BACKGROUND
brassica napus  is an amphidiploid species developed from b. rapa  and b. oleracea .
b. rapa and b. oleracea are self-incompatible, but b. napus is self-compatible.
elucidating how self-incompatibility  was lost and self-compatibility  was acquired has profound consequences for understanding the origin of b. napus as well as trait changes during the evolutionary process of plant polyploidization.
self-incompatibility in brassica is controlled sporophytically by a single multi-allelic locus called s locus   <cit> .
the s locus consists of at least two genes: the stigma determinant s-locus receptor kinase gene   <cit>  and the pollen determinant s-locus protein  <dig> gene /s-locus cysteine-rich protein  gene  <cit>  .
the s locus is also termed the ‘s haplotype’ because s-locus genes are transmitted to progeny as one unit  <cit> .
the srk-scr interaction is haplotype-specific and only occurs between the receptor and ligand encoded in the same s-locus haplotype .
s haplotypes can be divided into two classes.
class-ii haplotypes are generally recessive to class-i haplotypes in pollen, but they are co-dominant in the stigma  <cit> .
more than  <dig> and 50 s haplotypes occur in b. rapa and b. oleracea, respectively  <cit> ; only three and four haplotypes, respectively, are class ii  <cit> .
self-incompatible b. napus strains have been developed via introgression from b. oleracea and b. rapa <cit>  or via the resynthesis of b. napus from b. oleracea and b. rapa <cit> .
thus, natural b. napus is usually thought to have lost s haplotypes, resulting in sc during evolution.
however, latent s alleles are widespread in cultivated sc b. napus <cit> , and s haplotypes are widely distributed in cultivated b. napus lines  <cit> .
the most predominant s genotype is class-i s haplotype bns- <dig> on the a genome ) and class-ii s haplotype bns- <dig> on the c genome ).
an insertion of  <dig>  kb in the promoter region of bnscr- <dig> previously resulted in no gene expression, but the non-functional class-i scr on the a genome suppressed the expression of the recessive bnscr- <dig> on the c genome, resulting in sc  <cit> .
however, sc in b. napus with two class-ii s haplotypes has rarely been observed.
the b. napus self-incompatible line s- <dig> contains two class-ii s haplotypes, bns- <dig> on the a genome  and bns- <dig> on the c genome  <cit> .
in s- <dig>  si is recessive in most accessions but dominant in some genetic backgrounds  <cit> ; si is determined by bns- <dig> <cit> .
the suppression of bnscr- <dig> by the non-functional bns- <dig> in most lines with dominant sc explains their dominant sc.
accessions with recessive sc usually have only two class-ii s haplotypes, one on the a genome  and the other on the c genome   <cit> .
furthermore, one recessive gene previously co-segregated with the s-locus scr gene and was reported to control recessive sc in bing <dig>  <cit> , while at least two genes controlled the recessive sc of 97wen <dig>  <cit> .
it is puzzling that bing <dig> and 97wen <dig> are self-compatible but can maintain the si of s- <dig>
to uncover the basis of recessive sc in natural b. napus,  <dig> b. napus accessions were screened to identify stable recessive sc and had the s genotype bns- <dig> on the a genome and bns- <dig> on the c genome in this study.
genetic analysis, gene expression, and gene cloning suggest that diverse causes underlie recessive sc in b. napus.
RESULTS
screening b. napuswith recessive sc
of the 30 f <dig> hybrids derived from crossing si line s- <dig> as a mother with  <dig> sc lines,  <dig> were stably si, with an average sci of < <dig> in both wuhan and lanzhou .
thus,  <dig> male parents  maintained the si of s- <dig> and displayed stable recessive sc.table  <dig>
plants si phenotype of 30 f
1
hybrids developed from s- <dig> as the mother line
si: self-incompatible, sci <2; psi : 2 ≤ sci <10; sc: self-compatible, sci ≥10; +: stable recessive sc accessions.
sci was calculated as the number of seeds per flower.
shaplotypes in recessive sc b. napus
the s haplotypes of the  <dig> accessions were identified with the primer combinations in table  <dig>  there was no amplification by the class-i specific primer pair ps5/ps <dig>  but amplification did result from class-ii specific primers ps3/ps <dig> , indicating that the  <dig> accessions only had class-ii s haplotypes.table  <dig>
primers for
s
haplotype identification
s
bosrk-15
brsrk-60
brscr-29
brsrk-29s
haplotype identification.
control  is westar in ps5/ps <dig> and s- <dig> in the other five primer pairs.
primer pairs srk15-3/srk15- <dig>  srka-l/srka-r, and sp11a-l/sp11a-r were designed to amplify class-ii s-locus genes bosrk- <dig>  brsrk- <dig>  and brscr- <dig>  respectively.
