged from 66.35% to 71.50% and averaged 68.61% (Figure 9). The highest average starch content had populations from Derived flints (70.43%), and the lowest populations from Montenegrin flints (67.53%). Among landraces some interesting forms with high level of starch content were identified: Dent flints pop. 709 (71.10%), Derived flints pop. 190 (71.50%) and pop. 186 (70.95%), Dent type of USA Corn

It could be concluded that many tested landraces show an exceptional kernel quality based on the oil, protein and starch content, and this makes them suitable to be used for futher

Abbreviations: MF-Montenegrin flints; BED-Bosnian early dent; KFD-Kosmet flinty dents;MaF-Macedonian flints; ERME-Eight rawed corn type of NE America; DF-Derived flints; MEF-Mediterranean flint, SKF-Small kernel flint; ERSD-Eight rowed soft dents; RF-Romanian flints; LEFlarge-earted flints, WFCM- White flinty dents Moravac; DUSA-Dent type of USA corn belt dents; DSEUSA-Dent type of southern east USA; DD-Derived dents; SD-Serbian dents; FD-Flinty dents; DF-Denty flints.

Fig. 7. Protein content of landraces of agro ecological groups

Fig. 8. Oil content of agroecological groups of landraces, abbreviations are same as on Fig. 7

Genetic Diversity of Maize Landraces as Sources of Favorable Traits 107

a Pi concentration 30% greater than Pi mean and will be used for further breeding genotypes

Landraces were developed through a complex adaptation process of different orginal genotypes to diverse climatic and soil conditions and therefore are the most accessible part of maize biodiversity. Genetic diversity of landraces have important role in maize breeding and could have a significant impact on the improvment of plants as a valuable source of useful traits. A considerable diversity among landraces from MRI genbank has been observed with morphological and molecular markers. The landraces are autochthonous source of potentially useful traits and alleles for improvement of the existing modern varieties. Fourteen most drought tolerant landraces were selected for further breeding programme aimed to broaden the genetic base of the elite breeding material. Presence of a wide genetic diversity of landraces based on their oil, protein, starch and phytate content, makes them more than suitable to be

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**7. Conclusion**

**8. References**

Fig. 9. Starch content of agroecological groups of landraces, abbreviations are same as on Fig.7

Many of the problems associated with P in maize grain are not due to concentration of the total P per se, but rather to the fact that most of the P is bound in phytate. Therefore, it would be desirable to increase the amount of available P and reduce the amount of phytate in maize grain. Approaches to resolve problems of the bad nutritive quality of grain phytate include engineering of crops to express high levels of *phytase* enzyme in seeds (Brinch-Pedersen et al, 2002), or through recurrent selection that uses the indigenous quantitative genetic variation (Raboy, 2009). Genetic variability in phytate contents of 54 landraces was observed, with values in the range from 1,147 to 4,13 and the average of 2.91 g kg-1 (Drinic et al, 2009). Twenty six population had high, twenty intermediate and eight low phytate content. Populations beloging to Derived flints had the lowest (1.95 g kg-1) while populations from Flinty dents had the highest (3.43 g kg-1) phytate content ,(Figure 10).

Fig. 10. Phytate and Pi contents of agroecological groups of landraces, abbreviations are same as on Fig.7

Populations from Mediterranean flints had the lowest (0.46 g kg-1) and populations from Dent type of southern east USA the highest (0.82 gkg-1) Pi content. One of the populations, i.e. population 216 was determined to have the lowest phytate concentration of 1.14 g kg-1, a Pi concentration 30% greater than Pi mean and will be used for further breeding genotypes with low phytate content and good agronomic traits.

