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Common buckwheat (Fagopyrum esculentum) is a heteromorphic self-incompatible (SI) species with two types of floral architecture: thrum (short style) and pin (long style). The floral morphology and intra-morph incompatibility are controlled by a single genetic locus, S. However, the molecular mechanisms underlying the heteromorphic self-incompatibility of common buckwheat remain unclear. To identify these mechanisms, we performed proteomic, quantitative reverse-transcription PCR, and linkage analyses. Comparison of protein profiles between the long and short styles revealed a protein unique to the short style. Amino-acid sequencing revealed that it was a truncated form of polygalacturonase (PG); we designated the gene encoding this protein FePG1. Phylogenetic analysis classified FePG1 into the same clade as PGs that function in pollen development and floral morphology. FePG1 expression was significantly higher in short styles than in long styles. It was expressed in flowers of a short-homostyle line but not in flowers of a long-homostyle line. Linkage analysis indicated that FePG1 was not linked to the S locus; it could be a factor downstream of this locus. Our finding of a gene putatively working under the regulation of the S locus provides useful information for elucidation of the mechanism of heteromorphic self-incompatibility.

High temperature disrupts the targeting of SRK to the plasma membrane, resulting in breakdown of the stigmatic self-incompatibility response in self-incompatible Arabidopsis transformants., Commercial seeds of Brassicaceae vegetable crops are mostly F1 hybrids, the production of which depends on self-incompatibility during pollination. Self-incompatibility is known to be weakened by exposure to elevated temperatures, which may compromise future breeding and seed production. In the Brassicaceae, self-incompatibility is controlled by two genes, SRK and SCR, which function as female and male determinants of recognition specificity, respectively. However, the molecular mechanisms underlying the breakdown of self-incompatibility under high temperature are poorly understood. In this study, we examined the self-incompatibility phenotypes of self-incompatible Arabidopsis thaliana SRK-SCR transformants under normal (23 °C) and elevated (29 °C) temperatures. Exposure to elevated temperature caused defects in the stigmatic, but not the pollen, self-incompatibility response. In addition, differences in the response to elevated temperature were observed among different S haplotypes. Subcellular localization revealed that high temperature disrupted the targeting of SRK to the plasma membrane. SRK localization in plants transformed with different S haplotypes corresponded to their self-incompatibility phenotypes, further indicating that defects in SRK localization were responsible for the breakdown in the self-incompatibility response at high temperature. Our results provide new insights into the causes of instability in self-incompatibility phenotypes.

SCR/SP11 encodes the male determinant of recognition specificity of self-incompatibility (SI) in Brassica species and is sporophytically expressed in the anther tapetum. Based on dominance relationships in pollen and nucleotide sequence similarity, the S haplotypes in Brassica have been classified as class I or class II, with class-I S haplotypes being dominant over class-II S haplotypes. Here, we revealed that S-22 in B. rapa belonging to class I is recessive to class-II S-44 and class-I S-36 in pollen, whereas it is dominant over S-60, S-40, and S-29 based on pollination tests. SCR/SP11 of S-22 (SCR-22) was sequenced, revealing that the deduced amino-acid sequence of SCR-22 has the longest C-terminal domain among the SCR/SP11 sequences. The expression of SCR-22 was found to be suppressed in S-22/S-44 and S-22/S-36 heterozygotes. Normal transcription of SCR-44 was considered to be due to the transcription suppression of Smi sRNA of the S-22 haplotype and a very low methylation state of the SCR-44 promoter region in the tapetum of S-22/S-44 heterozygotes. In SCR-22, only the cytosine residue located at the –37 bp position of the promoter region was hypermethylated in the tapetum of S-22/S-44 heterozygotes, and few methylated cytosines were detected in the promoter and coding regions of SCR-22 in S-22/S-36 heterozygotes. SCR-22 was also expressed in microspores in S-22 homozygotes but not in S-22/S-44 and S-22/S-36 heterozygotes. These results suggest that a mechanism different from class-II SCR/SP11 suppression may operate for the suppression of recessive class-I SCR-22 in S heterozygotes., Self-incompatibility: Rethinking hierarchy Studies in cabbage plants shed new light on self-incompatibility mechanisms to avoid self-fertilization. In Brassica, self-incompatibility is controlled by the expression of dominant and recessive genetic variants in the S-locus region. These variants encode proteins that mediate the rejection of self-pollen, but little is known about how the dominance relationship is established between them. Takeshi Nishio at Tohoku University in Japan and colleagues have examined the sequence, expression pattern and dominance relationships of the gene encoding the pollen-coat protein SCR-22. Despite sharing features with dominant SCR variants, SCR-22 can act in a recessive manner. Interestingly, unlike other recessive SCR variants, the suppression of SCR-22 expression does not depend on the addition of methyl groups that prevent transcription factor binding. This finding suggests the dominance hierarchy is governed by different mechanisms.

Self-incompatibility in the Brassicaceae family is governed by two-linked highly polymorphic genes located at theSlocus,SRKandSCR. Previously, theSRKandSCRgenes ofArabidopsis lyratawere introduced intoArabidopsis thalianatransformants to generate self-incompatible lines. However, it has not been reported that theSRKandSCRgenes ofBrassicaspecies confer self-incompatibility inA. thaliana. In this study, we attempted to construct self-incompatibleA. thalianatransformants expressing the self-recognition activity ofBrassica rapaby introducing theBrSCRgene along with a chimericBrSRKgene (BrSRK chimera, in which the kinase domain ofBrSRKwas replaced with that ofAlSKRb). We found thatBrSRK chimeraandBrSCRofB. rapa S-9andS-46haplotypes, but not those ofS-29,S-44, andS-60haplotypes, conferred self-recognition activity inA. thaliana. We also investigated the importance of amino acid residues involved in the BrSRK9–BrSCR9 interaction usingA. thalianatransformants expressing mutant variants ofBrSRK-9 chimeraandBrSCR-9. The results showed that some of the amino acid residues are essential for self-recognition. The method developed in this study for the construction of self-incompatibleA. thalianatransformants showingB. rapaself-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae.