the  <dig> sc accessions and si line s- <dig> yielded a fragment with the same size  which amplified by primer pair srk15-3/srk15- <dig> .
this fragment was 100% identical to bnsrk- <dig> , indicating that the  <dig> accessions carried bns- <dig>  on the c genome.
primer pair srka-l/srka-r produced a fragment of ~1000-bp only in s- <dig>  while sp11a-l/sp11a-r amplified a 303-bp dna fragment in each of the  <dig> accessions .
the 303-bp fragment was 100% identical to brscr- <dig> , which is named bns- <dig> in b. napus <cit> .
to confirm that the  <dig> sc accessions had bns- <dig>  on the a genome, primer pair srk29-1a/srk29-1b was designed based on a fragment deletion of bnsrk- <dig> compared with sequences bnsrk- <dig> and bnsrk- <dig>  the  <dig> sc accessions yielded identical 300-bp fragments, but s- <dig> did not .
this fragment was 100% identical to brsrk- <dig> , confirming that bns- <dig>  was on the a genome.
gene expression of srkand scr
to detect relationships between s-locus genes expression and si phenotype, specific primers based on s-locus genes were designed for qrt-pcr .
stigmas from parents and f <dig> hybrids, bnsrk- <dig> and bnsrk- <dig> expressed normally  at a mean value of  <dig>  .
thus, srk genes expression is co-dominant in stigma.figure  <dig>
srk
and
scr
expression in parental lines  and f
1
hybrids.
semi-quantitative real-time pcr  quantitative real-time pcr, error bars represent standard error .
in anther of self-incompatible s- <dig> and f <dig> hybrids, bnscr- <dig> transcripts were present at high levels , while bnscr- <dig> and/or bnscr- <dig> were nearly undetectable .
bnscr- <dig> was only expressed in anthers from three males  , with expression values of  <dig> ,  <dig> , and  <dig>  , respectively; no expression was detected in other males .
similarly, bnscr- <dig> was expressed in  <dig>   <dig>  and  <dig> , with values of  <dig> ,  <dig> , and  <dig> , respectively , but little or no expression in other lines  .
thus, bnscr- <dig> expression is necessary for the si of s- <dig> and f <dig> hybrids, and diverse expression patterns of the scr genes occur across sc accessions.
cloning and sequence analysis of s-locus genes
sequence mutations in the bns- <dig> and bns-7 s-locus genes are thought to be responsible for recessive sc.
thus,  <dig> sc accessions were used to clone srk from the a and c genomes.
primer combinations bnsrk7-2a/bnsrk7-2b  produced a 2590-bp sequence that contained the full bnsrk- <dig> cds.
sequence alignment showed that  <dig> sc accessions were 100% identical to brsrk- <dig> , with four different base pairs in line c <dig> dispersed in exons  <dig> and  <dig> .
primer pair bnsrk6-1a/bnsrk6-1b produced a 2577-bp fragment with the full-length bnsrk- <dig> cds from each sc accession.
these fragments shared 100% sequence identity with bnsrk- <dig> .figure  <dig>
structure of
s
-locus genes and nucleotide sequence alignment.
arrows sites show  <dig> base pairs diversity dispersed in exons  <dig> and  <dig> of c <dig>
as only three male sc accessions  expressed bnscr- <dig> and bnscr- <dig> , cdna from their anthers was used as template to clone the full cds of scr.
fragments 340-bp and 292-bp in length were obtained with primer pairs bnscr7-2/bnscr7- <dig> and bnscr6-1a/bnscr6-1b , respectively, with 100% identity to brscr- <dig>  and bnscr- <dig> , respectively.
primer combinations were developed to amplify genomic dna including the 5′ promoter regions of bnscr- <dig> and bnscr- <dig>  primer combination bnscr7-3/bnscr7- <dig> amplified a 1784-bp fragment of bnscr- <dig> that included 1367-bp 5′ upstream of the translation initiation site .
all bnscr- <dig> sequences from the  <dig> sc accessions were 100% identical.
combination of the cds and genomic dna sequences revealed that bnscr- <dig> is 367-bp long and contains two exons and one intron.
primer combinations bnscr6-4a/bnscr6-4b and bnscr6-2a/bnscr6-2b were developed to amplify 1193-bp and 1268-bp fragments of bnscr- <dig>  respectively.
a 2320-bp fragment was obtained by combining the two fragments.
this fragment encompasses 1176-bp of sequence 5′ upstream of the translation initiation site.
all obtained bnscr- <dig> sequences were 100% identical.
combining the cds and genomic dna sequences indicated that bnscr- <dig> is 377-bp long, with two exons and one intron.
