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Gamma-rays are the most widely used mutagenic radiation in plant mutation breeding, but detailed characteristics of mutated DNA sequences have not been clarified sufficiently. In contrast, newly introduced physical mutagens, e.g., heavy-ion beams, have attracted geneticists’ and breeders’ interest and many studies on their mutation efficiency and mutated DNA characteristics have been conducted. In this study, we characterized mutations induced by gamma rays and carbon(C)-ion beams in rice (Oryza sativa L.) mutant lines at M5 generation using whole-genome resequencing. On average, 57.0 single base substitutions (SBS), 17.7 deletions, and 5.9 insertions were detected in each gamma-ray-irradiated mutant, whereas 43.7 single SBS, 13.6 deletions, and 5.3 insertions were detected in each C-ion-irradiated mutant. The structural variation (SV) analysis detected 2.0 SVs (including large deletions or insertions, inversions, duplications, and reciprocal translocations) on average in each C-ion-irradiated mutant, while 0.6 SVs were detected on average in each gamma-ray-irradiated mutant. Furthermore, complex SVs presumably having at least two double-strand breaks (DSBs) were detected only in C-ion-irradiated mutants. In summary, gamma-ray irradiation tended to induce larger numbers of small mutations than C-ion irradiation, whereas complex SVs were considered to be the specific characteristics of the mutations induced by C-ion irradiation, which may be due to their different radiation properties. These results could contribute to the application of radiation mutagenesis to plant mutation breeding.

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.

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

For identification of genes responsible for varietal differences in flowering time and leaf morphological traits, we constructed a linkage map of Brassica rapa DNA markers including 170 EST-based markers, 12 SSR markers, and 59 BAC sequence-based markers, of which 151 are single nucleotide polymorphism (SNP) markers. By BLASTN, 223 markers were shown to have homologous regions in Arabidopsis thaliana, and these homologous loci covered nearly the whole genome of A. thaliana. Synteny analysis between B. rapa and A. thaliana revealed 33 large syntenic regions. Three quantitative trait loci (QTLs) for flowering time were detected. BrFLC1 and BrFLC2 were linked to the QTLs for bolting time, budding time, and flowering time. Three SNPs in the promoter, which may be the cause of low expression of BrFLC2 in the early-flowering parental line, were identified. For leaf lobe depth and leaf hairiness, one major QTL corresponding to a syntenic region containing GIBBERELLIN 20 OXIDASE 3 and one major QTL containing BrGL1, respectively, were detected. Analysis of nucleotide sequences and expression of these genes suggested possible involvement of these genes in leaf morphological traits.

The recognition specificity of the pollen ligand of self-incompatibility (SP11/SCR) was investigated using Brassica rapa transgenic plants expressing SP11 transgenes, and SP11 of Raphanus sativus S-21 was found to have the same recognition specificity as that of B. rapa S-9. In a set of three S haplotypes, whose sequence identities of SP11 and SRK are fairly high, R. sativus S-6 showed the same recognition specificity as Brassica oleracea S-18 and a slightly different specificity from B. rapa S-52. B. oleracea S-18, however, showed a different specificity from B. rapa S-52. Using these similar S haplotypes, chimeric SP11 proteins were produced by domain swapping. Bioassay using the chimeric SP11 proteins revealed that the incompatibility response induction activity was altered by the replacement of Region III and Region V. Pollen grains of Brassica transgenic plants expressing chimeric SP11 of the B. oleracea SP11-18 sequence with Region III and Region V from B. rapa SP11-52 (chimeric BoSP11-18[52]) were partially incompatible with the B. rapa S-52 stigmas, and those expressing the R. sativus SP11-6 sequence with Region III and Region V from B. rapa SP11-52 (chimeric RsSP11-6[52]) were completely incompatible with the stigmas having B. rapa S-52. However, the transgenic plant expressing chimeric RsSP11-6(52) also showed incompatibility with B. oleracea S-18 stigmas. These results suggest that Regions III and Region V of SP11 are important for determining the recognition specificity, but not the sole determinant. A possible process of the generation of a new S haplotype is herein discussed.

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.

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.

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.

Spinocerebellar ataxia 6 (SCA6) is an autosomal dominant spinocerebellar degeneration caused by the expansion of the polymorphic CAG repeat in the human alpha1A voltage-dependent calcium channel subunit gene (CACNL1A4 gene). We have analyzed 60 SCA6 individuals from 39 independent SCA6 Japanese families and found that the CAG repeat length is inversely correlated with the age of onset (n = 58, r = -0.51, P < 0.0001). SCA6 chromosomes contained 21-30 repeat units, whereas normal chromosomes displayed 6-17 repeats. There was no overlap between the normal and affected CAG repeat number. The anticipation of the disease was observed clinically in all eight parent-child pairs that we examined; the mean age of onset was significantly lower (P = 0.0042) in children than in parents. However, a parent-child analysis showed the increase in the expansion of CAG repeats only in one pair and no diminution in any affected cases. This result suggests that factors other than CAG repeats may produce the clinical anticipation. A homozygotic case could not demonstrate an unequivocal gene dosage effect on the age of onset.