In our previous study, we identified a QTL for cold tolerance at the booting stage of ‘Ukei840’, the cold tolerance of which is derived from one of the most tolerant cultivars, ‘Lijiangxintuanheigu’, in Yunnan province of China, in a 1.2-Mb region between RM7000 and RM3719 markers on chromosome 3. In the present study, we delimited the previously identified QTL to a region of about 35 kb by SSR and dot-blot-SNP marker analysis and nucleotide sequencing using a newly produced near-isogenic line having the ‘Lijiangxintuanheigu’ allele in the QTL region. Comparison of nucleotide sequences of the published ‘Nipponbare’ and ‘Hitomebore’ genome sequences and our previously determined sequence of ‘Ukei840’ revealed nine SNPs in the delimited 35-kb region harboring six genes. LOC-Os03g59190 containing two SNPs in its coding region was a retrotransposon-like sequence having long terminal repeats, which has at least seven other copies in the rice genome. Expression levels of the other five genes in spikelets at the booting stage were not different between ‘Ukei840’ and ‘Hitomebore’. There was one SNP in the coding region of Os03g0806700 which causes amino acid substitution in ‘Ukei840’. However, in Os03g0806700, the genotype of ‘Ukei840’ is the same as that of cold sensitive Indica cultivars. Among the other six SNPs in intergenic regions, one SNP was present in a locus for small RNAs. Possible candidates for the cold-tolerance gene derived from ‘Lijiangxintuanheigu’ are discussed.

Radish, belonging to the family Brassicaceae, has a self-incompatibility which is controlled by multiple alleles on the S locus. To employ the self-incompatibility in an F1 breeding system, identification of S haplotypes is necessary. Since collection of S haplotypes and determination of nucleotide sequences of SLG, SRK, and SCR alleles in cultivated radish have been conducted by different groups independently, the same or similar sequences with different S haplotype names and different sequences with the same S haplotype names have been registered in public databases, resulting in confusion of S haplotype names for researchers and breeders. In the present study, we developed S homozygous lines from radish F1 hybrid cultivars in Japan and determined the nucleotide sequences of SCR, the S domain and the kinase domain of SRK, and the SLG of a large number of S haplotypes. Comparing these sequences with our previously published sequences, the haplotypes were ordered into 23 different S haplotypes. The sequences of the 23 S haplotypes were compared with S haplotype sequences registered by different groups, and we suggested a unification of these S haplotypes. Furthermore, dot-blot hybridization using SRK allele-specific probes was examined for developing a standard method for S haplotype identification.

Radish (Raphanus sativus L.) has a self-incompatibility system similar to those of Brassica rapa and Brassica oleracea. Self-recognition specificities of pollen and stigmas are determined by multiple alleles of SP11 and SRK, respectively, both of which are located on the S locus. As in B. rapa and B. oleracea, stigmas in radish also have S glycoprotein encoded by SLG closely linked to SP11 and SRK. The set of alleles of SP11, SRK, and SLG is called S haplotype, and S haplotypes are numbered as S-1, S-2, S-3, etc. Although radish belongs to a genus different from Brassica, nucleotide sequences of SP11, SRK, and SLG alleles of radish and Brassica are intermingled in phylogenetic trees of SP11, SRK, and SLG, respectively, indicating that diversification of these alleles predates speciation of these genera. SP11, SRK, and SLG alleles of some S haplotypes in radish are highly similar to those of some S haplotypes in Brassica, and one S haplotype in radish has been revealed to have the same recognition specificity as that of one S haplotype in B. rapa. Comparison of nucleotide sequences of SP11 and SRK alleles and recognition specificities between similar S haplotypes of radish and Brassica may provide information useful for understanding the molecular structures of SP11 and SRK proteins. The self-incompatibility system is used for seed production in F1 hybrid cultivars of radish, in which characterization of each S haplotype, especially the strength of the self-incompatibility phenotype, is required. However, the numbering of S haplotypes in radish varies by researchers, and nucleotide sequence information on S haplotypes is thus confusing. The importance of unification of S haplotype nomenclature is herein discussed.

Monosomic addition lines (MALs) are useful materials not only for cytogenetic and molecular genetic studies but also for plant breeding as gene sources. In our previous study, two MALs in the tribe Brassiceae were developed, one being Raphanus sativus lines with alien chromosomes of Brassica rapa (B. rapa-monosomic addition lines; BrMALs) and the second being those with alien chromosomes of Brassica oleracea (B. oleracea-monosomic addition lines; BoMALs). We developed species-specific DNA markers from the genomic sequences of B. rapa and B. oleracea comparing them with those of R. sativus, and identified chromosomes added in BrMALs and BoMALs using these markers. It was revealed that eight types of BrMALs have seven chromosomes of B. rapa and seven types of BoMALs have six chromosomes of B. oleracea. Furthermore, chromosome breakage and homoeologous recombination were suggested to have occurred in some MALs. The developed species-specific DNA markers are considered to be useful for producing MALs and also for assessing chromosome abnormality in MALs.

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥ 300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

Stable genic male sterility (GMS), which is not influenced by environmental factors, has not been used for F1 hybrid seed production because male-sterile inbred lines cannot be developed and male-sterile plants must be selected from segregating populations every time. However, the stability of male sterility may provide a reliable system for F1 seed production without contamination of selfed seeds. A genic male-sterile mutant in rice (Oryza sativa L.), C204, which was selected from progeny of the cultivar ‘Koshihikari’ irradiated by gamma rays, has shorter and whiter anthers than those of ‘Koshihikari’ and has no pollen grains. Segregation analysis of C204 suggested the male sterility of this mutant to be controlled by a recessive allele of a single gene. Linkage analysis of a mutated gene responsible for the male sterility revealed the gene to be in a region of ca. 75 kb on the long arm of chromosome 9. The nine genes predicted in the 75-kb region were sequenced, and compared with the published Nipponbare genome sequences. A single-base deletion was found in the first exon of a C204 allele of Os09g0493500, which encodes an NAD-dependent epimerase/dehydratase family protein, resulting in a frameshift causing a premature stop codon. A dot-blot single nucleotide polymorphism marker for detection of the single-base deletion in Os09g0493500 was developed. We herein propose an F1 hybrid seed production system using stable GMS with a simple selection method of GMS plants.