taken together, these observations show that the full genomic dna sequences, including the 5′ promoter regions of scr, have no sequence variation in  <dig> sc accessions, regardless of differences in expression.
genetic analysis of recessive sc
the f <dig> populations and two bc <dig> populations from six males were used for genetic analysis of recessive sc.
segregation of the si/sc phenotype demonstrated that the sc of three males  was controlled by a single locus .
however, χ <dig> values > <dig>  were observed in populations  ×  <dig>   ×  <dig>   ×  <dig>  and  f <dig>  revealing that the sc of lines  <dig>   <dig>  and  <dig> was not controlled by a single locus.
separate analyses of four males  conducted over two years returned similar results .table  <dig>
genotypes and phenotypes in segregating populations
a
χ
2
1300
s
bns-7
1300
s
bns-7
f2
f2
f2
f2
f2
f2
f2
f2
f2
f2
si: self-incompatible, sci <2; sc: self-compatible, sci ≥ <dig>
aprimer combinations srka-l/srka-r and srk29-1a/srk29-1b were used to detect bns- <dig> and bns- <dig>  respectively.
bnumber of investigated plants with the si/sc phenotype.
primer combinations srka-l/srka-r and srk29-1a/srk29-1b , were used to determine the inheritance of s <dig> and sbns- <dig> and to clarify the role of s genotypes in determining the si phenotype.
all s1300s <dig> plants were completely si in all progeny from six accessions; all s1300sbns- <dig> plants were si in progenies from lines  <dig> and  <dig>  however, si plants of genotype sbns-7sbns- <dig> were observed in all populations, and some plants with genotype s1300sbns- <dig> were sc in a population descended from four males  .
these results suggest that factor independent of the s locus are involved in recessive sc, but that the s haplotype bns- <dig> in the a genome is necessary for si.
discussion
recessive sc in b. napushas diverse causes
si has been used for hybrid breeding in b. rapa and b. oleracea.
as b. napus is an oil crop, its hybrids should be fertile for harvesting seeds.
on the other hand, a si line must be propagated on a large scale to produce many hybrid seeds.
the si of line s- <dig> is recessive in most accessions but dominant in some genetic backgrounds  <cit> .
therefore, it has been utilized for three-component hybrid breeding via si f <dig> hybrids  <cit> .
recessive sc lines thus play a key role in hybrid breeding.
here,  <dig> accessions were screened to identify stable recessive sc and had the s genotype bns-7/bns- <dig>  in 8/ <dig> accessions, expression of bnscr- <dig> and bnscr- <dig> was nearly absent, but srk genes were expressed at normal levels .
srk genes in these accessions contained no cds mutations, with the exception of 4-bp in bnsrk- <dig> in line c <dig> .
these data indicate that sc is caused by the loss of function of bnscr- <dig> and bnscr- <dig> in the pollen of these eight accessions.
however, bnscr- <dig> and bnscr- <dig> harbor no structural mutations in the promoters of these lines, rendering the mechanism of this loss of function unclear.
sequence insertion/deletion causing loss-function of scr was previously reported to cause sc.
the sc b. rapa cultivar yellow sarson, which has a self-compatible class-i s haplotype , contains an 89-bp deletion in the scr promoter region; this deletion resulted in the production of no transcript, which caused the loss of function in the s-f <dig> homozygote.
the expression of recessive class-ii scr- <dig> was suppressed in the s-f2/s- <dig> heterozygote by non-functional class-i scr-f <dig> <cit> .
no expression of the class-i dominant scr on the a genome resulted from the insertion of ~ <dig>  kb in the promoter region; this insertion’s suppression of the recessive locus bnscr- <dig> on the c genome explains the sc of b. napus accessions with the s genotype of bns-1/bns- <dig> <cit> .
sc in a. thaliana is also caused by a 213-bp inversion in the male-specific gene scr that inhibits the transcription of scr <cit> .
however, we were surprised that srk and scr were expressed normally in three sc accessions in the present investigation .
the scr and srk cdss in these lines had no mutations relative to brs- <dig> and bos- <dig>  implying that other factors contribute to sc by taking part in translational repression.
therefore, diverse causes result in recessive sc in b. napus.
factor independent of the slocus contribute to sc in b. napus
other factor independent of the s locus may control recessive sc, based on our observations that s1300s <dig> plants are completely si, while sbns-7sbns- <dig> and s1300sbns- <dig> plants segregated si phenotypes in their progeny .
these observations are consistent with those of ekuere et al.
<cit> , who identified a latent s allele in at least two oilseed rape cultivars; the s phenotype of these latent alleles was masked by a suppressor system common to oilseed rape.
a modifier was also proposed to cause transient si in a. thaliana <cit> .
liu et al.
