many post - zygotic reproductive barrier forms have been reported in plants : hybrid weakness , hybrid necrosis , and hybrid chlorosis . the latter has been observed often in the f1 generation from crosses among wheat ( triticum aestivum l. ) and its relatives [ 26 ] .
research for distribution of these genes contributed greatly to the study of the origin of wheat .  
hybrid chlorosis in f2 generation has been reported only in rice ( oryza sativa l. )   and interspecific crosses among melilotus species .
sato et al .   incidentally found a case of hybrid chlorosis in the f2 population from a cross between two japanese native cultivars : j-147 and j-321 .
its first symptom was discoloration of the second or third leaf ( figure 1 ) .
the phenomenon was caused by a set of mutually independent duplicated recessive genes , named hca-1 and hca-2 by sato and morishima . according to the new gene nomenclature system for rice
, we changed our description of the gene symbols , as shown in table 1 .
( 1 ) the hca2 - 1 gene is widely distributed in native japonica - type cultivars , whereas many indica - type cultivars carry its dominant allele , hca2 - 2 .
this gene is probably rare because the occurrence of f2 chlorosis has not been reported in crosses between taichung 65 , which carries hca2 - 1 , and many cultivars except for j-147 .
the mode of inheritance differs between wheat hybrid chlorosis and that of rice , but the distribution of causal genes is related to varietal differentiation in both cases .  
we are interested in genes conferring the post - zygotic reproductive barrier in rice , and we have mapped these genes in the rice genome with the aid of dna markers [ 1114 ] .
we produced hybrids from crosses between j-147 and several cultivars to verify the results of sato and morishima . from them , we incidentally found chlorotic plants in the f2 population from the cross between j-147 and a philippine indica - type cultivar ir24 .
we have never seen chlorotic plants in f2 population from the cross between ir24 and rice cultivars except j-147 .
moreover , no reports in the literature describe chlorotic plants in an f2 population from the cross with ir24 .
in general , much dna polymorphism exists between japonica - type and indica - type cultivars .
this report describes linkage analysis of hca1 and hca2 using progeny from the f2 population between j-147 and ir24 assisted by dna markers .
then the molecular mechanism causing f2 chlorosis , as deduced from the location of the two genes , is discussed .
five rice cultivars were used for this study : j-147 , akihikari , asominori , milyang 23 , and ir24 .
yoshimichi fukuta of japan international research center for agricultural sciences provided akihikari and milyang 23 .
akihikari and milyang 23 are the parents of a set of recombinant inbred ( ri ) lines developed by fukuta et al . .
asominori and ir24 are the parents of another set of ri lines developed by tsunematsu et al . .
no report in the relevant literature describes the appearance of chlorotic plants in progeny from a cross between asominori and ir24 , or from a cross between akihikari and milyang 23 , although hybrid breakdown phenomena were reported for the cross between asominori and ir24 [ 17 , 18 ] , and in the cross between akihikari and milyang 23 .
j-147 , akihikari , and asominori are generally categorized as japonica - type , whereas milyang 23 and ir24 are generally categorized as indica - type .
j-147 was crossed with four cultivars : akihikari , asominori , milyang 23 , and ir24 .
chlorotic plants were segregated in the f2 population from the cross between j-147 and ir24 ( see section 3 ) .
the normal plants were transplanted to a paddy field in the experimental farm of kagoshima university to harvest self - pollinated seeds .
approximately 80 plants in each of 16 f3 lines were grown in the nursery for the segregation of chlorotic plants . among them , normal plants in the f3 lines in which chlorotic plants segregated were transplanted in the same way as the f2 generation .
approximately 80 plants in each f4 line were also grown in the nursery for segregation of the chlorotic plants .
f4 lines in which only hca1 or hca2 gene was expected to segregate were subjected to linkage analysis using dna markers .
preliminary analysis using a small number of plants detected the approximate locations of hca1 and hca2 .
then , we selected the f4 lines segregating chlorotic plants in which one locus is fixed for a recessive chlorosis - causing allele , whereas the other locus is heterozygous and the heterozygous chromosomal region encompasses the locus that is extended most .
after the linkage analysis , the f2 populations from the cross between j-147 and ir24 were grown again .
dna was extracted from each plant and subject to genotyping of the most closely linked dna markers with hca1 and hca2 loci to verify that hca1 - 1 and hca2 - 1 were sufficient to cause hybrid chlorosis in the f2 population from the cross between j-147 and ir24 . 