An F2 population was developed from a cross between a mur-cytoplasmic male sterile broccoli line and a restorer Chinese kale line. Phenotypic analysis of F2 plants indicated that the pollen fertility is controlled by two genes and segregated in a duplicate gene interaction mode with a ratio of 15:1. A total of 236 single nucleotide polymorphism (SNP) markers were developed utilizing 1,448 primers designed for production of expressed sequence tag (EST)-SNP markers of Raphanus sativus and analyzed by the dot-blot technique in 205 F2 individuals. A linkage map was constructed with a total of 142 markers and these markers were assigned to nine linkage groups together with simple sequence repeat markers mapped previously on the published linkage maps of Brassica oleracea. The linkage map spanned 909 cM with an average marker distance of 6.4 cM. A fertility restorer locus (Rfm1) was mapped on LG1, corresponding to chromosome 3, along with a flower color locus at a distance of 25 cM. SNP markers flanking the Rfm1 locus were BoCL2642s at a distance of 2.5 cM on one side and BoCL2901s at a distance of 7.5 cM on the other side. All the SNP markers showed homology with Arabidopsis thaliana and Brassica rapa genome sequences. Three pentatricopeptide repeat genes of the P-subfamily, particularly expressed in buds of the restorer line, were identified and these genes could be potential candidate fertility restorer genes.

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.

Low temperature at the booting stage of rice causes male sterility resulting in severe yield loss. Cold tolerance has long been an important objective in rice breeding. We identified a quantitative trait locus (QTL) for cold tolerance on the long arm of chromosome 3 from the cold-tolerant breeding line 'Ukei 840' by using F(2) and BC(1)F(2) populations from crosses between 'Ukei 840' and 'Hitomebore'. The cold tolerance of 'Ukei 840' is derived from the Chinese cultivar 'Lijiangxintuanheigu'. The effect of this QTL on cold tolerance was confirmed by developing 'Hitomebore' chromosome segment substitution lines having 'Lijiangxintuanheigu' alleles on chromosome 3. By producing recombinants in chromosome 3, the QTL region for cold tolerance was delimited to the region of about 1.2-Mb region between RM3719 and RM7000. All lines heterozygous for the QTL showed seed fertilities as low as that of 'Hitomebore', suggesting that the 'Lijiangxintuanheigu' allele for cold tolerance in the QTL region is recessive. Determination of a 1.2-Mb nucleotide sequence of 'Ukei 840' and comparison with the published genomic sequence of 'Nipponbare' showed 254 SNPs, of which 11 were in coding regions of genes, seven in five genes being non-synonymous. SNPs were detected in the 5-kb upstream regions of 89 genes, but no differences of gene expression levels were detected between alleles of these genes. Although further delimitation is required to identify the gene responsible for cold tolerance of 'Lijiangxintuanheigu', SNP markers developed here will be useful for marker-assisted selection in a breeding program using 'Lijiangxintuanheigu' as a donor of cold tolerance.

The self-incompatibility (SI) system of the Brassicaceae is based on allele-specific interactions among haplotypes of the S locus. In all tested self-incompatible Brassicaceae, the S haplotype encompasses two linked genes, one encoding the S -locus receptor kinase (SRK), a transmembrane kinase displayed at the surface of stigma epidermal cells, and the other encoding its ligand, the S -locus cysteine-rich (SCR) protein, which is localized in the pollen coat. Transfer of the two genes to self-fertile Arabidopsis thaliana allowed the establishment of robust SI in several accessions, indicating that the signaling cascade triggered by this receptor–ligand interaction and the resulting inhibition of “self” pollen by the stigma have been maintained in extant A. thaliana . Based on studies in Brassica species, the membrane-tethered kinase MLPK, the ARM repeat-containing U-box protein ARC1, and the exocyst subunit Exo70A1 have been proposed to function as components of an SI signaling cascade. Here we tested the role of these molecules in the SI response of A. thaliana SRK-SCR plants. We show that the A. thaliana ARC1 ortholog is a highly decayed pseudogene. We also show that, unlike reports in Brassica , inactivation of the MLPK ortholog AtAPK1b and overexpression of Exo70A1 neither abolish nor weaken SI in A. thaliana SRK-SCR plants. These results do not support a role for these molecules in the SI response of A. thaliana .

Pyrus displays gametophytic self-incompatibility controlled by a single highly polymorphic gene complex termed S locus, which comprises a stylar-expressed gene (S-RNase) tighlty linked with a pollen expressed gene, that determines the specificity of the self-incompatibility locus. Deduced amino acid sequence of ‘Meigetsu’ S 8 -RNase in Pyrus pyrifolia and ‘Kuerlexiangli’ S 28 -RNase in P. sinkiangensis showed 100% identity. S 3 -RNase in Malus spectabilis was also found to be similar to S 8 -RNase in P. pyrifolia with 96.9% identity in the deduced amino acid sequence. The intron, which is generally highly polymorphic between alleles, was also remarkably well conserved within these allele pairs. The intron of PpS 8 -RNase showed 95.3 and 91.9% identity with PsS 28 -RNase and MsS 3 -RNase, respectively. Pollen tube growth in styles, pollen tube length in artificial media containing different S-RNases and segregation of S haplotypes in F1 plants revealed commonality of the recognition specificity between PpS 8 -RNase and PsS 28 -RNase and between PpS 8 -RNase and MsS 3 -RNase. Results suggested that PpS 8 -RNase, PsS 28 -RNase and MsS 3 -RNase have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found between PpS 8 -RNase and MsS 3 -RNase do not alter the recognition specificity.