<cit>  demonstrated that transient si is caused by a hypomorphic allele of pub <dig> that regulates srk transcript levels, and suggested that disruption or down-regulation of the s-locus recognition genes was a major mechanism for the switch to self-fertility in a. thaliana.
although genetic analyses are not completely consistent with our previous results  <cit> , si plants with genotype sbns-7sbns- <dig> and sc plants with genotype s1300sbns- <dig> are useful for mapping and characterizing the other factor or suppressor in this system.
in the brassicaceae, self-recognition involves srk-scr interaction and signal transmission.
any factor that suppresses mrna expression of srk or scr or disrupts subsequent signal transduction would cause the breakdown of si.
several proteins have been shown to affect si response in brassica, such as the armadillo repeat-containing protein arc <dig>  <cit> , the thioredoxin h-like proteins thl <dig> and thl <dig>  <cit> , and m-locus protein kinase mlpk  <cit> .
however, mlpk, arc <dig>  and exo70a <dig> orthologs do not contribute to the si response in a. thaliana srk-scr transformants  <cit> .
the arc1-related u-box gene atpub <dig>  which is highly expressed in the stigma, does not function in si either  <cit> .
further investigations will be needed to determine whether the factor proposed in the present study consist of these proteins.
dominant/recessive relationships in recessive class-ii shaplotypes
in general, s haplotypes in brassica exhibit dominant/recessive relationships in pollen and co-dominant relationships in stigma  <cit> .
some s haplotypes are hierarchically dominant; in b. rapa, the order is s9 > s44 > s60 > s40 > s <dig> <cit> .
of these s haplotypes, s <dig>  s <dig>  s <dig> are dominant in some cases but recessive in others, based on the expression level of scr <cit> .
the si line s- <dig> and  <dig> recessive sc accessions have a common s haplotype  on the c genome, but different s haplotypes on the a genome .
bnscr- <dig>  was only expressed in s- <dig> and the 11 f <dig> hybrids, while bnscr- <dig>  and bnscr- <dig>  were nearly undetectable .
in pollen, brscr- <dig> is dominant over brscr- <dig> <cit> .
the si of f <dig> hybrids may be due to the dominant, functional bnscr- <dig> on the a genome, to which bnscr- <dig> is recessive.
dominant/recessive relationships between class-i and class-ii s haplotypes are regulated by dna methylation of the promoter of the recessive scr gene  <cit> ; this methylation is triggered by a trans-acting small non-coding rna  <cit> .
however, the mechanism underlying the dominant/recessive relationships between two class-ii s haplotypes has not been reported to date.
previously, boscr- <dig> was  <dig> % identical at the amino-acid level to brscr- <dig>  but only  <dig> % identical to brscr- <dig> <cit> .
if the dominant/recessive relationship between two s haplotypes in b. rapa and b. oleracea is present in b. napus, as observed by okamoto et al.
<cit> , then bnscr- <dig> and bnscr- <dig> may be co-dominant, with both dominant to bnscr- <dig>  however, we did not detect bnscr- <dig> transcripts in the si line s- <dig>  and neither bnscr- <dig> nor bnscr- <dig> was expressed in the anthers of eight sc accessions; the other three sc accessions clearly expressed bnscr- <dig> and bnscr- <dig> .
our observations cannot be explained by any dominant/recessive relationship among the class-ii bnscr- <dig>  bnscr- <dig>  and bnscr- <dig>  the si line s- <dig> and the recessive sc accessions employed here are unique materials for dissecting the dominant/recessive relationship and its mechanisms in class-ii s haplotypes.
much progress has been made toward elucidating the mechanism of sc in b. napus, but many questions persist, such as the roles of srk and scr in self-recognition, the dominant/recessive relationships between recessive class-ii s haplotypes, and the identities and functions of other factors involved in si singling.
our study provides insight into the complex mechanisms of sc in b. napus, laying the groundwork to characterize the novel factor affecting s-locus gene expression and si signaling.
dissecting these pathways will help elucidate the mechanisms of recessive sc and further our understanding of the evolution of plants from diploid to autoploid species and the changes in self-fertility during polyploidization.
CONCLUSIONS
the recessive self-compatible accessions screened in this study had two common class-ii s haplotypes: bns- <dig> on the c genome and bns- <dig> on the a genome.
our observations of different bnscr- <dig> and bnscr- <dig> expression patterns across sc accessions, the reliance of si on the expression of bnscr- <dig> rather than srk genes, and the contributions to si phenotypes of factor independent of the s locus according to the inheritance of segregating populations suggest that diverse causes underlie recessive sc in b. napus, yielding insight into these complex mechanisms and laying the groundwork to characterize the novel factor affecting s-locus gene expression and si signaling.

