摘要
陈凤祥等(1993,1998)报道了甘蓝型油菜隐性上位互作核不育系统并提出了该不育系统的遗传模式,提出不育性状由二对隐性不育重叠基因和一对隐性上位抑制基因互作控制。该不育系统具有不育性彻底、稳定且恢复源广等优点。然而由于该不育系的育性受多对基因互作控制,分离后代基因型复杂,给选育带来很大困难,限制了该不育系统在杂交育种中的大规模应用。为了进一步解决隐性上位互作核不育统在生产应用中的难题,我们认为有必要寻找与育性相关基因紧密连锁的共显性标记,通过标记辅助筛选达到快速高效改良和选育两型系和临保系的目的,从而生产出农艺性状和品质特点符合大面积制种需要的遗传稳定的全不育系。为此我们以陕西省农垦科教中心提供的纯合两型系和临保系及全不育系构建基因池,用于目标基因连锁标记的筛选；利用三基因互作核不育杂种沪油杂1号通过多代兄妹交保存的两型系和可育株自交后代分离群体为材料定位Bnms3基因；同时将全不育系与临保系回交获得BCl群体定位Bnrf基因。从本课题组SG图谱以及拟南芥基因组共线性区域和油菜BAC文库等公共资源获得相关序列信息并开发标记,利用BSA(集群分离法)法寻找与育性基因Bnms3位点和上位抑制基因Bnrf紧密连锁的分子标记,构建目标基因区域的连锁图谱。取得的实验结果和主要结论如下：
隐性不育基因Bnms3位点的定位研究及共显性标记的开发1) Bnms3基因初定位及目标连锁群的获得：从前人的研究结果中筛选到与育性表型共分离的标记共5个(SC1、SC5、SEP4B、SEP7和SEP8),均为显性标记。根据5个标记在DH(Sollux×Gaoyou)群体中的分析结果,SEP7被定位于本课题组构建SG图谱A10连锁群,进一步从该连锁群上获得连锁标记PBI和ZAAS133,其中PBI为共显性标记。最终依据连锁标记在500单株的小群体中的连锁分析结果,将Bnms3基因定位于SG-A10连锁群,并初步获得与目标基因连锁的标记共7个,除PBI为共显性标记外,其余均为显性标记。因此我们仅选择PBI和SEP7(定位于SG-A10)以及He等(2008)刚发表的AR23和AR48(显性标记)共4个标记对Bnms3基因进行初步定位,将目标基因定位于AR23和AR48之间,遗传距离分别为0.4cM和1.0cM,共显性标记PBI的遗传图距为2.1 cM。
2) Bnms3基因的精细定位：依据初定位结果,从模式作物拟南芥基因组获得共线性区域,进一步从芸薹属BAC库获得7个位于目标区段的BAC片段并设计了45对引物,BSA分析表明有15对引物显示多态性,最终6个(Ms3-6、Ms3-9、Ms3-10、Ms3-20、Ms3-32和Ms3-44)被证明与目标基因紧密连锁,涉及3个BAC。Bnms3基因被定位于连锁标记Ms3-32和Ms3-6之间,定位区域由1.4cM缩小为0.5cM,对应同源拟南芥基因组物理距离为280Kb,比发表的Bnms3二侧最近标记(AR23和AR48)470Kb缩小了190 Kb。
3)共显性标记的获得：至此,所有已公开发表的Bnms3连锁标记均为显性标记,而本研究针对Bnms3区域BAC序列开发标记,获得3个分别位于目标基因两侧的共显性标记：PBI、MS3-9和MS3-32,距离Bnms3基因分别为2.1 cM、0.4cM和0.1 cM。共显性标记的获得将对分子标记辅助选育起到至关重要的作用。
4)常规品种Bnms3位点基因型的检测：利用共显性标记(PBI和Ms3-32)对72个代表性品种的Bnms3位点基因型进行检验,结果表明普通品种中Bnms3位点均为显性纯合(BnMS3MS3),这解释了为什么所有普通油菜品种均为其恢复系的原因。
二、隐性上位基因Bnrf的初定位及共显性连锁标记的开发
从本课题组SG图谱上筛选到3个(MR166、SR0282和R118)与Bnrf基因连锁的分子标记并初步构建了目标基因的连锁图谱,同时将他人研究获得的标记(ESPSC1、ESPSC2和XSC5)整合到了连锁图谱中,最终将目标基因定位于SG-A7连锁群9.2cM的区间内,距离Bnrf基因两侧最近的标记是ESPSC1和MR166,遗传距离分别为6.0cM和3.2cM,其中SR0282被证实是共显性标记,其他均为显性标记。
Chen et al (1993,1998) reported that the sterility of RGMS was controlled by two recessive genes (Bnms3 and Bnms4) and one recessive epistatic suppression gene (Bnrf) interactively. This sterile system has several remarkable advantages, a stable, complete male sterility, which makes sure for yielding high rate F1 hybrid seeds; a complete open flower petal, which benefits for getting a good seeds set. Of them, the most important one is that almost all the normal rapeseed cultivars are its restorers, which provides possibility to produce huge number of F1 combinations for breeding purpose. Therefore, this RGMS was regarded as one of the most potential sterile system in oilseed rape nowadays. However, due to the polygene control system, resulting in complex genotypes in segregation generations, the improvement for sterile lines becomes difficult if without advanced technique support in practical breeding procedures. So it is necessary to develop a set of specific markers, which linked either to sterile or epistatic genes for creating or improving a two-type lines and temporary maintainer lines, and thereby to get geneticly stable full sterile lines finally. To achieve this goals, firstly we used the DNA samples from sterile and fertile plants of two-type lineand also DNA's from temporary maintainer line and full sterility lines provided by Shan Xi province for marker screening. Secondly, the linked markers were confirmed and fine mapping for Bnms3 and Bnrf genes with large segregation population generated from sib-mating (9012A×9012B) and by full self-mating (9012B×9012B) of fertile plants, which from Huyouza 1 hao (F1 hybrid) originally. The locus specific PCR primers were developed on the basis of the syntenic region of the Arabidopsis genome, the Brassica BAC sequences and the published reference markers. The main results and conclusions are as following