Limiting the ingestion of protein is the fundamental idea in the diet therapy for patients with chronic renal failure. Two mutations involved in the content of major rice storage proteins useful for developing low easy-to-digest protein rice variety have been isolated. The glb1 mutation causes the deficiency of alpha-globulin, and the Lgc1 mutation reduces the glutelin content. By combining the glb1 and the Lgc1 mutations, it is possible to reduce the easy-to-digest protein content by approximately 50%. The Lgc1 mutation has been shown to be caused by a 3.5-kb deletion between the glutelin structural genes, GluB4 and GluB5, while the molecular basis of glb1 mutation has been less understood. PCR analysis of the glb1 mutation revealed a 62.8-kb deletion, including the structural gene of alpha-globulin. Based on these lines of information, we generated PCR markers that make it possible to detect the glb1 and Lgc1 mutations. Using those PCR markers, we genotyped F(2) plants segregating for the glb1 mutation and the Lgc1 mutation and confirmed the consistency of genotype and phenotype. Because the PCR marker sets can distinguish heterozygotes, they will be very useful in developing new varieties of low easy-to-digest protein rice.

Most Rosaceae fruit trees such as Japanese plum and sweet cherry have a gametophytic self-incompatibility (GSI) system controlled by a single S locus containing at least two linked genes with multiple alleles, i.e., S-RNase as a pistil determinant and SFB (S-haplotype-specific F-box gene) as a candidate for the pollen S determinant. For identification of S genotypes, many methods based on polymerase chain reaction (PCR) utilizing polymorphism in length of the S-RNase and SFB gene have been developed. In this study, we developed two dot-blot analysis methods for S-haplotype identification utilizing allele-specific oligonucleotides based on the SFB-HVa region, which has high sequence polymorphism. Dot-blotting of allele-specific oligonucleotides hybridized with digoxigenin-labeled PCR products allowed S genotyping of plants with nine S haplotypes (S-a, S-b, S-c, S-e, S-f, S-h, S-k, S-7 and S-10) in Japanese plum and ten S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) in sweet cherry (dot-blot-S-genotyping). In addition, dot-blotting of PCR products of SFB probed with the allele-specific oligonucleotides, occasionally utilizing competitive hybridization, was successful in screening for a desirable S haplotype in sweet cherry (dot-blot-S-screening).

DNA polymorphism of the Wx gene in glutinous rice cultivars was investigated by PCR-RF-SSCP and heteroduplex cleavage analysis using Brassica petiole extract, and the nucleotide sequence variations were identified. Most japonica-type glutinous rice was found to have a 23-bp duplication in the second exon, which causes loss of the function of granule-bound starch synthase (GBSS) encoded by the Wx gene. Without the 23-bp duplication, there was an insertion of 7,764 bp in the ninth exon of the wx allele of 'Oragamochi'. Expression analysis of the wx allele using RT-PCR and Northern blot analysis revealed that transcripts of the 'Oragamochi' wx allele are about 1-kb shorter and that the deduced amino acid sequence of the transcript lacks a motif important for GBSS. Therefore, this insertion was considered to be the cause of the glutinous trait of 'Oragamochi'. This 7,764-bp insertion had long terminal repeats, a primer binding site, and a polypurine tract, but no sequence homologous with gag and pol, suggesting that it is a non-autonomous element. Furthermore, it had a structure similar to Dasheng and may be a member of Dasheng.

Brassica napus is an amphidiploid species with the A genome from Brassica rapa and the C genome from Brassica oleracea. Although B. rapa, B. oleracea and artificially synthesized amphidiploids with the AC genome are self-incompatible, B. napus is self-compatible. Six S genotypes were identified in B. napus, five of which had class I S haplotypes from one species and a class II S haplotype from the other species, and mutations causing self-compatibility were identified in three of these S genotypes. The most predominant S genotype (BnS-1;BnS-6), which is that of cv. 'Westar', had a class I S haplotype similar to B. rapa S-47 (BrS-47) and a class II S haplotype similar to B. oleracea S-15 (BoS-15). The stigmas of 'Westar' rejected the pollen grains of both BrS-47 and BoS-15, while reciprocal crossings were compatible. Insertion of a DNA fragment of about 3.6 kb was found in the promoter region of the SP11/SCR allele of BnS-1, and transcripts of SP11/SCR were not detected in 'Westar'. The nucleotide sequence of the SP11 genomic DNA of BnS-6 was 100% identical to that of SP11 of BoS-15. Class I SP11 alleles from one species showed dominance over class II SP11 alleles from the other species in artificially synthesized B. napus lines, suggesting that the non-functional dominant SP11 allele suppressed the expression of the recessive SP11 allele in 'Westar'. Two other S genotypes in B. napus also had non-functional class I S haplotypes together with recessive BnS-6. These observations suggest independent origins of self-compatibility in B. napus.

Common buckwheat plants have heteromorphic self-incompatibility. Using two self-fertilizing lines, we revealed that there are two distinct systems of sell-compatibility, one using a self-compatible allele, S', the other using modifier genes located outside the S locus and suppressing the functions of the S-locus genes. S h appears to have been produced by recombination in the S supergene (gi s I P PA/gi S I p PA), in which each gene is functional. The modifier genes control the intensity of self-incompatibility and have three distinctive features: (1) Pollen shows cross-compatibility with styles of all flower types, although the compatibility is influenced by the genetic background of those plants. (2) Flower morphology of F 1 plants is controlled by the genotype of the S locus. (3) F 1 plants show high self-compatibility, although the level of self-compatibility is influenced by genetic background. The use of these genes for buckwheat breeding is discussed.