 brassica napus is an amphidiploid species developed from b. rapa and b. oleracea. b. rapa and b. oleracea are self-incompatible, but b. napus is self-compatible. elucidating how self-incompatibility was lost and self-compatibility was acquired has profound consequences for understanding the origin of b. napus as well as trait changes during the evolutionary process of plant polyploidization. self-incompatibility in brassica is controlled sporophytically by a single multi-allelic locus called s locus. the s locus consists of at least two genes: the stigma determinant s-locus receptor kinase gene and the pollen determinant s-locus protein gene /s-locus cysteine-rich protein gene. the s locus is also termed the ‘s haplotype’ because s-locus genes are transmitted to progeny as one unit. the srk-scr interaction is haplotype-specific and only occurs between the receptor and ligand encoded in the same s-locus haplotype. s haplotypes can be divided into two classes. class-ii haplotypes are generally recessive to class-i haplotypes in pollen, but they are co-dominant in the stigma. more than and 50 s haplotypes occur in b. rapa and b. oleracea, respectively; only three and four haplotypes, respectively, are class ii. self-incompatible b. napus strains have been developed via introgression from b. oleracea and b. rapa or via the resynthesis of b. napus from b. oleracea and b. rapa. thus, natural b. napus is usually thought to have lost s haplotypes, resulting in sc during evolution. however, latent s alleles are widespread in cultivated sc b. napus, and s haplotypes are widely distributed in cultivated b. napus lines. the most predominant s genotype is class-i s haplotype bns- on the a genome ) and class-ii s haplotype bns- on the c genome ). an insertion of  kb in the promoter region of bnscr- previously resulted in no gene expression, but the non-functional class-i scr on the a genome suppressed the expression of the recessive bnscr- on the c genome, resulting in sc. however, sc in b. napus with two class-ii s haplotypes has rarely been observed. the b. napus self-incompatible line s- contains two class-ii s haplotypes, bns- on the a genome and bns- on the c genome. in s- si is recessive in most accessions but dominant in some genetic backgrounds; si is determined by bns-. the suppression of bnscr- by the non-functional bns- in most lines with dominant sc explains their dominant sc. accessions with recessive sc usually have only two class-ii s haplotypes, one on the a genome and the other on the c genome. furthermore, one recessive gene previously co-segregated with the s-locus scr gene and was reported to control recessive sc in bing, while at least two genes controlled the recessive sc of 97wen. it is puzzling that bing and 97wen are self-compatible but can maintain the si of s- to uncover the basis of recessive sc in natural b. napus, b. napus accessions were screened to identify stable recessive sc and had the s genotype bns- on the a genome and bns- on the c genome in this study. genetic analysis, gene expression, and gene cloning suggest that diverse causes underlie recessive sc in b. napus.  screening b. napuswith recessive sc of the 30 f hybrids derived from crossing si line s- as a mother with sc lines, were stably si, with an average sci of < in both wuhan and lanzhou. thus, male parents maintained the si of s- and displayed stable recessive sc.table plants si phenotype of 30 f 1 hybrids developed from s- as the mother line si: self-incompatible, sci <2; psi: 2 ≤ sci <10; sc: self-compatible, sci ≥10; +: stable recessive sc accessions. sci was calculated as the number of seeds per flower. shaplotypes in recessive sc b. napus the s haplotypes of the accessions were identified with the primer combinations in table  there was no amplification by the class-i specific primer pair ps5/ps but amplification did result from class-ii specific primers ps3/ps, indicating that the accessions only had class-ii s haplotypes.table primers for s haplotype identification s bosrk-15 brsrk-60 brscr-29 brsrk-29s haplotype identification. control is westar in ps5/ps and s- in the other five primer pairs. primer pairs srk15-3/srk15- srka-l/srka-r, and sp11a-l/sp11a-r were designed to amplify class-ii s-locus genes bosrk- brsrk- and brscr- respectively. the sc accessions and si line s- yielded a fragment with the same size which amplified by primer pair srk15-3/srk15-. this fragment was 100% identical to bnsrk-, indicating that the accessions carried bns- on the c genome. primer pair srka-l/srka-r produced a fragment of ~1000-bp only in s- while sp11a-l/sp11a-r amplified a 303-bp dna fragment in each of the accessions. the 303-bp fragment was 100% identical to brscr-, which is named bns- in b. napus. to confirm that the sc accessions had bns- on the a genome, primer pair srk29-1a/srk29-1b was designed based on a fragment deletion of bnsrk- compared with sequences bnsrk- and bnsrk- the sc accessions yielded identical 300-bp fragments, but s- did not. this fragment was 100% identical to brsrk-, confirming that bns- was on the a genome. gene expression of srkand scr to detect relationships between s-locus genes expression and si phenotype, specific primers based on s-locus genes were designed for qrt-pcr. stigmas from parents and f hybrids, bnsrk- and bnsrk- expressed normally at a mean value of. thus, srk genes expression is co-dominant in stigma.figure srk and scr expression in parental lines and f 1 hybrids. semi-quantitative real-time pcr quantitative real-time pcr, error bars represent standard error. in anther of self-incompatible s- and f hybrids, bnscr- transcripts were present at high levels, while bnscr- and/or bnscr- were nearly undetectable. bnscr- was only expressed in anthers from three males, with