不結球白菜雄不稔性之遺傳分析、基因精細定位及基因體關聯性之研究

Genetic Analysis, Gene Fine-mapping, and Genome-wide Association Studies on Male Sterility in Brassica rapa ssp. chinensis

林子凱(Tzu-Kai Lin)

指導教授 ： 林順福

國立臺灣大學/生物資源暨農學院/農藝學研究所/博士(2020年)

https://doi.org/10.6342/NTU202002822

摘要

白菜類作物是重要的蔬菜來源之一，為了應用雄不稔性生產具雜種優勢的商業F1種子，急需豐富雄不稔基因的遺傳資源。本研究在小油菜（Brassica rapa ssp. chinensis var. oleifera Makino）地方品種中發現具雄不稔性的自發性突變體，利用後裔檢定進行系統性的遺傳分析，結果顯示該性狀符合單一基因座上三個複等位基因控制的遺傳模式，將該基因命名為BrMs，而此三個複等位基因間的顯隱性關係為BrMsa > BrMsb > BrMsc，其中控制雄不稔性表現者為BrMsb，此遺傳模式為不結球白菜上首見，透過genotyping-by-sequencing (GBS)策略針對不同雄不稔等位基因建構之F2及BC1分離族群進行基因定位，不同雄不稔等位基因均被定位於Chr07染色體之相同區域而驗證遺傳分析的結論。
依據GBS粗定位之區間標誌（bin markers）設計為單一核苷酸多型性（single nucleotide polymorphism, SNP）分子標誌，並以包含1,590株的F2分離族群進行驗證，其中有二個SNP 分子標誌分別位於雄不稔基因的二側，與基因之遺傳距離各均為0.3 cM而將BrMs限縮於約400 kb範圍內，為比較不同雄不稔基因間之差異，結球白菜雄不稔基因Ms的序列特徵化增幅區域（sequence characterized amplified region, SCAR）分子標誌也成功的轉換為SNP 分子標誌，並分析相同F2族群，連鎖分析顯示Ms基因雖亦屬Chr07連鎖群但與本研究之BrMs基因位於不同區域，然而透過基因體比較分析發現大油菜雄不稔基因BnRf位於本研究BrMs基因粗定位範圍內，進一步依據BnRf之基因序列，分析本研究雄不稔系與恢復系其BnRf 同源基因之差異而開發了一個Indel分子標誌，以該分子標誌偵測前述F2族群之923個植株，結果顯示該分子標誌基因分型的結果與外表型完全一致。
為了進行BrMs基因的精細定位，將雄不稔系NH80-A與恢復系TA95進行全基因體定序，針對BrMs粗定位區間比較二品系DNA序列之差異，並開發了9個SNP分子標誌用以分析4,415株 F2族群，基因精細定位的結果顯示BrA7_6746K與BrA7_6749K等二個SNP分子標誌與雄不稔外表型共分離，然而依據BnRf 而設計的Indel分子標誌進行基因分型時，發現2個體其外表型與分子標誌的基因型不一致，推測該個體可能為複合基因內重組的結果，透過基因精細定位將BrMs限縮於24 kb範圍內，該區域僅包含Bra014987、Bra014988及Bra014989等3個預測基因（putative genes），可做為據圖選殖（map-based cloning）之參考。
正常稔性個體其BrMs基因型大都不具BrMsb等位基因，為了開發可鑑別BrMsa與BrMsc等位基因之分子標誌，利用140個B. rapa自交系與NH80-A進行測交而分析其BrMs基因型，並針對整個族群進行全基因體關連性分析（genome-wide association study, GWAS），結果於Chr07染色體上探勘出17個SNPs與BrMs基因具關聯性，挑選4個設計為SNP分子標誌，其中TqBrA7_6750K基因分型的結果顯示其可有效鑑別BrMsa與BrMsc等位基因。
本研究解析本土小油菜細胞核雄不稔性之遺傳機制，研究結果顯示其遺傳行為具商業應用潛力，並可擴大應用於白菜類作物，BrMs基因定位之結果及開發的分子標誌，除可做為基因選殖之參考外，亦有助於透過分子育種加速選育白菜類作物之雄不稔品系。

