Introduction {#s1}
============

Self-incompatibility (SI) is a widespread genetic system in many flowering plants which serves to prevent self-fertilization and maintain genetic diversity. It is based on self/non-self pollen--pistil recognition interactions followed by inhibition of self-pollen hydration, germination, or pollen tube growth.

In the Brassicaceae, SI is sporophytically controlled by a multiallelic locus termed the *S* locus ([@CIT0002]). Male and female determinants have been identified as SP11/SCR (*S*-locus protein 11/*S*-locus cysteine-rich) ([@CIT0038]; [@CIT0049]) and SRK (*S* receptor kinase) ([@CIT0047]), respectively. When a compatible pollen grain lands on the stigma, it swells and a pollen tube is allowed to grow, whereas when self-pollen attaches to the stigma, SP11/SCR binds specifically to the extracellular domain of SRK of the same *S*-haplotype ([@CIT0050]), which triggers an SI signalling pathway to reject self-pollen. Another stigmatically expressed gene located at the *S* locus, *S* locus glycoprotein (*SLG*) ([@CIT0028]; [@CIT0048]), has been shown to enhance the recognition process between self-pollen and stigma ([@CIT0047]); however, this function of *SLG* remains controversial ([@CIT0040]). A recent study suggested that the plants in the Brassicaceae genus Levenworthia use paralogous *SRK* and *SP11/SCR* genes, *Lal2* (*Leavenworthia alabamica SRK-related 2*) and *SCRl* (*SCR-like*), for self/non-self recognition in SI, but the function of their orthologues in other Brassicaceae genera also remains unknown ([@CIT0005]).

Many studies focusing on the downstream components involved in this type of SI signalling pathway have been performed and, thus far, two components have been identified as positive effectors. ARC1 (arm repeat containing 1) was identified by a yeast two-hybrid screen using the kinase domain of SRK as the bait ([@CIT0010]; [@CIT0042]). ARC1 is a U-box protein with E3 ubiquitin ligase activity ([@CIT0041]), and has been shown to interact with Exo70A1, a putative component of the exocyst complex required for compatible pollination ([@CIT0036]). MLPK (*M*-locus protein kinase) was identified by positional cloning as the gene responsible for the self-compatible mutation of *Brassica rapa* var. Yellow sarson ([@CIT0024]). MLPK is a membrane-anchored cytoplasmic protein kinase and interacts directly with SRK to transduce SI signalling ([@CIT0017]). However, the importance of these two components in the Brassicaceae SI mechanism remains controversial ([@CIT0018]; [@CIT0015]).

In the Brassicaceae, it has been known that SI can be overcome under some physiological and environmental conditions such as plant age ([@CIT0032]; [@CIT0013]), stigmatic chemical treatments (e.g. ether, KOH, and NaCl) ([@CIT0051]; Tao and Yong, 1986; [@CIT0023]), and high temperature ([@CIT0022]; [@CIT0033]). CO~2~ gas (3--5%) treatment ([@CIT0026]) is the most effective way to overcome SI. Today, most cultivated lines of crucifer vegetables, such as cabbage, broccoli, Chinese cabbage, and radish are F~1~ hybrids whose seeds are produced by mix-planting two self-incompatible inbred parental lines. In this economical F~1~ hybrid breeding system, CO~2~ gas treatment has been used to suppress SI and allow self-fertilization, thereby providing large-scale seed propagation of parental lines. This method has been used all over the world for many years, but the molecular mechanism leading to SI breakdown by CO~2~ gas treatment is entirely unknown.

Previous studies suggested that not all lines respond equally to CO~2~, and there are variations in SI response to CO~2~ (CO~2~ sensitivity) in the Brassicaceae ([@CIT0027]; [@CIT0029]). Preliminary genetic analysis using lines with different CO~2~ sensitivity in radish (*Raphanus sativus*) suggested that high CO~2~ sensitivity was controlled by a recessive gene independent of the *S*-locus ([@CIT0029]). In another genetic analysis using CO~2~-sensitive and CO~2~-insensitive lines of Chinese cabbage (*B. rapa*), high CO~2~ sensitivity was suggested to be controlled by a dominant gene ([@CIT0014]); however, no responsible genes have been identified from these studies so far.

In this study, new inbred lines of *B. rapa* with different CO~2~ sensitivity, a CO~2~-sensitive line (HA-11621) and a CO~2~-insensitive line (HA-11623), were selected and analysed. X-ray microanalysis suggested that SI breakdown in the CO~2~-sensitive line was accompanied by significant calcium accumulation at the pollen--stigma interface. Independent pre-treatment of pollen or pistil with CO~2~ gas before pollination showed no effect on the SI reaction, suggesting that some physiological process that occurs after pollination is necessary for SI to be overcome. Genetic analyses using F~1~ and F~2~ progeny of a CO~2~-sensitive×CO~2~-insensitive cross suggested that CO~2~ sensitivity is a semi-dominant and quantitative trait. Furthermore, quantitative trait locus (QTL) analyses identified two major responsible loci, *BrSIO1* and *BrSIO2*, which function additively in overcoming SI during CO~2~ treatment.