#### **7. Conclusion**

106 The Molecular Basis of Plant Genetic Diversity

Fig. 9. Starch content of agroecological groups of landraces, abbreviations are same as on Fig.7

Many of the problems associated with P in maize grain are not due to concentration of the total P per se, but rather to the fact that most of the P is bound in phytate. Therefore, it would be desirable to increase the amount of available P and reduce the amount of phytate in maize grain. Approaches to resolve problems of the bad nutritive quality of grain phytate include engineering of crops to express high levels of *phytase* enzyme in seeds (Brinch-Pedersen et al, 2002), or through recurrent selection that uses the indigenous quantitative genetic variation (Raboy, 2009). Genetic variability in phytate contents of 54 landraces was observed, with values in the range from 1,147 to 4,13 and the average of 2.91 g kg-1 (Drinic et al, 2009). Twenty six population had high, twenty intermediate and eight low phytate content. Populations beloging to Derived flints had the lowest (1.95 g kg-1) while populations from Flinty dents had the highest (3.43 g kg-1) phytate content ,(Figure 10).

Fig. 10. Phytate and Pi contents of agroecological groups of landraces, abbreviations are

Populations from Mediterranean flints had the lowest (0.46 g kg-1) and populations from Dent type of southern east USA the highest (0.82 gkg-1) Pi content. One of the populations, i.e. population 216 was determined to have the lowest phytate concentration of 1.14 g kg-1,

same as on Fig.7

Landraces were developed through a complex adaptation process of different orginal genotypes to diverse climatic and soil conditions and therefore are the most accessible part of maize biodiversity. Genetic diversity of landraces have important role in maize breeding and could have a significant impact on the improvment of plants as a valuable source of useful traits. A considerable diversity among landraces from MRI genbank has been observed with morphological and molecular markers. The landraces are autochthonous source of potentially useful traits and alleles for improvement of the existing modern varieties. Fourteen most drought tolerant landraces were selected for further breeding programme aimed to broaden the genetic base of the elite breeding material. Presence of a wide genetic diversity of landraces based on their oil, protein, starch and phytate content, makes them more than suitable to be used for further maize breeding improvement programs.

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*1,2Italia*

**Genetic Biodiversity and Phylogenetic**

*1Dipartimento di Chimica e Biologia, Università di Salerno, Fisciano, Salerno*

Biodiversity can be defined as the totality of living forms present on earth, with the wealth of genetic information that they have (Grassi et al., 2006). It can be viewed at various levels, from ecosystems, down to species, populations, genomes and genes. Populations are representative of the species and may contain a large part of their genetic biodiversity. They are the basis of allopatric and sympatric speciation phenomena, due to a reduction of gene flow, caused by the presence of new geographical barriers, the former, and to auto- or allo-

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**1. Introduction**

polyploidy events, the latter.

(AFLPs, RAPDs, etc.).

**Studies in Poplar by Means of the**

**Metallothionein Multigene Family**

Angela Cicatelli1, Renato Lupi2, Francesco Sala2,

*2Dipartimento di Biologia, Università di Milano, Milano*

*Chinese Academy of Forestry, Wan Shou Shan, Beijing*

Han Yfan3 and Stefano Castiglione1

*3Research Institute of Forestry*

*3People's Republic of China*

### **Genetic Biodiversity and Phylogenetic Studies in Poplar by Means of the Metallothionein Multigene Family**

Angela Cicatelli1, Renato Lupi2, Francesco Sala2, Han Yfan3 and Stefano Castiglione1 *1Dipartimento di Chimica e Biologia, Università di Salerno, Fisciano, Salerno 2Dipartimento di Biologia, Università di Milano, Milano 3Research Institute of Forestry Chinese Academy of Forestry, Wan Shou Shan, Beijing 1,2Italia 3People's Republic of China*

#### **1. Introduction**

112 The Molecular Basis of Plant Genetic Diversity

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populations of alfalfa by random amplification of genomic DNA samples.

expression of the mitochondrial chimeric sequence R in S-type male sterility in

Biodiversity can be defined as the totality of living forms present on earth, with the wealth of genetic information that they have (Grassi et al., 2006). It can be viewed at various levels, from ecosystems, down to species, populations, genomes and genes. Populations are representative of the species and may contain a large part of their genetic biodiversity. They are the basis of allopatric and sympatric speciation phenomena, due to a reduction of gene flow, caused by the presence of new geographical barriers, the former, and to auto- or allopolyploidy events, the latter.