關鍵字

不結球白菜 ； 雄不稔性 ； 遺傳分析 ； 遺傳連鎖圖譜 ； 基因定位 ； 單一核苷酸多型性分子標誌 ； 育種

並列摘要

Consisting of many crop subspecies, Brassica rapa is cultivated as a very important source of vegetables. Despite male sterility has been widely used in commercial production of hybrid seeds in brassica crops, but not in B. rapa due to lack of useful genetic resources with male sterile genes. We discovered a spontaneous mutant from a local variety of rape (B. rapa ssp. chinensis var. oleifera Makino) in Taiwan.
Results from a series of progeny tests indicated that male sterility in B. rapa ssp. chinensis fits a one gene with multi-allelic model. The gene designated as BrMs is composed of three alleles with BrMsa dominant to BrMsb, and BrMsb dominant to BrMsc (BrMsa > BrMsb > BrMsc). Among three alleles, BrMsb conferring male sterility and the genotype with BrMsbMsb and BrMsbMsc would be sterile. This is the first report of such genetic model in non-heading Chinese cabbage. The model was confirmed according to the results of gene mapping under the genotyping-by-sequencing (GBS) strategy, which was that the male sterility loci were mapped at the same region of chromosome 07 using BC1 and F2 populations involved different BrMs alleles.
SNP markers were designed based on GBS bin markers co-segregating with BrMs trait to evaluate genetic distance between BrMs and markers loci based on 1,590 F2 plants derived from male sterile (NH80-A) and restorer line (TA95). Two flanking SNP markers, BrA7_6632K and BrA7_7625K, were only 0.3 cM away from the upstream and downstream of BrMs locus respectively, narrowing the male sterility gene down to a 400 kb region. A SNP marker, syau_scr04_SNP, developed from sequence characterized amplified region (SCAR) marker closely linked to Ms locus in Chinese cabbage was used to investigate the same F2 population. The result of linkage analysis suggested that BrMs and Ms were located on the same A07 chromosome but at different loci. Interestingly, comparative genomic analysis showed that a male sterile gene in Brassica napus, BnRf, was within the 400 kb region of the BrMs locus. The BnRf orthologs of B. rapa ssp. chinensis were sequenced, and one indel marker (BrMs_indel) was developed for genotyping the same F2 population. Male sterility phenotypes of all 923 investigated plants were completely co-segregated with the marker genotypes.
To perform gene fine-mapping for the BrMs, the whole genome sequencing of the male sterile line NH80-A and restorer line TA95 was accomplished. Within the interval detected from primary BrMs gene mapping, the variations of DNA sequences between two lines were developed into 9 SNP markers. These SNP markers and BrMs_indel marker were investigated in 4,415 plants of an F2 population derived from NH80-A × TA95. The genotyping results showed that two SNP markers, BrA7_6746K and BrA7_6749K, were co-segregated with male sterility. Two out of 4,415 plants showed no co-segregation between male sterility phenotypes and BrMs_indel genotypes, and it might be resulted from the intragenic recombination within the BrMs gene complex. In addition, the results of fine mapping had located BrMs to a 24 kb physical region. This area contains only 3 putative genes, Bra014987, Bra014988 and Bra014989, providing solid information for map-based cloning.
Despite BrMsb conferring male sterility in B. rapa, most male fertile varieties retain BrMsa or BrMsc but not BrMsb allele in the locus. In order to develop molecular markers to distinguish BrMsa and BrMsc alleles, 140 B. rapa inbred lines were crossed with NH80-A to test their BrMs genotype, and followed by performing genome-wide association studies (GWAS). Seventeen SNPs on the Chr07 chromosome associated with BrMs were found, and four of them were selected to develop SNP markers. Genotyping result of examined B. rapa accessions indicated that TqBrA7_6750K marker could be used to discriminate BrMsa, and BrMsc alleles effectively.
This study explored the genetic mechanism of the new genic male sterility mutation in non-heading Chinese cabbage. The mutated gene has commercial application potential and can be extended to the other B. rapa crops. The results of gene fine mapping and marker development, not only have provided a solid foundation for BrMs gene cloning, but also speeded up the development of male sterile lines of B. rapa crops through molecular breeding.

並列關鍵字

non-heading Chinese cabbage ； male sterility ； genetic analysis ； genetic map ； gene mapping ； SNP marker ； plant breeding

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