figure 2 shows a flow chart depicting the breeding of the plant materials for mapping hca1 and hca2 and for verification that hca1 - 1 and hca2 - 1 were sufficient to cause hybrid chlorosis in the f2 population from the cross between j-147 and ir24 .
the dna of plant materials except for the f2 population was extracted using the process explained by dellaporta et al .   with some modifications .
the dna of the f2 population was extracted according to the experimental protocols of the rice genome project ( rgp ) ( http://rgp.dna.affrc.go.jp/e/rgp/protocols/index.html , written in japanese ) with some modifications .
the pcr conditions for indel and ssr markers used for this study were 95c for 10  min , 40 cycles of 94c for 30  s , 55c for 30  s , and 72c for 30  s with subsequent final extension of 72c for 1  min . the pcr mixture ( 5  l ) contained 1  l of template dna , 200  mm of each dntp , 0.2  m of primers , 0.25 units of taq polymerase ( amplitaq gold ; applied biosystems ) , and 1  buffer containing mgcl2 .
the pcr products were analyzed using electrophoresis in 10% ( 29  :  1 ) polyacrylamide gel with subsequent ethidium bromide staining
details of pcr - based dna marker design were reported in our previous papers [ 13 , 14 ] .
the f2 populations of the four cultivars with j-147 all produced both normal plants and chlorotic plants ( table 2 ) .
the ratios of normal plants and chlorotic plants were fitted to 15  :  1 , the expected ratio deduced from the segregation of two independent recessive genes .
these results indicated that j-147 carries hca1 - 1 gene and that akihikari , asominori , milyang 23 , and ir24 carry the hca2 - 1 gene .  
indica - type cultivars and japonica - type cultivars have frequently shown dna polymorphism between them .
this is true for asominori and ir24 , and for akihikari and milyang 23 . using the dna polymorphism between the two pair of cultivars ,
dna marker - based linkage maps were constructed [ 15 , 16 ] . results of our recent study indicated much polymorphism between indica - type cultivars and j-147 , and little polymorphism between japonica - type cultivars and j-147 . therefore , the progeny from the cross between indica - type cultivars and j-147 were more suitable for mapping genes .
we selected the progeny from the cross between ir24 and j-147 as a mapping population for hca1 and hca2 because the linkage map constructed from the cross between asominori and ir24 covered almost the whole genome , whereas that constructed from akihikari and milyang 23 had some large gaps , suggesting that some chromosomal regions exist with no dna polymorphism .
seeds of normal plants in the f2 population were harvested to produce the f3 generation .
the segregation of chlorotic plants was examined for 16 f3 lines from each f2 plant .
chlorotic plants segregated in six lines . in the six lines , the ratios of normal plants : chlorotic plants were all fitted to 15  :  1 . in the f3 generation , the expected ratio of lines fixed for normal plants , those showing 3 normal : 1 chlorotic segregation , and others showing a 15 normal : 1 chlorotic segregation , was 7  :  4  :  4 . however , no lines showing 3 normal : 1 chlorotic segregation appeared .
then , six f3 lines in which segregated chlorotic plants were transplanted to a paddy field , and the seeds of normal plants were harvested to produce f4 generation . the segregation of chlorotic plants was examined for 211 f4 lines from each f3 plant .