During the last two decades many molecular tools based on PCR (Polymerase Chain Reaction) have been developed and used to calculate and evaluate, through bioinformatics, genetic biodiversity of all living organisms and of plants in particular (Lowe et al., 2004). These molecular tools comprise DNA sequencing of single genes, or of entire genomes (*e.g.* rice, poplar, wheat, grape, etc.), locus specific (SSRs and SNPs) and random genome analysis (AFLPs, RAPDs, etc.).

Poplar, after man and rice, was one of the first organisms with a fully sequenced plant genome (Tuskan et al., 2006). Moreover it has also been a subject study of many plant biologists and physiologists due to its commercial and economic importance, and to its relevance for riparian ecosystems. Many populations and germplasm collections of different poplar species have been investigated (Brundu et al., 2008; Castiglione et al., 1993; Castiglione et al., 2010; Fossati et al., 2003) using random and locus specific molecular analysis to evaluate their genetic biodiversity. Poplar is a perennial flowering plant, native to most of the northern hemisphere, known to play valuable ecological roles: in fact, it is considered a trustworthy health indicator of riparian ecosystems and a promising phytoremediation tree of polluted soils (Castiglione et al., 2009; Sebastiani et al., 2004; Yadav

Genetic Biodiversity and Phylogenetic Studies in

Poplar by Means of the Metallothionein Multigene Family 115

genetic variability. Moreover, the rDNA gene family is known to be affected by concerted evolution and gene conversion events, which may alter the phylogenetic signals carried by the analyzed sequences. Despite these limitations, molecular evolution of plants is mainly inspected using rDNA and/or cpDNA alone, which possibly lead to evident mistakes in phylogeny reconstruction due to biases present at the molecular level (Alvarez & Wendel, 2003; Doyle & Gaut, 2000; Small et al., 2004). Undoubtedly, the drawing of accurate and resolved phylogenetic trees may be supported using additional molecular markers, such as nuclear –and thus mendelian-inherited– single-copy genes and/or multigene families. In particular, multigene families are very attractive since they offer the possibility to sample independent loci (*i.e.*, not associated in linkage units) that share a deeply related evolutionary framework. This is a crucial feature as far as hybridization and introgression events are concerned: thus, the use of such nuclear genes should be considered for reconstructing the phylogeny of poplar and also for assessing the genetic variability within the studied genus. Actually, several analyses of plant gene families, with particular emphasis on *P. trichocarpa*, the poplar model system, have been carried out in the recent past in order to understand their evolutionary history, the functional diversification of their members and their detailed expression in poplar (Lan et al., 2009; Petre et al., 2011). At present, however, little information is available about phylogenetic relationships among poplar species by means of nuclear genes analysis (Fladung & Buschbom, 2009). Hence, in order to shed light on the unresolved problematic issues, we have undertaken a deep investigation of the nuclear-encoded metallothionein (MT), a gene family involved in response to plant stress. In particular, MTs are known to have protective roles against plant wounds of pathogenic origins and senescence (Kohler et al., 2004), and to be involved in metal homeostasis (Cobbett & Goldsbrough, 2002), being upregulated in foliar tissue, together with polyamines, to enhance poplar tolerance towards heavy metal-contaminated soils (Castiglione et al., 2007; Cicatelli et al., 2010; 2011), or in water deficit (Berta et al., 2009). Nonetheless, no comprehensive study on MT gene family has been performed so far, even if it shows interesting and potentially attractive features. In fact MT multigene family is characterized by: (a) a quite high but manageable number of members (six), at least in *P. trichocarpa* (personal observation); (b) a peculiar organization of specific cysteine residues, whose function is cooperatively binding metal ions (Kille et al., 1991), thus allowing easy arrangement of orthologous MTs into three types, namely MT1, MT2 and MT3 (Cherian & Chan, 1993; Cobbett & Goldsbrough, 2002); (c) presence of a full-exon coding for a specific region, called "spacer", which show remarkable levels of genetic variability (Buchanan-Wollaston, 1994; Zhou & Goldsbrough, 1994). Indeed these spacers could be rich of singlenucleotide polymorphism (SNP). SNP including single-base changes or indels (insertion or deletion), at specific nucleotide positions, has been shown to be the most abundant class of DNA polymorphism in many organisms (Brookes, 1999; Cho et al., 1999). SNP variation analysis and SNP marker development from candidate genes could provide valuable information regarding their evolution and effects on complex traits. Very recently, Fladung and Buschbom (2009) have used partial DNA sequence of six nuclear genes to draw phyologenetic relationships among a limited number of poplar species using SNPs. This study substantiate the importance of this class of molecular markers as a very promising molecular tool to study the evolution or measure the genetic variability of plants, in general, and within *Populus,* in particular. The present study is part of a wider and ample collaborative project between Italy and the People's Republic of China focused on poplar.