In the course of evolution, a gene is often duplicated in tandem, resulting in a functional redundancy. The analysis of function of these genes by raising double mutant might be difficult because they are very tightly linked. We described here a mutant of such a tandem duplicated gene. glu1 is a gamma-ray-induced rice mutant, which lacks an acidic subunit of glutelin, a major seed storage protein. We found that glu1 harbors a 129.7-kb deletion involving two highly similar and tandem repeated glutelin genes, GluB5 and GluB4. The deletion eliminated the entire GluB5 and GluB4 gene except half of the first exon of GluB5. GluB5 and GluB4 have the same amino acid sequence in the acidic subunit, suggesting that only the mutation involving both GluB5 and GluB4 results in the lack of the glutelin acidic subunit deleted in glu1. Our finding suggests that gamma-ray can be an effective mutagen to analyze tandem repeated and functionally redundant genes.

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum.

DNA methylation is essential for normal development and plays important roles in regulating gene expression in plants. Analysis of the key enzymes catalyzing DNA methylation is important to understand epigenetic phenomena. In this study, three putative methyltransferase genes, BrMET1a, BrMET1b, and BrCMT, were isolated from a genome library of Brassica rapa. Structural conservation of the amino acid sequence between BrMET1a/BrMET1b and AtMET1 and that between BrCMT and AtCMT3 suggests that they may function as DNA methyltransferase. BrMET1a was expressed in vegetative and reproductive organs, while BrMET1b was expressed only in pistils, indicating that these two genes have different functions. BrCMT was expressed especially in stamens at the stage of 2-4 days before anthesis. We isolated three DNA methyltransferase genes in Brassica rapa and indicated differences of expression patterns of these DNA methyltransferase genes and expression levels in different tissues and developmental stages, suggesting that these genes might play important roles in epigenetic gene regulation in B. rapa.

Selection of mutants by the reverse genetic approach is a useful tool for gene function analysis and crop improvement. TILLING (Targeting Induced Local Lesions in Genomes), in which mutants are selected from the progeny of EMS-treated plants by the SNP analysis using heteroduplex cleavage enzyme from celery, has been applied to various plant species. Since gamma-ray-induced point mutations have not been identified by the reverse genetic approach, we attempted to select mutants from a population of the gamma-ray-irradiated rice. A heteroduplex between wild-type DNA and mutant DNA was cleaved by an endonuclease extracted from petioles of Brassica rapa, and cleaved DNA was detected using agarose gel electrophoresis. M2 plants derived from 2,130 M1 plants irradiated by gamma rays were used for the screening of mutants having nucleotide changes in 25 regions from 1.0 kb to 1.5 kb. Six mutations were identified and the rate of mutation induced by gamma rays was estimated to be one mutation per 6,190 kb. Four of the mutations were single nucleotide substitutions and two were 2-bp and 4-bp deletions. These results suggest that the point mutation rate with gamma rays was quite lower than that with EMS, but that the rate of knockout mutations among detected mutations generated by the gamma-ray irradiation was higher than that by the EMS treatment.

From a series of studies on single nucleotide polymorphisms (SNPs) in the bovine growth hormone (GH) gene of Japanese Black cattle, the type-C (127(Val) and 172(Met) ) that is specific for this breed has been intensively focused upon because of the economic importance for carcass traits, such as intramuscular oleic acid contents. In the present study, we intended to analyze the 3-D structure of GH of haplotype C, and developed a novel method to detect the type C gene. Three-D analysis of the type C protein showed that the amino acid residues (127(Val) and 172(Met) ), which are present in the third and fourth helixes, respectively, and are important for binding with GH receptors, are shifted to deeper positions in the molecule compared with that for type A (127(Leu) and 172(Thr) ), implying the alteration of binding interaction with receptors. A novel, efficient and cost-effective method (Dot-blot-SNP technique) for type C genotyping was successfully established, of which the basal method was a reported genotyping of SNPs for a large number of plants, reducing the cost to 10% or less of direct sequencing.

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (πS and πN) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of πN:πS for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS 46 (S 46 in B. rapa) and BoS 7 (S 7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS 47 and BoS 12 , and BrS 8 and BoS 32 , having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS 46 and BoS 15 showed incompatibility with a BoS 7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS 46 and BoS 7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS 46 and BoS 7 . These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.

In higher plants, the self-incompatibility mechanism is important for inhibition of self-fertilization and facilitation of out-crossing. In Brassicaceae, the self-incompatibility response is mediated by allele-specific interaction of the stigma-localized S-locus receptor kinase (SRK) with the pollen coat-localized ligand (SCR/SP11). All self-incompatible Brassicaceae plants analyzed have been found to have the SRK and SCR/SP11 genes in the S-locus region. Although Arabidopsis thaliana is self-compatible, transformation with functional SRK-SCR genes from self-incompatible Arabidopsis species confers the self-incompatibility phenotype to A. thaliana. The allele-specific interaction between SRK and SCR activates the downstream signaling cascade of self-incompatibility. Yeast two-hybrid analysis with a kinase domain of SRK as bait and genetic analysis suggested several candidate components of self-incompatibility signaling in Brassica. Recently, A. thaliana genes orthologous to the identified genes for Brassica self-incompatibility signaling were evaluated by using a self-incompatible transgenic A. thaliana plant and these orthologous genes were found not to be involved in self-incompatibility signaling in the transgenic A. thaliana. In this review, we describe common and different aspects of S-locus genomic regions and self-incompatibility signaling between Brassica and Arabidopsis.

Self-incompatibility (SI) in Brassica is sporophytically controlled by the multi-allelic S locus. SI phenotypes of the stigma and pollen in an S heterozygote are determined by the two S haplotypes it carries; the two haplotypes may be co-dominant or exhibit a dominant/recessive relationship. Because the S receptor kinase (SRK) gene of the S locus was recently shown to determine the S haplotype specificity of the stigma, we wished to investigate whether SRK also plays a role in the dominance relationships between S haplotypes. We crossed plants carrying an SRK 28 transgene with plants homozygous for one of five S haplotypes that are either co-dominant with, or recessive to, S 28 haplotype in the stigma, and analyzed the SI phenotypes of the progeny. In all cases, the SI phenotype of the stigma of plants carrying the SRK 28 transgene could be predicted by the known dominance relationships between the S haplotype(s) and the S 28 haplotype. Moreover, in the S 43 homozygote carrying the SRK 28 transgene where the S 43 phenotype in the stigma was masked by the presence of the SRK 28 , the transcript level of SRK 28 was found to be much lower than that of SRK 43 . All these results suggest that the dominance relationships between S haplotypes in the stigma are determined by SRK, but not by virtue of its relative expression level.