the line named tf4 27 - 10 was a progeny from an f3 line tf3 27 . in tf4 27 - 10 , 70 normal plants and 10 chlorotic plants segregated , showing the maximum chlorotic plant ratio among the 10 tf3 27-derived f4 lines tested in our first experiment in the f4 generation and significantly deviated from 15  :  1 ratio .
another line named tf4 23 - 5 was the progeny of an f3 line tf3 23 . in tf4 23 - 5
, 80 normal plants and 7 chlorotic plants segregated , fitted to a 15  :  1 ratio .
we performed a preliminary linkage analysis using a bulked dna composed of 20 normal plants from tf4 27 - 10 and another bulked dna composed of 20 normal plants from tf4 23 - 5 , and 39 dna marker scattered on all the 12 chromosomes .
because of successive self - pollination , the ratio of heterozygous chromosomal region reduced to approximately 0.25 in the f4 generation .
hca1 and/or hca2 are expected to be located on heterozygous chromosomal region of the lines in which chlorotic plants segregated . among the dna markers , four showed heterozygosity in both lines
possible linkage was observed between chlorosis and kgs1739 , a dna marker located on the short arm of chromosome 12 : no homozygotes of ir24 allele were detected .
then we analyzed 7 chlorotic plant and 54 normal plants in tf4 27 - 10 line individually .
results showed that the cosegregation of hca1 and the more distal marker rm27421   was detected : all 7 chlorotic plants in this line were homozygous for j-147 allele at the rm27421 locus , whereas all 54 normal plants were heterozygous or homozygous for the ir24 allele at the locus .
based on rflp - based linkage analysis using the f2 population from the cross between nipponbare and kasalath , nagamura et al .   reported that the distal regions of the short arms of rice chromosomes 11 and 12 are highly conserved as a duplicated chromosomal segment .
confirmed the high degree of conservation of duplicated segments in these regions using four other mapping populations .
moreover , wu et al .   generated physical maps covering most of the duplicated regions .
these experimentally obtained results and the fact that the hybrid chlorosis was controlled by a couple of recessive duplicate genes led us to the idea that hca2 might be located on the distal region of the short arm of chromosome 11 .
our preliminary analysis using an f4 line named tf4 29 - 30 showed the cosegregation of hca2 and e30794 , an sts marker designed by rgp ( http://rgp.dna.affrc.go.jp/e/publicdata/caps/index.html ) , located on the distal region of the short arm of chromosome 11 : all 20 chlorotic plants in this line were homozygous for ir24 allele at the e30794 locus , whereas all 68 normal plants were heterozygous or homozygous for j-147 allele at the locus . to construct linkage maps around the hca1 and hca2 loci , we selected f4 lines in which one locus is fixed for a recessive chlorosis - causing allele and the other locus is on the heterozygous region extending farthest . using these lines ( hca1 : tf4 33 - 21 , hca2 : tf4 23 - 19 , see figure 2 ) , we conducted linkage analysis of each gene using the dna markers shown in table 3 .
the dna marker sources were the international rice genome sequencing project , rgp , mccouch et al .
, chen et al . , monna et al . , and ichitani et al . .
the linkage analysis of hca1 gene and six dna markers using 13 chlorotic plants and 102 normal plants in tf4 33 - 21 showed that hca1 was located at the distal end of the short arm of chromosome 12 and that it cosegregated with rm27404 ( figure 3 ) : all 13 chlorotic plants in this line were homozygous for j-147 allele at the rm27404 locus , whereas all 102 normal plants were heterozygous or homozygous for ir24 allele at the locus .
the ratio of 102  :  13 did not fit to the expected ratio 3  :  1 .
cosegregation of hca1 and rm27404 and distorted segregation of the linked dna markers ( table 4 ) showed that the distorted segregation of hca1 resulted from gametophytic reproductive barrier gene(s ) , often found in the cross between indica - type and japonica - type ( see section 4 ) .