et al., 2010). Poplars, aspens and cottonwoods are part of the genus *Populus* that, along with the two other related genera *Salix* and *Chosenia*, belongs to the Salicacae family. It is now generally accepted that the genus *Populus* comprises 29 species, identified on the basis of diagnostic morphological characters and assigned to six different sections, namely Abaso, Aigeros, Leucoides, Populus, Tacamahaca and Turanga (Dickmann, 2001). However, correct identification and classification of poplar species are made difficult by: (a) the very high variability of their morphological traits; (b) their strong tendency towards intra-section hybridization, especially in sympatric ranges, resulting in hybrids with mixed and broadspectrum parental features; (c) the unusual possibility to inter-section hybridization (*e.g.,* between specimens belonging to the Aigeros and Tacamahaca sections - Dickmann, 2001). Moreover, it should be noticed that fertile F1 hybrids could backcross towards one/both parental species, or produce further generations, thus making species and hybrid identification even more problematic (Fossati et al., 2004; Lexer et al., 2005). Recently, several studies using molecular markers have been carried out in order to revise the assignment of poplar species to the above-listed sections and even to resolve their controversial taxonomy (Cervera et al., 2005; Hamzeh & Dayanandan, 2004). Indeed, due to discrepancies among phylogenetic trees reconstructed with nuclear ribosomal DNA (rDNA) or chloroplast genes, it was established that nuclear genome (nDNA) of *P. nigra* (Aigeros) is divergent from species belonging to Populus section, although its chloroplast DNA (cpDNA) is strongly associated to that of trembling poplars (Populus) (Hamzeh & Dayanandan, 2004). Thus, *P. nigra* should not be considered derived from the inter-section crossing between *P. deltoides* x *P. alba* (Smith, 1988), but might be classified in a brand-new and independent taxon (*i.e.*, Nigrae). Further studies, using Amplified Fragment Length Polimorfisms (AFLP), revealed that *P. mexicana*, the single species present in the Abaso section, is so divergent from all the other poplar sections that might be considered part of the *Salix* genus (Cervera et al., 2005). Moreover, AFLP data also showed that species belonging to the Populus section are characterized by the greatest levels of interspecific genetic variability, whereas the opposite trend has been observed for the sections Aigeros and Tacamahaca. Given that ancestral species have greater variation with respect to the derived ones, Populus is believed to be the oldest section, whilst Aigeros and Tacamahaca the most recent ones (Cervera et al., 2005; Eckenwalder, 1996). However, controversial conclusions have been drawn on the taxonomy of the Populus section using different molecular markers. On one hand, the analysis of rDNA sequences indicates that Populus section could be further sub-divided into two major clades: one comprising *P. alba*, *P. tremula* and *P. davidiana* (Korean aspen); the second one grouping together the American aspens *P. tremuloides* and P. *grandidentata* (Hamzeh & Dayanandan, 2004). On the other hand, AFLP data suggested that *P. tremula* and *P. tremuloides* are ecotypes still capable of hybridization and thus part of the same group, whilst *P. alba* represents a totally different taxon (Cervera et al., 2005). In addition, Tacamahaca section was surprisingly found to be a polyphyletic group, *i.e.,* a kind of meta-cluster comprising many small sub-groups, each containing closely related species (Hamzeh et al., 2004). Finally, American cottonwoods *P. trichocarpa* and *P. balsamifera*, which have been ever considered subspecies of a single taxon, were discriminated as different species when assessed by AFLP analysis (Cervera et al., 2005). Such inconclusive and contrasting results indicate that both rDNA and AFLP markers are not suitable to correctly classify poplar species and fully resolve their phylogenetic relationships. Indeed, it should be stressed that AFLP analysis is intended to get insight into the overall structure of genomes, thus being more properly used to determine interspecific