The MIP-MOD (for MOD-locus associated Major Intrinsic Protein) gene encodes an aquaporin-like product, and has been reported to be a candidate for the MOD gene which is required for the self-incompatibility response in Brassica rapa. In an antisense suppression experiment designed to investigate the role of MIP-MOD, we found that levels of MIP-MOD mRNA in the stigmas of fourteen antisense transgenics, as well as in the self-incompatible cultivar Osome (Osm), were much lower than in the stigmas of the self-incompatible S8 homozygous (S8) strain. Therefore, we analyzed the molecular structure of the MIP-MOD gene in three B. rapa strains: S8, Osm, and the self-compatible var. Yellow Sarson (YS). Nucleotide sequence analysis of the MIP-MOD genes isolated from the three strains revealed that all three encode the same amino acid sequence and that YS and Osm contain the same MIP-MOD allele, designated MIP-MOD(YS). Analysis of other self-incompatible B. rapa strains that are homozygous for the MIP-MOD(YS) allele indicated that high levels of MIP-MOD transcripts are not essential for the self-incompatibility response. Furthermore, a MOD mutant generated by gamma-irradiation was found to contain a wild-type MIP-MOD gene that is expressed at normal levels. These data suggest that MIP-MOD is not MOD itself. We suggest that this gene should be renamed MLM (for MIP gene linked to MOD).

Self-incompatibility (SI) in Brassica is regulated by a single multi-allelic locus, S, which contains highly polymorphic stigma-expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self-incompatible S18 and S60 homozygotes of Brassica oleracea have an in-frame stop codon and a 23 bp deletion resulting in a frame-shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes.

DNA sequences of SLG and SRK , believed to function in self-pollen recognition of self-incompatibility, were analysed and compared between many S haplotypes of Brassica oleracea . Some different S haplotypes have highly similar SLGs (more than 97.5% amino acid sequence identity), while their SRKs are less similar (less than 88% identity). Different lines having the same recognition specificity often show a high degree of sequence variation in SLG (88.5% identity in the S 2 haplotype), but their SRKs are very similar to each other (97.3% identity). S haplotypes, in which SLG protein is undetectable by Western blot analysis, have stop codons in the middle of the coding region of SLG , or lack an SLG gene in the genome, suggesting that these haplotypes do not synthesize the functional SLG protein. The SRK alleles in these haplotypes are normal. The function of SLG and SRK in self-incompatibility and the evolution of these alleles are discussed.

DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes.

Self-incompatibility in Brassica is controlled by a single multi-allelic locus ( S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene ( SLG ) and an S receptor kinase gene ( SRK ). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris . Sequence comparisons of a large number of SLG alleles and SLG -related genes revealed the following points. ( i ) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG 14 of B. oleracea and SLG 25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. ( ii ) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison ( SLG 8 and SLG 46 of B. campestris ) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. ( iii ) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG / SRK are presented.

Summary Monofactorial gametophytic self-incompatibility in the family Rosaceae is controlled by the single multi-allelic S-locus. In Japanese pear, a naturally occurring self-compatible mutant was found as a sport of a self-incompatible cultivar with an S-genotype of S2,S4. The S,4 allele has been shown to be non-functional in the style of the mutant, but functional in pollen. The present study shows the S2-RNase gene to be transcribed normally in the style of the mutant, but that the S4-RNase gene transcript is not detectable. Furthermore, the S4-RNase gene could not be detected in the mutant by genomic Southern blot, indicating that the mutant lacks the gene. The extent of deletion in the mutant was estimated to be more than 4 kbp, and spans the entire length of the S4-RNase gene. These findings provide evidence that the S-RNase gene is essential for the self-incompatibility reaction in the style, but is not involved in controlling the self-incompatibility behavior of pollen.

Nine mutant lines lacking glutelin subunits were selected from M2 seeds of about 10000 M1 plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F2 plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease.

Rice mutants containing low levels of the 16-kDa allergenic protein, which is the main allergen in the rice grain for patients of atopic dermatitis due to the intake of rice, were screened, and 4 independent mutant lines with small amounts of this protein were found by SDS gel electrophoresis and immunoblot analysis. These mutants were grouped into two types. Two mutant lines, 85KG-4 and 86RG-18, contained low levels of the 16k-Da and 26-kDa polypeptides and a high level of the 57-kDa polypeptide. The 16-kDa polypeptide content of these mutants was about half that of the original cultivars. Homozygous lines were developed, and these showed normal growth and seed set. The other 2 mutant lines, 87KG-970 and 89WPKE-149, showed traces of the 16-kDa and 26 kDa polypeptides and contained a high level of the 13 kDa polypeptide. The homozygous plants of this type were sterile. All of the mutant lines had floury endosperms. Genetic analysis suggested that low 16-kDa polypeptide content is controlled by a single recessive gene. Attempts to separate of the genes for low 16-kDa polypeptide content and floury endosperm by crossing with the original cultivar were unsuccessful, suggesting the tight linkage of these two genes or pleiotropism of a single mutated gene. The relationship between low 16-kDa polypeptide content and the floury character and the possible use of the mutant as a low allergen rice are discussed.