1. Fine mapping of the Bnms3 gene
1) Screening markers linked to the Bnms3 gene:Based on published reports, we obtained 7 markers linkaged with the Bnms3 gene (SC1,SC5,SEP4B,SEP7, SEP8, AR23 and AR48), Of them, SEP7 was also mapped to SG (Sollux×Gaoyou) map in linkage group 10 (A10) Further, two markers nearby SEP7were proved to be also linked to Bnms3 (PBI and ZAAS133), PBI was a co-dominant marker. Thus, according to our result, Bnms3 gene was located on A10 of B. rape instead of C9 of B. oleracea as previously published. Finaly we choose 4 markers (PBI SEP7,AR23 and AR48) for future mapping Bnms3 gene in large population of 1896 plants.The Bnms3 gene was genetically mapped in a flanking region of 1.4 cM.Bnms3 gene was 0.4 cM from marker AR23 in one side and 1.0 cM from marker AR48 in another side.
2) Fine mapping of the Bnms3 gene:According to the 4 marker mapping result of Bnms3 gene, we future developed 45 genome region specific primers from the syntenic search of the BAC clone in Brassica database between marker AR23 and AR48 and found 15 of them showing clear polymorphism between fertile and sterile plants. At last,6 markers (Ms3-6、Ms3-9、Ms3-10、Ms3-20、Ms3-32 and Ms3-44) showing clear co-segregation with the Bnms3 gene were selected for fine mapping in population of 1896 plants. The results future narrowed Bnms3 gene in a flanking region of 0.5 cM, with 0.1 cM from marker Ms3-32 and 0.4 cM from marker Ms3-6 in respective sides. Syntenic analysiswith Arabidopsis genome, a physical distance of 280 Kb between Ms3-6 and Ms3-32 was identified, which was 190 Kb less than published interval between flanking markers of Bnms3 gene.
3) Co-dominant marker:Two of six co-segregation markers (Ms3-9 and Ms3-32) with Bnms3 showed co-dominant nature, with 0.4 cM and 0.1 cM to Bnms3 respectively. So far, all the published markers were dominant in nature.
4) Identification of Bnms3 alleles in normal varieties:Using co-dominant marker PBI and Ms3-32,72 representative rapeseed cultivars worldwide were analyzed for allelic detection. The results indicated that all the checked cultivars were homozygosis locus for Bnms3 gene of BnMS3MS3. This result explained the reason for why almost all the normal rapeseed cultivars can be used as restorers.
2. Fine mapping of the Bnrf gene
Initially, more than 200 markers evenly distributed on SG-map were screened with sterile and fertile pools from temporary maintainer line and full sterility lines (1:1), searching for Bnrf linked markers. As a result, three markers (MR 166, R118 and sR0282) from A7 showed tightly linked with Bnrf gene. These markers together with another three published dominant markers (ESPSC1、ESPSC2 and XSC5) were used for co-segregation analysis in a F2 population of 864 plants. The result indicated that Bnrf gene was located on linkage group 7 (A7) of B. napus, which agreed with previously published result. The genetic distance between Bnrf gene to flanking markers MR166 and ESPSC1 were 3.2 and 6.0, respectively. One co-dominant marker sR0282 was detected, which located 11.9 cM away from Bnrf gene.
All the co-dominant markers obtained from this research are positively contributed in present hybrid breeding.
引文
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