et al., 2010). Poplars, aspens and cottonwoods are part of the genus *Populus* that, along with the two other related genera *Salix* and *Chosenia*, belongs to the Salicacae family. It is now generally accepted that the genus *Populus* comprises 29 species, identified on the basis of diagnostic morphological characters and assigned to six different sections, namely Abaso, Aigeros, Leucoides, Populus, Tacamahaca and Turanga (Dickmann, 2001). However, correct identification and classification of poplar species are made difficult by: (a) the very high variability of their morphological traits; (b) their strong tendency towards intra-section hybridization, especially in sympatric ranges, resulting in hybrids with mixed and broadspectrum parental features; (c) the unusual possibility to inter-section hybridization (*e.g.,* between specimens belonging to the Aigeros and Tacamahaca sections - Dickmann, 2001). Moreover, it should be noticed that fertile F1 hybrids could backcross towards one/both parental species, or produce further generations, thus making species and hybrid identification even more problematic (Fossati et al., 2004; Lexer et al., 2005). Recently, several studies using molecular markers have been carried out in order to revise the assignment of poplar species to the above-listed sections and even to resolve their controversial taxonomy (Cervera et al., 2005; Hamzeh & Dayanandan, 2004). Indeed, due to discrepancies among phylogenetic trees reconstructed with nuclear ribosomal DNA (rDNA) or chloroplast genes, it was established that nuclear genome (nDNA) of *P. nigra* (Aigeros) is divergent from species belonging to Populus section, although its chloroplast DNA (cpDNA) is strongly associated to that of trembling poplars (Populus) (Hamzeh & Dayanandan, 2004). Thus, *P. nigra* should not be considered derived from the inter-section crossing between *P. deltoides* x *P. alba* (Smith, 1988), but might be classified in a brand-new and independent taxon (*i.e.*, Nigrae). Further studies, using Amplified Fragment Length Polimorfisms (AFLP), revealed that *P. mexicana*, the single species present in the Abaso section, is so divergent from all the other poplar sections that might be considered part of the *Salix* genus (Cervera et al., 2005). Moreover, AFLP data also showed that species belonging to the Populus section are characterized by the greatest levels of interspecific genetic variability, whereas the opposite trend has been observed for the sections Aigeros and Tacamahaca. Given that ancestral species have greater variation with respect to the derived ones, Populus is believed to be the oldest section, whilst Aigeros and Tacamahaca the most recent ones (Cervera et al., 2005; Eckenwalder, 1996). However, controversial conclusions have been drawn on the taxonomy of the Populus section using different molecular markers. On one hand, the analysis of rDNA sequences indicates that Populus section could be further sub-divided into two major clades: one comprising *P. alba*, *P. tremula* and *P. davidiana* (Korean aspen); the second one grouping together the American aspens *P. tremuloides* and P. *grandidentata* (Hamzeh & Dayanandan, 2004). On the other hand, AFLP data suggested that *P. tremula* and *P. tremuloides* are ecotypes still capable of hybridization and thus part of the same group, whilst *P. alba* represents a totally different taxon (Cervera et al., 2005). In addition, Tacamahaca section was surprisingly found to be a polyphyletic group, *i.e.,* a kind of meta-cluster comprising many small sub-groups, each containing closely related species (Hamzeh et al., 2004). Finally, American cottonwoods *P. trichocarpa* and *P. balsamifera*, which have been ever considered subspecies of a single taxon, were discriminated as different species when assessed by AFLP analysis (Cervera et al., 2005). Such inconclusive and contrasting results indicate that both rDNA and AFLP markers are not suitable to correctly classify poplar species and fully resolve their phylogenetic relationships. Indeed, it should be stressed that AFLP analysis is intended to get insight into the overall structure of genomes, thus being more properly used to determine interspecific