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 POLLINATION AND POLLEN TUBE GROWTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 Interaction s between the M ale G ameto phyte and Pistil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 SelfIncom patibili ty Systems: Gameto phytic and S poro phyti c Determin ation of Pollen Phenoty pe . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 DEVELOPMENTAL S TUDIES OF REPRODUCTIVE STRUCTURES . . . . . . . . .. . . ... . . . . 398 Tissues of the Pistil with Special Reference to Pollination Functions 398 The Male Gametophyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 MOLECULAR ANALYSIS OF SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 1 The S-M ultigene F amily o j Brassica. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 402 Allelism at the S-Lo cus and the SLG Gene""""""""""""""""""""""""" 402 The S-Locus Related (SLR) Genes 405 Molecular Complexity oj the S-Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 EXPRESSION OF THE SLG GENE IN PIS TILS AND ANTHERS . . . . . . . . . . . . . . . . . . . . . . . 409 Expression in Cruc ifers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 409 Express ion in Tr ansgen ic Tob acco and the Rel at ionship of G ameto phytic and S poro phyt ic Incompatibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 10 GENETIC INTERACTIONS IN SELF-INCOMPATIBILITY RESPONSES . . . . . . . . . . . . . . 4 1 2 The Analysis o j Se lf-Com patible Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2 The An alysis o j Transgenic Pl ants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 3 GENETIC ABLATION O F FLORAL TISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 As a Tool Jor the Study o j Pl ant Develo pmen t . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . 415 As a Tool Jar Cro p Im pro vement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . """ 416

Interlocus gene conversion is considered a crucial mechanism for generating novel combinations of polymorphisms in duplicated genes. The importance of gene conversion between duplicated genes has been recognized in the major histocompatibility complex and self-incompatibility genes, which are likely subject to diversifying selection. To theoretically understand the potential role of gene conversion in such situations, forward simulations are performed in various two-locus models. The results show that gene conversion could significantly increase the number of haplotypes when diversifying selection works on both loci. We find that the tract length of gene conversion is an important factor to determine the efficacy of gene conversion: shorter tract lengths can more effectively generate novel haplotypes given the gene conversion rate per site is the same. Similar results are also obtained when one of the duplicated genes is assumed to be a pseudogene. It is suggested that a duplicated gene, even after being silenced, will contribute to increasing the variability in the other locus through gene conversion. Consequently, the fixation probability and longevity of duplicated genes increase under the presence of gene conversion. On the basis of these findings, we propose a new scenario for the preservation of a duplicated gene: when the original donor gene is under diversifying selection, a duplicated copy can be preserved by gene conversion even after it is pseudogenized.

In self-incompatibility, a number of S haplotypes are maintained by frequency-dependent selection, which results in trans-specific S haplotypes. The region of several kilobases (∼40–60 kb) from SP6 to SP2, including self-incompatibility-related genes and some adjacent genes in Brassica rapa, has high nucleotide diversity due to the hitchhiking effect, and therefore we call this region the “S-locus complex.” Recombination in the S-locus complex is considered to be suppressed. We sequenced regions of >50 kb of the S-locus complex of three S haplotypes in B. rapa and found higher nucleotide diversity in intergenic regions than in coding regions. Two highly similar regions of >10 kb were found between BrS-8 and BrS-46. Phylogenetic analysis using trans-specific S haplotypes (called interspecific pairs) of B. rapa and B. oleracea suggested that recombination reduced the nucleotide diversity in these two regions and that the genes not involved in self-incompatibility in the S-locus complex and the kinase domain, but not the S domain, of SRK have also experienced recombination. Recombination may reduce hitchhiking diversity in the S-locus complex, whereas the region from the S domain to SP11 would disfavor recombination.

DNA methylation and histone modification play important roles in regulating gene expression. The DDM1 gene in Arabidopsis thaliana (AtDDM1) is required for the maintenance of DNA methylation level and histone H3 methylation pattern. We isolated DDM1 homologs of Brassica rapa, BrDDM1a and BrDDM1b, which have 84.4% and 84.1% deduced amino acid sequence identities with AtDDM1, respectively. Both the BrDDM1a and BrDDM1b genes were found to be expressed in vegetative and reproductive tissues. B. rapa ddm1-RNAi transgenic plants with reduced levels of BrDDM1a/BrDDM1b expression showed genome-wide and non-tissue-specific demethylation. These results suggest that the BrDDM1a and BrDDM1b genes are orthologs of AtDDM1 and are required for the maintenance of DNA methylation as is AtDDM1. Despite genome-wide demethylation, developmental abnormalities were not found in the ddm1-RNAi transgenic plants. Dominance relationships of SP11/SCR alleles, the determinant of pollen recognition specificity in Brassica self-incompatibility, in S heterozygotes in B. rapa were not influenced by the low level of the BrDDM1 expression. Transcriptional reactivation of retrotransposon-like sequences observed in the ddm1-RNAi transgenic plants indicates that BrDDM1a and BrDDM1b participate in silencing of retrotransposons. Hypomethylation states of the ddm1-RNAi transgenic plants were inherited by plants of the next generation even by plants which had lost the RNAi construct by segregation. Remethylation was observed in a few progenies. Efficiencies of remethylation in the progenies without the RNAi construct were different between 18S rDNA, BoSTF12a/15a, and BrTto1 sequences.

Identification of single nucleotide polymorphisms (SNPs) in a large number of genes will enable estimation of the number of genes having different alleles in a population. In the present study, SNPs between 21 rice cultivars including 17 Japanese cultivars, one upland rice, and three indica cultivars were analyzed by PCR-RF-SSCP. PCR-RF-SSCP analysis was found to be a more efficient method for detecting SNPs than mismatch-cleavage analysis, though both PCR-RF-SSCP and mismatch-cleavage are useful for screening SNPs. The number of DNA fragments showing polymorphism between Japanese cultivars was 134 in the 1,036 genes analyzed. In 137 genes, 638 DNA polymorphisms were identified. Out of 52 genes having polymorphisms in the exons, one had a frame-shift mutation, three had polymorphism causing amino acid insertions or deletions, and 16 genes had missense polymorphisms. The number of genes having frame-shift mutations and missense polymorphisms between the 17 Japanese cultivars was estimated to be 41 and 677 on average, respectively, and those between japonica and indica to be 425 and 6,977, respectively. Chromosomal regions of cultivars selected in rice breeding processes were identified by SNP analysis of genes.