Materials and methods {#s2}
=====================

Plant materials {#s3}
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Two inbred lines of *B. rapa* (2*n*=20), a CO~2~-sensitive line (HA-11621) and a CO~2~-insensitive line (HA-11623), were established at Tohoku Seed Co., Ltd, and grown in the greenhouse with 16h light and 8h dark conditions at 20 °C. Both lines show stable SI under normal (open-air) condition but have different CO~2~ sensitivity; SI in HA-11621 breaks down following treatment with 4.5% CO~2~ whereas SI in HA-11623 is unaffected. HA-11621 and HA-11623 are reciprocally compatible, and their F~1~ progeny were obtained under normal conditions. Buds (1--2 d before flowering) from a randomly chosen F~1~ were used for F~2~ production. Young petals and stamens were removed from the bud, and the immature pistil was pollinated with pollen grains from mature flowers of the same plant (bud pollination). Pollinated pistil was then covered with a paper bag for 3 d and seeds from the pistil were harvested. More than 20 pistils were pollinated, and harvested seeds were used as the F~2~ population. A total of 110 F~2~ plants were used for phenotypic and genetic analysis.

Cryo-scanning electron microscopy and energy-dispersive X-ray analysis {#s4}
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Flowers were self- or cross-pollinated and incubated for 1.5h with or without 4.5% CO~2~ gas. These pollinated and non-pollinated pistils were submersed in liquid nitrogen slush and frozen under vacuum. While under vacuum, the sample was transferred to the microscope cryo stage (ALTO 1000, Gatan), and then the stage temperature was increased to --95 °C to remove frost that had settled on top of the specimen as a result of condensation. When all surface frost had been removed by sublimation, as verified by electron microscopy, the temperature was reduced to --140 °C. Imaging was performed using an ETD (Everhart--Thornley detector) by Quant 250 scanning electron microscopy (FEI). The chamber pressure was 30 Pa and the accelerating voltage was 15kV. EDX (energy-dispersive X-ray spectroscopy) analysis of the element assay was performed on selected papilla cells using INCA X-ray analysis software (Oxford Instruments, <http://www.oxinst.com/Pages/home.aspx>, last accessed 14 December 2013), with the detector's processing time set at 2. X-ray data were collected with 4.5 nA probe current for 2min. Each 2--3 pistils were used in one experiment and three individual experimental sets were performed.

Evaluation of reaction level of SI to CO~2~ (RLSICO~2~) {#s5}
-------------------------------------------------------

Three to five flowers were cut at the peduncle and stood on a 1% (w/v) solid agar plate. Flowers were self-pollinated, placed into a CO~2~ incubator, and treated with 4.5% CO~2~ for 4h at 23 °C. After 1 d at room temperature, pistils were fixed in ethanol:acetic acid (3:1) overnight, softened in 1 N NaOH at 60 °C for 2h, then stained with 0.01% (w/v) decolorized aniline blue in 2% K~3~PO~4~ for 6h. Pollen tube behaviour was observed under a fluorescent microscope (Axiophot 2, Zeiss). CO~2~ sensitivity was measured using the RLSICO~2~ index. RLSICO~2~ was classified into five categories, based on the number of pollen tubes penetrating into the stigma: 1, 0 pollen tubes; 2, 1--5 pollen tubes; 3, 6--15 pollen tubes; 4, 16--30 pollen tubes; and 5, \>30 pollen tubes. Three replicates were performed on each plant on different days. Non-CO~2~-treated self-pollinated flowers were used as controls. In all cases, no pollen tubes penetrated into the control stigmas.

Genotyping of *S*-haplotypes {#s6}
----------------------------

*S*-haplotypes of *B. rapa* were identified using primers PS5 (5′-ATGAAAGGCGTAAGAAAAACCTA-3′) and PS15 (5′-CCG TGTTTTATTTTAAGAGAAAGAGCT-3′) ([@CIT0030]) to amplify a fragment of the *SLG* gene. PCR-RFLP (restriction fragment length polymorphism) was used to distinguish the two *S*-haplotypes based on differential digest with the restriction enzyme *Kpn*I (TaKaRa, Japan). Digested DNA was electrophoresed on a 1.5% agarose gel.

Molecular markers and detection of DNA polymorphism {#s7}
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To screen for markers that show polymorphism between *B. rapa* lines, primers specific for simple sequence repeat (SSR) markers from different sources \[UK, prefixes Ra, Na, Ol, and ENA ([@CIT0021]; <http://brassica.bbsrc.ac.uk>); Japan, prefixes BRMS, KBr, and EST (Suwabe et al., [@CIT0043], [@CIT0044], [@CIT0046]; <http://vegmarks.nivot.affrc.go.jp>, NIVTS); China, prefix sau_um ([@CIT0009]); and Korea, prefix AMCP ([@CIT0035])\] were used. SSR, RFLP, and insertion/deletion (InDel) markers (prefixes XT and Bra) were also designed based on the *Brassica* database (BRAD) (<http://brassicadb.org/brad/>, last accessed 14 December 2013) ([Supplementary Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1) available at *JXB* online).