expression values of,, and, respectively; no expression was detected in other males. similarly, bnscr- was expressed in and, with values of,, and, respectively, but little or no expression in other lines. thus, bnscr- expression is necessary for the si of s- and f hybrids, and diverse expression patterns of the scr genes occur across sc accessions. cloning and sequence analysis of s-locus genes sequence mutations in the bns- and bns-7 s-locus genes are thought to be responsible for recessive sc. thus, sc accessions were used to clone srk from the a and c genomes. primer combinations bnsrk7-2a/bnsrk7-2b produced a 2590-bp sequence that contained the full bnsrk- cds. sequence alignment showed that sc accessions were 100% identical to brsrk-, with four different base pairs in line c dispersed in exons and. primer pair bnsrk6-1a/bnsrk6-1b produced a 2577-bp fragment with the full-length bnsrk- cds from each sc accession. these fragments shared 100% sequence identity with bnsrk-.figure structure of s -locus genes and nucleotide sequence alignment. arrows sites show base pairs diversity dispersed in exons and of c as only three male sc accessions expressed bnscr- and bnscr-, cdna from their anthers was used as template to clone the full cds of scr. fragments 340-bp and 292-bp in length were obtained with primer pairs bnscr7-2/bnscr7- and bnscr6-1a/bnscr6-1b, respectively, with 100% identity to brscr- and bnscr-, respectively. primer combinations were developed to amplify genomic dna including the 5′ promoter regions of bnscr- and bnscr- primer combination bnscr7-3/bnscr7- amplified a 1784-bp fragment of bnscr- that included 1367-bp 5′ upstream of the translation initiation site. all bnscr- sequences from the sc accessions were 100% identical. combination of the cds and genomic dna sequences revealed that bnscr- is 367-bp long and contains two exons and one intron. primer combinations bnscr6-4a/bnscr6-4b and bnscr6-2a/bnscr6-2b were developed to amplify 1193-bp and 1268-bp fragments of bnscr- respectively. a 2320-bp fragment was obtained by combining the two fragments. this fragment encompasses 1176-bp of sequence 5′ upstream of the translation initiation site. all obtained bnscr- sequences were 100% identical. combining the cds and genomic dna sequences indicated that bnscr- is 377-bp long, with two exons and one intron. taken together, these observations show that the full genomic dna sequences, including the 5′ promoter regions of scr, have no sequence variation in sc accessions, regardless of differences in expression. genetic analysis of recessive sc the f populations and two bc populations from six males were used for genetic analysis of recessive sc. segregation of the si/sc phenotype demonstrated that the sc of three males was controlled by a single locus. however, χ values > were observed in populations  ×   ×   ×  and f revealing that the sc of lines and was not controlled by a single locus. separate analyses of four males conducted over two years returned similar results.table genotypes and phenotypes in segregating populations a χ 2 1300 s bns-7 1300 s bns-7 f2 f2 f2 f2 f2 f2 f2 f2 f2 f2 si: self-incompatible, sci <2; sc: self-compatible, sci ≥ aprimer combinations srka-l/srka-r and srk29-1a/srk29-1b were used to detect bns- and bns- respectively. bnumber of investigated plants with the si/sc phenotype. primer combinations srka-l/srka-r and srk29-1a/srk29-1b, were used to determine the inheritance of s and sbns- and to clarify the role of s genotypes in determining the si phenotype. all s1300s plants were completely si in all progeny from six accessions; all s1300sbns- plants were si in progenies from lines and however, si plants of genotype sbns-7sbns- were observed in all populations, and some plants with genotype s1300sbns- were sc in a population descended from four males. these results suggest that factor independent of the s locus are involved in recessive sc, but that the s haplotype bns- in the a genome is necessary for si. discussion recessive sc in b. napushas diverse causes si has been used for hybrid breeding in b. rapa and b. oleracea. as b. napus is an oil crop, its hybrids should be fertile for harvesting seeds. on the other hand, a si line must be propagated on a large scale to produce many hybrid seeds. the si of line s- is recessive in most accessions but dominant in some genetic backgrounds. therefore, it has been utilized for three-component hybrid breeding via si f hybrids. recessive sc lines thus play a key role in hybrid breeding. here, accessions were screened to identify stable recessive sc and had the s genotype bns-7/bns- in 8/ accessions, expression of bnscr- and bnscr- was nearly absent, but srk genes were expressed at normal levels. srk genes in these accessions contained no cds mutations, with the exception of 4-bp in bnsrk- in line c. these data indicate that sc is caused by the loss of function of bnscr- and bnscr- in the pollen of these eight accessions. however, bnscr- and bnscr- harbor no structural mutations in the promoters of these lines, rendering the mechanism of this loss of function unclear. sequence insertion/deletion causing loss-function of scr was previously reported to cause sc. the sc b. rapa cultivar yellow sarson, which has a self-compatible class-i s haplotype, contains an 89-bp deletion in the scr promoter region; this deletion resulted in the production of no transcript, which caused the loss of function in the s-f homozygote. the expression of recessive class-ii scr- was suppressed in the s-f2/s- heterozygote by non-functional class-i scr-f. no expression of the class-i dominant scr on the a genome resulted from the insertion of ~  kb in the promoter region; this insertion’s suppression of the recessive locus bnscr- on the c genome