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, both of which are encoded in the S locus. The nucleotide sequence analyses of many SRK and SP11/SCR alleles have identified several interspecific pairs of S haplotypes having highly similar sequences between B. oleracea and B. rapa. These interspecific pairs of S haplotypes are considered to be derived from common ancestors and to have maintained the same recognition specificity after speciation. In this study, the genome structures of three interspecific pairs of S haplotypes were compared by sequencing SRK, SP11/SCR, and their flanking regions. Regions between SRK and SP11/SCR in B. oleracea were demonstrated to be much longer than those of B. rapa and several retrotransposon-like sequences were identified in the S locus in B. oleracea. Among the seven retrotransposon-like sequences, six sequences were found to belong to the ty3 gypsy group. The gag sequences of the retrotransposon-like sequences were phylogenetically different from each other. In Southern blot analysis using retrotransposon-like sequences as probes, the B. oleracea genome showed more signals than the B. rapa genome did. These findings suggest a role for the S locus and genome evolution in self-incompatible plant species.

Mutations in the S locus of a self-compatible cultivar Yellow Sarson in Brassica rapa, which has a self-compatible class-I S haplotype, S-f2, were investigated. S-28 in Brassica oleracea was found to be a member of an interspecific pair with S-f2 in B. rapa. The original S haplotype of S-f2 was identified to be S-54 in B. rapa. Sequence comparison of alleles in S-f2 with those in S-54 and B. oleracea S-28 revealed insertion of a retrotransposon-like sequence in the first intron of SRK and 89-bp deletion in the promoter region of SP11. No transcripts of SRK and SP11 were detected in S-f2 homozygotes, suggesting that the insertion and the deletion in SRK and SP11, respectively, caused the loss of the function of these genes. Promoter assay using transgenic plants indicated that the SP11 promoter of S-f2 has no activity. Heterozygotes of S-f2 and a normal class-II S haplotype, S-60, in B. rapa were found to be self-compatible. Interestingly, transcription of SP11-60 was revealed to be suppressed in the S-f2/S-60 heterozygotes, suggesting that an untranscribed class-I SP11 allele suppresses the expression of a recessive class-II SP11 allele in the anthers of S heterozygotes. Similar phenomenon was observed in heterozygotes of a self-compatible class-I S haplotype and a self-incompatible class-II S haplotype in B. oleracea.

DNA polymorphism of randomly selected genes in rice cultivars was analyzed by the polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) technique. Single DNA fragments were amplified from genomic DNA of the Nipponbare cultivar by 671 primer pairs among the 1000 primer pairs tested. PCR-RF-SSCP analysis using the 671 primer pairs detected polymorphism in 108 DNA fragments between 17 japonica paddy-rice cultivars. An average of 36.9 DNA fragments showed polymorphism between any pair of japonica paddy-rice cultivars. The nucleotide sequences of the polymorphic DNA fragments were determined for 50 alleles of 45 genes together with Nipponbare alleles. In these genes, 142 SNPs and 32 insertions/deletions were identified. Among these 174 sequence variations, 71 were in exons, 78 in introns, and 25 in unassigned regions. There were 28 alleles which had sequence variations in the exons. One allele had a 1-bp deletion in the exon causing a frame-shift mutation, 15 alleles had missense mutations, and the other 12 alleles had synonymous changes and/or sequence variations in 3' untranslated regions. The number of genes having sequence variations between the rice cultivars and the functional implications of the identified SNPs are herein discussed.

The nucleotide sequence of an 86.4-kb region that includes the SP11, SRK, and SLG genes of Brassica rapa S-60 (a class-II S haplotype) was determined. In the sequenced region, 13 putative genes were found besides SP11-60, SRK-60, and SLG-60. Five of these sequences were isolated as cDNAs, five were homologues of known genes, cDNAs, or ORFs, and three are hypothetical ORFs. Based on their nucleotide sequences, however, some of them are thought to be non-functional. Two regions of colinearity between the class-II S-60 and Brassica class-I S haplotypes were identified, i.e., S flanking region 1 which shows partial colinearity of non-genic sequences and S flanking region 2 which shows a high level of colinearity. The observed colinearity made it possible to compare the order of SP-11, SRK, and SLG genes in the S locus between the five sequenced S haplotypes. It emerged that the order of SRK and SLG in class-II S-60is the reverse of that in the four class-I S haplotypes reported so far, and the order of SP11, SRK and SLG is the opposite of that in the class-I haplotype S-910. The possible gene designated as SAN1 ( S locus Anther-expressed Non-coding RNA like-1), which is located in the region between SP11-60 and SRK-60, has features reminiscent of genes for non-coding RNAs (ncRNAs), but no homologous sequences were found in the databases. This sequence is transcribed in anthers but not in stigmas or leaves. These features of the genomic structure of S-60 are discussed with special reference to the characteristics of class-II S haplotypes.

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. In Brassica, it is controlled by a single multi-allelic locus, S, and it is believed that two highly polymorphic genes in the S locus, SLG and SRK, play central roles in self-recognition in stigmas. SRK is a putative receptor protein kinase, whose extracellular domain exhibits high similarity to SLG. We analyzed two pairs of lines showing cross-incompatibility (S2 and S2-b; S13 and S13-b). In S2 and S2-b, SRKs were more highly conserved than SLGs. This was also the case with S13 and S13-b. This suggests that the SRKs of different lines must be conserved for the lines to have the same self-recognition specificity. In particular, SLG2-b showed only 88.5% identity to SLG2, which is comparable to that between the SLGs of different S haplotypes, while SRK2-b showed 97.3% identity to SRK2 in the S domain. These findings suggest that the SLGs in these S haplotypes are not important for self-recognition in SI.

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S9 haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG9, SRK9, SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG9. Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.