Total genomic DNA was extracted from young leaves of two parental lines and F~2~ progeny using the cetyltrimethylammonium bromide (CTAB) method ([@CIT0025]). DNA polymorphism analysis with SSRs was carried out using PCR with fluorescent dyes, performed according to [@CIT0045] with some modifications. The M13 (--21) universal primer sequence (18bp) was fused to the 5′ end of the original forward primer, and the M13 (--21) universal primer was labelled with 6-FAM, NED, VIC, or PET fluorescent dye (Applied Biosystems, CA, USA). PCRs were performed in a 10 μl reaction volume containing 10ng of template DNA, 4.7 μM of labelled M13 (--21) universal primer and reverse primer, 0.3 μM of forward primer, 1× PCR buffer, 1× dNTP, 1× MgCl~2~, and 0.5U of rTaq (TOYOBO, Japan). Conditions for PCR were as follows: initial denaturation was carried out at 94 °C for 3min followed by 37 cycles at 94 °C for 30 s, 55 °C (slope of 0.5 °C s^--1^) for 30 s, 72 °C (slope of 0.5 °C s^--1^) for 30 s, and a final extension at 72 °C for 4min. A 1 μl aliquot of 50-fold diluted PCR product was added to 8.9 μl of Hi-Di™ Formamide and 0.1 μl of GeneScan™ 600 LIZ™ Size Standard (Applied Biosystems, USA) and applied to an ABI 3730 DNA Analyzer (Applied Biosystems). Data were analysed using ABI GeneMapper^®^ software.

For polymorphism analysis with RFLP and InDel markers, PCR was carried out in a 10 μl reaction volume with 5 pmol of forward and reverse primers instead of fluorescent dyes. For RFLP markers, amplified fragments were digested using restriction enzymes for 1h. Fragments of digested DNA were separated on a 2--4% agarose gel.

Linkage map construction and QTL analysis {#s8}
-----------------------------------------

A genetic map was constructed using JoinMap^®^ version 4 ([@CIT0054]) utilizing the double pseudo-testcross strategy with a log~10~ of odds (LOD) threshold of 6.0 for linkage group identification. The best marker order was calculated with the regression mapping algorithm, and marker order was retained from the first round only. Map distance units in centiMorgans (cM) were converted from recombination frequencies using the Kosambi mapping function ([@CIT0019]). Interval mapping (IM) was performed to identify putative QTLs using the established linkage map and the observed phenotypic traits. This method was run using MapQTL^®^ version 6 ([@CIT0055]). With this software, a *P* \< 0.05 LOD score significance threshold was calculated by creating a group-wide distribution of the data based on a 1000 permutation test. LOD peaks were used to estimate the position of QTLs on the map.

Statistical analysis {#s9}
--------------------

Box plots were prepared by Ekuseru-Toukei 2012 software (Social Survey Research Information Co., Ltd, Japan) to compare the phenotypic difference, as this plot type gives a good sense of environmental data distribution ([@CIT0053]). Kruskal--Wallis analysis of variance (ANOVA) by ranks was used between paired comparisons of markers to examine marker association.

Results {#s10}
=======

Phenotypic analysis of *B. rapa* lines in response to CO~2~ treatment {#s11}
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Two inbred lines of *B. rapa* with different CO~2~ sensitivity, a CO~2~-sensitive line (HA-11621) and a CO~2~-insensitive line (HA-11623), were used in this study. Flowers were self-pollinated by hand pollination and incubated in a CO~2~ incubator (4.5% CO~2~) for 4h. Both lines were self-incompatible under normal conditions (control) ([Fig. 1A](#F1){ref-type="fig"}, [B](#F1){ref-type="fig"}), whereas they showed significantly different responses to CO~2~ gas treatment ([Fig. 1C](#F1){ref-type="fig"}, [D](#F1){ref-type="fig"}). Specifically, in the CO~2~-sensitive line, many pollen tubes were seen to penetrate into papilla cells after CO~2~ treatment. This pollination test confirmed that the CO~2~-sensitive line had high sensitivity to CO~2~ while the CO~2~-insensitive line hardly responded to 4.5% CO~2~. Cross-pollination was performed as a positive control ([Fig. 1E](#F1){ref-type="fig"}, [F](#F1){ref-type="fig"}).

![Phenotype of inbred *Brassica rapa* lines used in this study. (A, B) Pollen tube behaviour after self-pollination of CO~2~-sensitive (HA-11621) and CO~2~-insensitive (HA-11623) lines under normal conditions (without CO~2~ treatment). No penetrated or elongated pollen tubes are observed in either line. (C, D) Pollen tube behaviour after self-pollination of CO~2~-sensitive and CO~2~-insensitive lines under 4.5% CO~2~ gas treatment. Pollen tubes could penetrate into the stigma and elongate through the style only in the CO~2~-sensitive line. (E, F) Cross-pollination as a positive control; the arrow shows pollen tubes which have penetrated. Pt, pollen tubes. Bar=1000 μm.](exbotj_ert438_f0001){#F1}