the linkage analysis of hca2 gene and seven dna markers using 23 chlorotic plants and 96 normal plants in tf4 23 - 19 showed that hca2 was located at the distal end of the short arm of chromosome 11 and that it cosegregated with rm25969 ( figure 3 ) : all 23 chlorotic plants in this line were homozygous for ir24 allele at the rm25969 locus , whereas 96 normal plants were heterozygous or homozygous for j-147 allele at the locus .
the ratio of 23 chlorotic plants : 96 normal plants fitted the expected ratio 1  :  3 .
the segregation of the tightly linked dna markers of hca2 also fitted the expected ratio 1  :  2  :  1 ( table 4 ) .
constructed an often - cited restriction fragment length polymorphism ( rflp ) marker - based high - density linkage map for rice , in which some rflp markers have been sequenced .
based on the nipponbare genome sequence , the relation between our linkage map and that by harushima et al .  
however , the ratio of normal plants to chlorotic plants was often distorted from the expected one ; that is , the chlorotic plants were often significantly fewer than expected .
therefore , we again produced the f2 generation from the cross between j-147 and ir24 to confirm that hca1 - 1 and hca2 - 1 are sufficient to cause chlorosis in f2 generation with the aid of tightly linked dna markers .
a total of 503 f2 plants were classified into 481 normal plants and 22 chlorotic plants .
the ratio 481  :  22 looked slightly skewed towards normal plants but fitted the expected ratio 15  :  1 (  = 3.033 , p = 0.08 ) .
genotypes at the rm25969 and rm27404 loci were analyzed for all f2 plants ( table 5 ) .
no homozygotes of j-147 allele at the rm27404 locus and ir24 allele at the rm25969 locus were present in normal plants .
in contrast , all chlorotic plants were homozygotes of j-147 allele at rm27404 locus and ir24 allele at the rm25969 locus .
this result indicated that hca1 - 1 and hca2 - 1 were sufficient to cause chlorosis in f2 population from the cross between j-147 and ir24 .
segregation of rm25969 fitted the expected ratio 1  :  2  :  1 , although that of rm27404 did not fit the expected ratio : homozygotes of ir24 allele were more numerous than expected , and homozygotes of j-147 allele were much fewer than expected .
the distorted segregation of chlorotic plants in f3 generation was thought to result from abnormal segregation at the chromosomal region around the hca1 locus .
in this study , hca1 and hca2 were located on the respective distal regions of the short arms of chromosomes 12 and 11 .
these regions are known to be highly conserved as a duplicated chromosomal segment [ 2426 ] . according to the rice genome annotation project ( http://rice.plantbiology.msu.edu/index.shtml ) , 30 genes with known functions
therefore , hca1 and hca2 are thought to be mutually homoeologous genes , and the loss - of - function mutations of these genes are thought to be the cause of hybrid chlorosis . based on the symptom
, the causal genes of hybrid chlorosis should be involved in chlorophyll synthesis or chloroplast metabolism .  
recently , two papers reported the reciprocal disruption of duplicate genes causing reproductive barrier in interspecific and intraspecific rice crosses .
yamagata et al .   reported that the reciprocal loss of duplicated gene encoding mitochondrial ribosomal protein l27 causes hybrid pollen sterility in f1 hybrids of o. sativa and o. glumaepatula .
( dpl1 ) and doppelganger2 ( dpl2 ) causing pollen sterility : independent disruption of dpl1 and dpl2 occurred , respectively , in indica - type and japonica - type .
recessive embryo lethality is caused by a disruption of duplicated histidinol - phosphate amino - transferase genes encoding for a protein that catalyzes an important pathway leading to histidine incorporated into proteins .
the hybrid chlorosis described in this study might offer a new example of a reproductive barrier caused by disruption of duplicate genes .
the segregation of hca2 and its tightly linked dna marker rm27404 was significantly distorted from the expected ratio : the frequencies of japonica - type alleles were smaller than expected .