explains the sc of b. napus accessions with the s genotype of bns-1/bns-. sc in a. thaliana is also caused by a 213-bp inversion in the male-specific gene scr that inhibits the transcription of scr. however, we were surprised that srk and scr were expressed normally in three sc accessions in the present investigation. the scr and srk cdss in these lines had no mutations relative to brs- and bos- implying that other factors contribute to sc by taking part in translational repression. therefore, diverse causes result in recessive sc in b. napus. factor independent of the slocus contribute to sc in b. napus other factor independent of the s locus may control recessive sc, based on our observations that s1300s plants are completely si, while sbns-7sbns- and s1300sbns- plants segregated si phenotypes in their progeny. these observations are consistent with those of ekuere et al., who identified a latent s allele in at least two oilseed rape cultivars; the s phenotype of these latent alleles was masked by a suppressor system common to oilseed rape. a modifier was also proposed to cause transient si in a. thaliana. liu et al. demonstrated that transient si is caused by a hypomorphic allele of pub that regulates srk transcript levels, and suggested that disruption or down-regulation of the s-locus recognition genes was a major mechanism for the switch to self-fertility in a. thaliana. although genetic analyses are not completely consistent with our previous results, si plants with genotype sbns-7sbns- and sc plants with genotype s1300sbns- are useful for mapping and characterizing the other factor or suppressor in this system. in the brassicaceae, self-recognition involves srk-scr interaction and signal transmission. any factor that suppresses mrna expression of srk or scr or disrupts subsequent signal transduction would cause the breakdown of si. several proteins have been shown to affect si response in brassica, such as the armadillo repeat-containing protein arc, the thioredoxin h-like proteins thl and thl, and m-locus protein kinase mlpk. however, mlpk, arc and exo70a orthologs do not contribute to the si response in a. thaliana srk-scr transformants. the arc1-related u-box gene atpub which is highly expressed in the stigma, does not function in si either. further investigations will be needed to determine whether the factor proposed in the present study consist of these proteins. dominant/recessive relationships in recessive class-ii shaplotypes in general, s haplotypes in brassica exhibit dominant/recessive relationships in pollen and co-dominant relationships in stigma. some s haplotypes are hierarchically dominant; in b. rapa, the order is s9 > s44 > s60 > s40 > s. of these s haplotypes, s s s are dominant in some cases but recessive in others, based on the expression level of scr. the si line s- and recessive sc accessions have a common s haplotype on the c genome, but different s haplotypes on the a genome. bnscr- was only expressed in s- and the 11 f hybrids, while bnscr- and bnscr- were nearly undetectable. in pollen, brscr- is dominant over brscr-. the si of f hybrids may be due to the dominant, functional bnscr- on the a genome, to which bnscr- is recessive. dominant/recessive relationships between class-i and class-ii s haplotypes are regulated by dna methylation of the promoter of the recessive scr gene; this methylation is triggered by a trans-acting small non-coding rna. however, the mechanism underlying the dominant/recessive relationships between two class-ii s haplotypes has not been reported to date. previously, boscr- was % identical at the amino-acid level to brscr- but only % identical to brscr-. if the dominant/recessive relationship between two s haplotypes in b. rapa and b. oleracea is present in b. napus, as observed by okamoto et al., then bnscr- and bnscr- may be co-dominant, with both dominant to bnscr- however, we did not detect bnscr- transcripts in the si line s- and neither bnscr- nor bnscr- was expressed in the anthers of eight sc accessions; the other three sc accessions clearly expressed bnscr- and bnscr-. our observations cannot be explained by any dominant/recessive relationship among the class-ii bnscr- bnscr- and bnscr- the si line s- and the recessive sc accessions employed here are unique materials for dissecting the dominant/recessive relationship and its mechanisms in class-ii s haplotypes. much progress has been made toward elucidating the mechanism of sc in b. napus, but many questions persist, such as the roles of srk and scr in self-recognition, the dominant/recessive relationships between recessive class-ii s haplotypes, and the identities and functions of other factors involved in si singling. our study provides insight into the complex mechanisms of sc in b. napus, laying the groundwork to characterize the novel factor affecting s-locus gene expression and si signaling. dissecting these pathways will help elucidate the mechanisms of recessive sc and further our understanding of the evolution of plants from diploid to autoploid species and the changes in self-fertility during polyploidization.  the recessive self-compatible accessions screened in this study had two common class-ii s haplotypes: bns- on the c genome and bns- on the a genome. our observations of different bnscr- and bnscr- expression patterns across sc accessions, the reliance of si on the expression of bnscr- rather than srk genes, and the contributions to si phenotypes of factor independent of the s locus according to the inheritance of segregating populations suggest that diverse causes underlie recessive sc in b. napus, yielding insight into these complex mechanisms and laying the groundwork to characterize the novel factor affecting s-locus gene expression and si signaling. 
other factor independent of the s locus are involved in recessive sc. brassica napusself-incompatibility s locus genesgene expressiongenetic analysisissue-copyright-statement© the author 2014 expression and cloning of srk and scr genes and genetic analysis were carried out to dissect bases of recessive sc in b.napus. in eight sc accessions, bnscr- and bnscr- were nearly undetectable and harbored no structural mutations in the promoters, while srk genes were expressed at normal levels and contained intact cds, with the exception of bnsrk- in line c srk and scr genes were expressed normally but their cdss had no mutations in three sc accessions. translational repression contributes to the recessive sc in three accessions, whose srk and scr genes were expressed normally and had identical cdss to brs- or bos- si in 11 f hybrids relies on the expression of bnscr- rather than srk genes. most b.napus accessions have dominant self-compatibility resulting from an insertion of  kb in the promoter region of bnscr- on the a genome, while recessive sc in b.napus has rarely been observed. eleven accessions were screened to identify stable recessive sc and had the s genotype bns- on the a genome and bns- on the c genome similarly to brs- and bos- respectively. the recessive sc in eight accessions is caused by the loss of function of bnscr- and bnscr- in pollen. therefore, diverse causes underlie recessive sc in b. napus, yielding insight into these complex mechanisms. plants of s genotype s1300s were completely si, while si phenotypes of sbns-7sbns- and s1300sbns- plants were segregated in bc and f populations. in self-incompatible s- and 11 f hybrids, srk genes and bnscr- transcripts were present at high levels, while expression of the bnscr- and bnscr- were absent. brassica napus is self-compatible, although its ancestor species brassica rapa and brassica oleracea are self-incompatible.
 brassica napus is self-compatible, although its ancestor species brassica rapa and brassica oleracea are self-incompatible. most b.napus accessions have dominant self-compatibility resulting from an insertion of  kb in the promoter region of bnscr- on the a genome, while recessive sc in b.napus has rarely been observed. expression and cloning of srk and scr genes and genetic analysis were carried out to dissect bases of recessive sc in b.napus.  eleven accessions were screened to identify stable recessive sc and had the s genotype bns- on the a genome and bns- on the c genome similarly to brs- and bos- respectively. in eight sc accessions, bnscr- and bnscr- were nearly undetectable and harbored no structural mutations in the promoters, while srk genes were expressed at normal levels and contained intact cds, with the exception of bnsrk- in line c srk and scr genes were expressed normally but their cdss had no mutations in three sc accessions. in self-incompatible s- and 11 f hybrids, srk genes and bnscr- transcripts were present at high levels, while expression of the bnscr- and bnscr- were absent. plants of s genotype s1300s were completely si, while si phenotypes of sbns-7sbns- and s1300sbns- plants were segregated in bc and f populations.  the recessive sc in eight accessions is caused by the loss of function of bnscr- and bnscr- in pollen. translational repression contributes to the recessive sc in three accessions, whose srk and scr genes were expressed normally and had identical cdss to brs- or bos- si in 11 f hybrids relies on the expression of bnscr- rather than srk genes. other factor independent of the s locus are involved in recessive sc. therefore, diverse causes underlie recessive sc in b. napus, yielding insight into these complex mechanisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brassica napusself-incompatibility s locus genesgene expressiongenetic analysisissue-copyright-statement© the author 2014 