Physiological changes in papilla cells after CO~2~ treatment {#s12}
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Previous work using X-ray microanalysis has revealed the accumulation of calcium at the stigmatic surface following compatible cross-pollination in *Brassica oleracea* ([@CIT0008]). X-ray mapping strongly indicated that a high concentration of calcium was localized at the points where the pollen grain made contact with the surface of the stigmatic papilla cell. The calcium accumulation was also observed in *B. rapa*, especially in compatible pollination ([@CIT0016]). To examine the physiological effect of CO~2~ on pollination reactions, this calcium accumulation was examined by using cryo-scanning electron microscopy fitted with an X-ray microanalysis system. When the CO~2~-sensitive line was cross-pollinated with the CO~2~-insensitive line, pollen grain hydrated and the pollen tube penetrated into the papilla cell, but when CO~2~-sensitive and CO~2~-insensitive lines were self-pollinated under normal conditions (without CO~2~ treatment), few pollen grains hydrated, and no pollen tube germination was observed in either line ([Fig. 2A](#F2){ref-type="fig"}, upper panel). After CO~2~ treatment, the cross-pollinated pollen grains did not show a significant difference, neither did the self-pollinated CO~2~-insensitive line. However, the self-pollinated CO~2~-sensitive line showed obvious changes: pollen hydration and germination were observed under CO~2~ treatment ([Fig. 2A](#F2){ref-type="fig"}, lower panel). The emission of elements (Kα) in the tip of the papilla cells was then analysed after selecting the pollinated papilla cells which faced nearly the same direction in relation to the X-ray detector. Emissions of P-Kα, S-Kα, K-Kα, and Ca-Kα were detected together with C and O, which are constituent elements of biological materials. The detected emission of Al-Kα originates from the stub that held the samples ([Fig. 2B](#F2){ref-type="fig"}). There was no significant difference in the elemental emissions between CO~2~-sensitive and CO~2~-insensitive lines before pollination. Ca^2+^ accumulation was observed after cross-pollination as previously reported ([@CIT0016]), and Ca-Kα emission was increased with CO~2~ treatment. After self-pollination, Ca-Kα emission was slightly increased in the CO~2~-sensitive line in the normal CO~2~ condition. This Ca-Kα increase became more pronounced (\~6-fold) in the CO~2~-sensitive line when it was self-pollinated in the high CO~2~ condition. In the CO~2~-insensitive line, no significant Ca-Kα increase was observed after self-pollination in normal or high CO~2~ conditions ([Fig. 2B](#F2){ref-type="fig"}). Although the biological significance of the accumulation of calcium at the pollen--stigma interface is not clear, it has been suggested that calcium plays some role in the successful development of the pollen tube tip into the region of expanded stigmatic wall ([@CIT0008]). The present results suggest that, in the CO~2~-sensitive line, high CO~2~ activates a compatible pollination pathway or disturbs an SI signalling pathway leading to Ca^2+^ accumulation at the pollen--stigma interface.

![Electron micrographs and X-ray microanalysis of *B. rapa*. (A) Cryo-scanning electron micrographs of pollinated papilla cells were taken 1.5h after pollination. Representative examples of the cross-pollinated (left column) and self-pollinated (middle column) CO~2~-sensitive line, and the self-pollinated (right column) CO~2~-insensitive line are shown. Without CO~2~ treatment (upper panels), only cross-pollen is accepted, and self-pollen grains maintain a spheroid shape without swelling in both lines. With 4.5% CO~2~ gas treatment (lower panels), pollen grains swell and germinate in the CO~2~-sensitive line (lower middle) but not in the CO~2~-insensitive line (lower right). Bar=25 μm. (B) Representative examples of energy-dispersive X-ray spectra of non-pollinated and pollinated papilla cell surfaces. Scanning positions for X-ray analyses are indicated by asterisks in (A). The emissions of Al-Kα, P-Kα, S-Kα, K-Kα, and Ca-Kα were detected at the papilla cell surfaces, and the intensity of Ca emission was increased after cross-pollination (left column). The increase of Ca emission was also observed after self-pollination with 4.5% CO~2~ gas treatment in the CO~2~-sensitive line (middle column) but not in the CO~2~-insensitive line (right column). These spectrum patterns are reproducible in three individual experiment sets. The emission of Al-Kα is mostly derived from the stub that held the samples.](exbotj_ert438_f0002){#F2}

The efficiency of CO~2~ treatment {#s13}
---------------------------------

A previous study suggested that the effect of CO~2~ on SI breakdown depends on the timing of treatment ([@CIT0027]). In the CO~2~-sensitive line, when self-pollinated flowers were immediately treated with 4.5% CO~2~ for 4h, SI could be overcome, and typically \>10 pollen tubes penetrated into the stigma ([Fig. 3A](#F3){ref-type="fig"}). When CO~2~ treatment started 3h after self-pollination, SI could still be overcome ([Fig. 3B](#F3){ref-type="fig"}). However, when CO~2~ treatment started 6h after self-pollination, the number of penetrating pollen tubes was decreased ([Fig. 3C](#F3){ref-type="fig"}). These results indicate that self-pollen inhibition in SI is biostatic, as previously suggested ([@CIT0037]), and can be reversed at least at 3h after pollination. However, at 6h after pollination, SI inhibition enters an irreversible phase that cannot be overcome by CO~2~ treatment.

![The efficiency of CO~2~ treatment of the CO~2~-sensitive line. (A) Self-pollinated stigmas were treated with 4.5% CO~2~ immediately after self-pollination, and pollen tube penetration and elongation were observed. (B) Self-pollinated stigmas were treated with 4.5% CO~2~ 3h after self-pollination. The pollen tube can still penetrate into papilla cells. (C) When CO~2~ treatment began 6h after self-pollination, the number of penetrating pollen tubes was decreased. Pt, pollen tubes. Bar=1000 μm.](exbotj_ert438_f0003){#F3}

Next, in order to narrow down the stage of SI affected by CO~2~ treatment, experiments with high CO~2~-pre-treated pollen or pistil from the CO~2~-sensitive line were performed ([Fig. 4](#F4){ref-type="fig"}). SI could not be overcome by pre-treatment of either tissue, and no pollen tube penetration could be observed even when both pollen and pistil were treated separately prior to pollination ([Fig. 4](#F4){ref-type="fig"}). SI could be overcome only when the self-pollinated pistil was treated with high CO~2~. These results suggest that some post-pollination physiological process is affected by high CO~2~, in the process of SI breakdown.

![Pollination assay using CO~2~-treated non-pollinated flowers of the CO~2~-sensitive line as either CO~2~-treated pistil or pollen. Pollen tube behaviour is observed after a 4h CO~2~ treatment immediately after self-pollination. (A) A pistil from a non-pollinated flower treated with CO~2~ for 4h was pollinated with pollen from the same plant which was not treated with CO~2~. (B) A pistil from a non-treated non-pollinated flower was pollinated with pollen from the same plant which was treated with CO~2~ for 4h. (C) A pistil from a CO~2~-treated non-pollinated flower (CO~2~-treated) was pollinated with pollen from the same plant which was treated with CO~2~ for 4h. (D) Self-pollinated flower treated with CO~2~ after pollination, positive control. Pt, pollen tubes. Bar=1000 μm.](exbotj_ert438_f0004){#F4}

*S*-allele characterization and phenotype of CO~2~ sensitivity in F~1~ and F~2~ {#s14}
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The *S*-haplotypes of the two parental inbred lines were first determined by amplifying their *SLG* genes ([@CIT0030]). The sequence data suggested that the *S*-haplotypes of the CO~2~-sensitive and CO~2~-insensitive lines were *S* ~55~ *S* ~55~ and *S* ~46~ *S* ~46~, respectively. To dissect genetically the gene(s) that determines sensitivity to CO~2~ treatment, six F~1~ plants (*S* ~46~ *S* ~55~) were produced by crossing CO~2~-sensitive and CO~2~-insensitive lines. These F~1~ plants exhibited an intermediate CO~2~ sensitivity phenotype where self-pollen tubes penetrated into the stigma under high CO~2~ treatment but there were fewer penetrating pollen tubes than observed in a self-pollination of the CO~2~-sensitive parent. An F~2~ population of 110 individuals derived from a bud-pollinated F~1~ plant was made and used for further genetic analyses of the CO~2~ sensitivity. F~2~ individuals were genotyped using PCR-RFLP to distinguish *SLG* alleles ([Supplementary Fig. S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1) at *JXB* online). *S* ~55~- and *S* ~46~-haplotypes were segregated in the F~2~ population according to Mendelian transmission ([Supplementary Table S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1)). Pollen tube behaviour after CO~2~ treatment varied among individuals and, in order to quantify the strength of CO~2~ sensitivity, the modified RLSICO~2~ (reaction level of SI to CO~2~) index was employed ([@CIT0029]), which calculates CO~2~ sensitivity based on the number of penetrating pollen tubes after self-pollination under high CO~2~ conditions (see the Materials and methods). The RLSICO~2~ of 110 F~2~ individuals is presented in [Supplementary Fig. S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1), and the summarized box-plot data are shown in [Fig. 5](#F5){ref-type="fig"}, together with the RLSICO~2~ of F~1~ and the parental inbred lines. F~1~ had an RLSICO~2~ score intermediate to the two parental lines, suggesting that the high CO~2~ sensitivity is a semi-dominant (incompletely dominant) trait in these inbred lines. Furthermore, the RLSICO~2~ of F~2~ individuals was continuously distributed and did not follow a simple one-locus biallelic Mendelian distribution ([Supplementary Fig. S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1)). These results suggest that CO~2~ sensitivity in the inbred lines used here could be a quantitative trait which is controlled by more than one gene.

![Box plots of CO~2~ sensitivity phenotypes. Data show the distribution of RLSICO~2~ with 25th, 50th, and 75th percentiles (horizontal bars), interquartile ranges (columns), and 1.5 interquartile ranges (error bars) of RLSICO~2~ from six CO~2~-sensitive (*S* ~55~ *S* ~55~) and six CO~2~-insensitive (*S* ~46~ *S* ~46~), individuals, six F~1~ individuals (*S* ~46~ *S* ~55~), and 110 F~2~ individuals (22 *S* ~46~ *S* ~46~, 64 *S* ~46~ *S* ~55~, and 24 *S* ~55~ *S* ~55~). *a* indicates a significant difference (*P* \< 0.01) between CO~2~-sensitive and CO~2~-insensitive lines.](exbotj_ert438_f0005){#F5}

Relationship between *S*-alleles and CO~2~ sensitivity {#s15}
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To investigate whether CO~2~ sensitivity is related to *S*-haplotypes, the 110 F~2~ individuals were grouped into three genotypes (*S* ~55~ *S* ~55~, *S* ~46~ *S* ~46~, and *S* ~46~ *S* ~55~). The RLSICO~2~ of each group is shown in [Fig. 5](#F5){ref-type="fig"}. In the three F~2~ groups, RLSICO~2~ scores were distributed from 1 to 5, and interquartile ranges overlapped, indicating that CO~2~ sensitivity is not linked to the *S*-locus in these two lines.

Reproductive tissue controlling CO~2~ sensitivity {#s16}
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From the F~2~ population, two *S* ~46~ *S* ~46~ homozygotes with different RLSICO~2~ were selected: F~2~-16, a CO~2~-insensitive line (RLSICO~2~=1±0); and F~2~-26, a CO~2~-sensitive line (RLSICO~2~=4.15±0.53). These two lines were used to examine the reproductive tissue controlling CO~2~ sensitivity. Reciprocal crosses were performed between these two SI lines with or without high CO~2~ gas treatment. Because all crosses under normal conditions (without CO~2~ treatment) were incompatible, only data from crosses performed in the high CO~2~ condition are shown in [Fig. 6](#F6){ref-type="fig"}. The cross between CO~2~-sensitive F~2~-26 pistil and CO~2~-insensitive F~2~-16 pollen was CO~2~ sensitive, showing many penetrating pollen tubes under high CO~2~. On the other hand, the cross between CO~2~-insensitive F~2~-16 pistil and CO~2~-sensitive F~2~-26 pollen was CO~2~ insensitive, showing no penetrating pollen tubes even under high CO~2~. These results suggest that CO~2~ sensitivity is controlled by genes expressed in the female tissue (pistil).

![Reciprocal crosses with CO~2~ treatment between two *S* ~46~ homozygous individuals from the F~2~ population with different RLSICO~2~. (A) CO~2~-sensitive F~2~ self-pollination. (B) CO~2~-insensitive F~2~ self-pollination. (C) A CO~2~-sensitive F~2~ pistil pollinated with pollen from a CO~2~-insensitive F~2~. (D) A CO~2~-insensitive F~2~ pistil pollinated with pollen from a CO~2~-sensitive F~2~. Pt, pollen tubes. Bar=1000 μm. *n*=3.](exbotj_ert438_f0006){#F6}

Marker analysis and construction of a linkage map {#s17}
-------------------------------------------------

In order to map QTLs that determine CO~2~ sensitivity, a linkage map was constructed for this F~2~ population. A total of 911 different genetic markers were examined in the two parental lines. To clarify the relationship between previously identified SI-related genes and CO~2~ sensitivity, *SLG* (an *S*-locus marker), *MLPK*, and *ARC1* were also selected. Though a very low level of polymorphism (14.7%) was detected for all types of markers, 123 polymorphic markers were selected, which include 113 SSRs, five RFLPs, and five InDel markers. These 123 markers were used for linkage mapping, and generated 10 linkage groups (A01--A10) at a LOD threshold value of 6.0 ([Fig. 7](#F7){ref-type="fig"}). The total length of the map was 947.5 cM, and the length of the linkage groups ranged from 63.2 cM (A10) to 168.4 cM (A03). The distance between markers varied from 0 to 29.3 cM, with an average interval of 7.7 cM. *SLG*, *MLPK*, and *ARC1* were mapped to A07, A03, and A04, respectively, which is consistent with previous reports ([@CIT0001]; [@CIT0011]).

![A linkage map of selected DNA markers from the F~2~ population. Map distances are shown to the left of vertical lines of the linkage group in cM, and marker names are shown to the right.](exbotj_ert438_f0007){#F7}

QTL analysis and association of markers with high CO~2~ sensitivity {#s18}
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Using the constructed linkage map, QTLs responsible for high CO~2~ sensitivity were analysed. Three QTLs were identified on linkage groups A03 and A05 based on a LOD threshold of 3.40 (1000 permutation test, *P* \< 0.05) ([Fig. 8](#F8){ref-type="fig"}, [Table 1](#T1){ref-type="table"}). These QTLs were tentatively named *Brassica rapa SI Overcome (BrSIO) 1--3*, and these results further supported the prediction that CO~2~ sensitivity of SI is controlled by a polygenic system. *BrSIO1* on A05 and *BrSIO2* on A03 are two major QTLs that explained 19.3% and 19.0% of phenotypic variation, respectively. *BrSIO3*, located near *BrSIO2*, accounted for 14.5% of the variance ([Table 1](#T1){ref-type="table"}).

###### 

Summary of CO~2~ sensitivity QTLs

  QTL        LG    Closest marker   QTL peak (cM)^*a*^   LOD    *R* ^2*b*^   Additive effect^*c*^
  ---------- ----- ---------------- -------------------- ------ ------------ ----------------------
  *BrSIO1*   A05   XT05-004         83.50                5.17   19.30        0.72
  *BrSIO2*   A03   BRMS-042-2       60.87                4.46   19.00        0.69
  *BrSIO3*   A03   KBrH110I17R      41.25                3.76   14.50        0.65

^*a*^ QTL peak position, detected by interval mapping, between two markers.

^*b*^ Amount of phenotypic variation explained by the QTL.

^*c*^ Additive effect of the CO~2~-sensitive HA-11621 allele on RLSICO~2~.

![QTL analysis results. The solid line indicates the LOD score and the dotted line indicates the QTL threshold (LOD=3.4) determined using a 1000 permutation test (*P* \< 0.05). The *x*-axis represents each linkage group (cM) and the *y*-axis indicates the QTL score. Two QTLs (*BrSIO2* and *3*) are detected in A03 and one in A05 (*BrSIO1*). Arrows show loci involved in SI stability reported by [@CIT0011].](exbotj_ert438_f0008){#F8}

To examine the significance of these QTLs, F~2~ progeny were classified into groups based on the genotypes of the linkage markers nearest these three newly identified loci, and the relationship of the loci to RLSICO~2~ in individual plants was analysed using Kruskal--Wallis ANOVA by ranks ([Table 2](#T2){ref-type="table"}). Alleles from CO~2~-sensitive (HA-11621) and CO~2~-insensitive (HA-11623) lines are presented as *S* and *I*, respectively. Almost all classifications using the closest linkage markers showed a higher RLSICO~2~ index in the *SS* group with significance at *P* \< 0.01, except marker BRMS-114, which showed significance at *P* \< 0.05.

###### 

Statistical analysis of QTL effect

![](exbotj_ert438_t0002)

^*a*^ *S*, CO~2~-sensitive HA-11621 allele; *I*, CO~2~-insensitive HA-11623 allele.

^*b*^ Individuals whose genotype was unidentified are excluded.

^*c*^ Kruskal--Wallis analysis comparing phenotype between genotype groups with individuals in the same groups.

^*d*^ Significance level: \*\**P* \<0.01; \**P* \<0.05.

Marker association was further examined with combinations of *BrSIO1* and *BrSIO2* since *BrSIO3* is a minor QTL closely linked to *BrSIO2*, making it difficult to identify it as an independent QTL. F~2~ progeny were classified into nine groups based on the genotypes of their closest linkage markers ([Table 3](#T3){ref-type="table"}). According to this classification, for example, the two above analysed *S* ~46~ *S* ~46~ homozygous lines with different RLSICO~2~, F~2~-16 (CO~2~-insensitive line) and F~2~-26 (CO~2~-sensitive line), were classified into group 8 and group 1, respectively. When groups with the same *BrSIO1* genotype were compared, the *BrSIO2 SS* group showed a higher RLSICO~2~ index compared with the *BrSIO2 II* group. Likewise, when the groups with the same *BrSIO2* genotype were compared, the *BrSIO1 SS* group showed a higher RLSICO~2~ compared with the *BrSIO1 II* group. Although the numbers of F~2~ individuals in each group were rather low, significance (*P* \< 0.05) was detected between groups 1 and 6, 2 and 6, and 2 and 8. These data suggest that *BrSIO1* and *BrSIO2* work additively in overcoming SI during CO~2~ treatment in the CO~2~-sensitive (HA-11621) line. No QTL was detected at genes known to affect SI stability (*MLPK*, *ARC1*, or the *S*-locus), indicating that CO~2~ sensitivity is determined by novel genes in the experimental lines used here.

###### 

QTL association for CO~2~ sensitivity

![](exbotj_ert438_t0003)

^*a*^ *S*, CO~2~-sensitive HA-11621 allele; *I*, CO~2~-insensitive HA-11623 allele.

^*b*^ Individuals whose genotype was unidentified are excluded.

^*c*^ Significance level, \**P* \<0.05.

Associated gene prediction by *in silico* comparative mapping {#s19}
-------------------------------------------------------------

Using the *B. rapa* genome sequence ([@CIT0006]), *BrSIO1* could be mapped to a 569kb region flanked by InDel marker XT05-004 and SSR marker BRMS-034, and *BrSIO2* to a 1469kb region flanked by SSR markers BRMS-042-2 and KBrH110I17R. These two regions include 121 and 280 genes annotated in the *Brassica* database (BRAD), respectively ([Supplementary Tables S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1), [S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1) at *JXB* online). Comparison of the *A. thaliana* genome with the Brassicaceae genome (reviewed by [@CIT0039]) suggests that *BrSIO1* has synteny on *A. thaliana* chromosome 2 and *BrSIO2* has synteny on both chromosomes 3 and 4. It is assumed that these two QTLs do not have the same genetic origin and could be two independent regions controlling high CO~2~ sensitivity. Based on reciprocal cross results, the CO~2~ sensitivity trait may be controlled by genes expressed in the female organ ([Fig. 8](#F8){ref-type="fig"}). A total of 121 and 280 annotated genes in *BrSIO1* and *BrSIO2* have 103 and 243 homologues in *A. thaliana*, respectively, and 54 and 141 of these genes are expressed in *A. thaliana* pistil (microarray data of carpel at stage 12, <http://affymetrix.arabidopsis.info/narrays/search.pl?f1=1&s1=ATGE_37>, last accessed 14 December 2013, [Supplementary Tables S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1), [S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1)).

Genes involved in related biological processes are often expressed cooperatively and their co-expression information is important for understanding biological systems ([@CIT0007]). ATTED-II (<http://atted.jp/>, last accessed 14 December 2013) is a gene co-expression database useful for identifying the potential partners working in the same biological processes ([@CIT0031]). Co-expression analysis was performed using ATTED-II with these 195 genes and it was found that *MAP kinase 6* (At2g43790 in *BrSIO1*) and *ethylene overproducer 1* (At3g511770 in *BrSIO2*) showed the strongest co-expression and *calmodulin-like 41* (At3g50770 in *BrSIO2*) has weak co-expression with *cytochrome c oxidase 10* (At2g44520in *BrSIO1*) and *beta glucosidase 28* (At2g44460 in *BrSIO1*). In addition to these co-expressed genes, these two regions encode highly homologous family member proteins, for example matrixin proteins (At2g45040 in *BrSIO1* and At4g16640 in *BrSIO2*) and senescence-associated proteins (At2g44670 in *BrSIO1* and At4g17670 in *BrSIO2*). All these can be candidate responsible genes, although the biological functions of these genes are mostly unknown.

Discussion {#s20}
==========

It has been \>40 years since [@CIT0026] first reported that SI could be overcome by CO~2~ and [@CIT0027] demonstrated the applicability of this technique to commercial use. Nowadays, seed companies have adopted this method to obtain inbred parental seeds of crucifer vegetables for large-scale commercial F~1~ hybrid seed production. However, there is still very limited understanding of the mechanism by which SI is overcome.

[@CIT0020] showed a shrunken and distorted papilla cell surface in the CO~2~-sensitive *B. rapa* cv. Hiratsuka, and suggested that these structural changes could cause SI to be overcome. The cryo-scanning electron microscopy data reported here did not show any structural changes in CO~2~-sensitive or CO~2~-sensitive lines ([Fig. 2A](#F2){ref-type="fig"}). Additionally, pre-treatment of non-pollinated pistils with high CO~2~ gas did not cause SI breakdown ([Fig. 4](#F4){ref-type="fig"}). Therefore, a completely different SI breakdown mechanism must be present, at least in the CO~2~-sensitive line. In contrast, massive Ca accumulation was observed at the pollen--stigma interface specifically in CO~2~-sensitive plants under high CO~2~ conditions ([Fig. 2B](#F2){ref-type="fig"}). [@CIT0004] were the first to describe the need for a high concentration of Ca^2+^ for pollen germination and pollen tube growth. The high concentration of Ca^2+^ could be needed for activating pectinase to loosen the papilla cell wall, allowing the pollen tube to penetrate ([@CIT0003]), or for keeping the pollen tube cell wall rigid enough not to burst ([@CIT0012]). Although causal relationships remain unclear, the data suggest that CO~2~ treatment induces a certain compatible reaction leading to Ca^2+^ accumulation at the pollen--stigma interface.

To date, several genetic studies have been performed to understand the mechanism of SI breakdown for breeding purposes. Niikura and Matsuura (2002) reported that in Japanese radish high CO~2~ sensitivity is controlled by a recessive gene that governs the construction and/or metabolism of the stigma, which reacts to CO~2~ without any changes in gene expression. In contrast, [@CIT0014] reported a dominant, *S*-haplotype-linked high CO~2~ sensitivity phenotype in *B. rapa*. In contrast to these previous reports, F~1~ plants had an intermediate CO~2~ sensitivity and the F~2~ population had a continuous frequency distribution of RLSICO~2~ in the present study ([Fig. 6](#F6){ref-type="fig"}). These results suggest that in the lines used for this study, CO~2~ sensitivity is a quantitative trait which is controlled by more than one gene.

Genetic linkage maps based upon frequency of recombination in segregating populations are fundamental and powerful tools for associating phenotypic trait-specific genetic regions. Linkage mapping can be used to understand the biological basis of complex traits and to dissect genetic determinants underlying the expression of agronomically important breeding traits ([@CIT0034]). Using QTL analysis, two major QTLs for high CO~2~ sensitivity were successfully identified ([Fig. 8](#F8){ref-type="fig"}). *BrSIO1* and *BrSIO2* had similar LOD scores and explained similar amounts of phenotypic variation (19.3% and 19%), and these could be two major factors controlling high CO~2~ sensitivity. Very recently, five QTLs associated with stability of SI in *B. rapa* have been identified. Two of them co-localized with *SLG* (A07) and *MLPK* (A03) and the other three were on A02, A06, and A10 ([@CIT0011]). CO~2~ sensitivity did not link with the *S*-locus in the present study ([Fig. 5](#F5){ref-type="fig"}) and none of the other reported loci co-localized with QTLs detected here ([Fig. 8](#F8){ref-type="fig"}), indicating that CO~2~ sensitivity of the lines in this study is determined by novel genes different from those known to affect SI stability. Genes in *BrSIO1* and *BrSIO2* regions have 103 and 243 homologues in *A. thaliana*, respectively, and 54 and 141 of these genes are expressed in *A. thaliana* pistil. *In silico* comparative analyses identified several co-expressing genes and highly homologous genes encoded in these two regions. All these can be candidate responsible genes; however, to dentify the genes responsible for high CO~2~ sensitivity in the QTL regions in *B. rapa* more accurately, it would be necessary to narrow down the regions by developing near-isogenic lines (NILs).

To maintain F~1~ seed quality, inbred lines with strong but CO~2~-sensitive SI are ideal for F~1~ hybrid breeding, and it is very important to understand the genetic relationships between SI-related genes and CO~2~ sensitivity phenotypes. These results could be useful for the marker-assisted selection of parental lines with both stable SI and high CO~2~ sensitivity.

Supplementary data {#s21}
==================

Supplementary data are available at *JXB* online.

[Figure S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). *S*-haplotype analysis of F~2~ plants by PCR-RFLP.

[Figure S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). RLSICO~2~ in CO~2~-sensitive and CO~2~-insensitive lines, and F~1~ and F~2~ progeny based on the number of penetrating pollen tubes after self-pollination under high CO~2~ conditions.

[Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). Genetic markers and their primers used for linkage analysis.

[Table S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). *S*-haplotype segregation in the F~2~ population.

[Table S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). Annotated genes and *Arabidopsis* homologues in *BrSIO1*.

[Table S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert438/-/DC1). Annotated genes and *Arabidopsis* homologues in *BrSIO2*.

###### Supplementary Data

The authors thank Ms Rina Nagai, Ms Hitomi Ishikawa, and Ms Yuko Yoshimura for their excellent technical assistance. This work was supported by a Grant-in-Aid for the Scientific Research on Innovative Areas (21112003, 23113002) and by Grants in Aid for Scientific Research (23570056, 21248014, 25252021) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and by the Japan Advanced Plant Science Network.

InDel

:   insertion/deletion

LG

:   linkage group

LOD

:   log of odds

QTL

:   quantitative trait locus

RFLP

:   restriction fragment length polymorphism

RLSICO~2~

:   reaction level of SI to CO~2~

SI

:   self-incompatibility

SSR

:   simple sequence repeat.