the segregation distortion of the distal end of the short arm of chromosome 12 was reported in the populations derived from the crosses between indica - type cultivars and japonica - type cultivars : ir24 and asominori , and milyang 23 and akihikari .
the peak of distortion was at an rflp marker xnpb193 (= g193 ) , and the frequencies of japonica - type alleles were smaller than expected in both crosses .
therefore , the segregation distortion observed in this study might result from the same genetic factor(s ) .
carriers of the weakness - inducing allele hwc2 - 1 , most of which are categorized as temperate japonica - type , share the same haplotype in the 200  kb region between the two dna markers , kgc4m5 and kgc4m52 .
the carriers of hwc2 - 2 have different haplotypes , most of which are distinct from those of hwc2 - 1 carriers .
these results suggested that hwc2 - 1 diffused in temperate japonica - types , dragging adjacent genes with it . the hca2 - 1 gene is mainly carried by japonica - type cultivars .
therefore , the haplotype analysis of hca2 might engender new findings related to japonica - indica differentiation .
we are undertaking haplotype analysis of hca2 using a core collection of world rice   and a minicollection of japanese rice landrace . before the experiment
, we expected that akihikari and asominori , both generally classified as japonica - type , carry hca2 - 1 gene whereas milyang 23 and ir24 , both generally classified as indica - type , carry the wild type hca2 - 2 .
based on that expectation , we produced an experimental design in which j-147 was crossed with a set of ri lines so that linkage between hca2 and dna markers was detectable by combining the genotype of each ri line of dna markers on the whole genome , which had been analyzed by the breeders of the ri lines , and the segregation of chlorotic plants in the f2 population between j-147 and each ri line .
the experimental design using ri lines for mapping a reproductive - barrier - related gene was successful in a hybrid weakness gene hwc2 .
however , contrary to our expectation , all four cultivars carry hca2 - 1 gene .
used a 1,536 snp panel genotyped across 395 rice diverse accessions to study genomic - wide pattern of polymorphism , to characterize population structure , and to infer the introgression history , revealing that most accessions exhibit some degree of admixture , with many individuals within a population sharing the same introgressed segment because of artificial selection .
therefore , a high probability exists that the hca2 locus and its surrounding chromosomal region of ir24 were introgressed from a japonica - type cultivar .
however , another possibility exists : loss - of - function mutation occurred at the hca2 locus independently in indica - type cultivars .
ichitani et al .   reported that j-147 shares the same banding patterns of 38 out of 39 pcr - based dna markers scattered on the whole genome with the three cultivars generally classified as temperate japonica - type .
a dna marker s1284 ( table 3 , figure 3 ) , located at 2.2  mbp from distal end of short arm of chromosome 11 ( irgsp pseudomolecules build05 ) , clearly discriminates japonica - type and indica - type , and j-147 carries the japonica - type allele .
the hca2 - 2 allele of j-147 might also be the result of the introgression of small chromosomal segment of the distal end of the short arm of chromosome 11 . to identify the causal genes
, we are undertaking closer linkage analysis and linkage disequilibrium analysis of both hca1 and hca2 genes .
identification of causal genes will contribute to the study of rice varietal differentiation , gene duplication , and chlorophyll synthesis or chloroplast metabolism .
 many postzygotic reproductive barrier forms have been reported in plants : hybrid weakness , hybrid necrosis , and hybrid chlorosis . in this study , 
 linkage analysis of the genes causing hybrid chlorosis in f2 generation in rice , hca1 and hca2 , was performed . 
 hca1 and hca2 are located respectively on the distal regions of the short arms of chromosomes 12 and 11 . 
 these regions are known to be highly conserved as a duplicated chromosomal segment . 
 the molecular mechanism causing f2 chlorosis deduced from the location of the two genes was discussed . 
 the possibility of the introgression of the chromosomal segments encompassing hca1 and/or hca2 was also discussed from the viewpoint of indica - japonica differentiation . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion