高粱A2类型CMS育性恢复基因的定位研究

以2个高粱A2类型雄性不育系及相应的保持系(A2V4/B2V4,A2TX622/B2TX622),2个恢复系(1383_2,晋粱5号)和3个组合(A2V4×1383_2,A2V4×晋粱5号,A2TX622×晋粱5号)的F1,F2群体为材料,采用SSR标记方法,分析A2类型雄性不育的育性恢复基因。结果发现,只有标记Xtxp65在A2TX622×晋粱5号的F2群体中与育性出现共分离现象,育性恢复基因与标记间距离为3.4 cM,位于连锁群J上;同样只有标记Xtxp141在A2V4×晋粱5号的F2群体中与育性出现共分离现象,该标记与育性恢复基因间的距离为13.4 cM,位于连锁群G上;在A2V4×1383_2的F2群体中,标记与育性未出现共分离现象。这说明,与这2个标记连锁的A2CMS育性恢复基因是位于同一个恢复系(晋粱5号)中的2个独立的位点;在A2恢复系材料中,至少有3个独立遗传的不同位点与A2雄性不育性的恢复有关。     

YM型小麦温敏雄性不育系不育基因的QTL定位

YM型小麦温敏雄性不育系的不育基因被定位在1Bs染色体片段上, 但已发现的相邻分子标记与该基因的遗传距离较大, 达10 cM以上。为寻找与该基因连锁更紧密的分子标记, 以YM型温敏雄性不育系ATM3314与恢复系中国春杂交的F₂代200株为作图群体, 从1Bs的22个SSR引物中筛选出5个在亲本和F₂代中分离的SSR引物, 构建了1个包含5个标记的1Bs局部遗传连锁图谱。结合F₂代个体的育性调查, 采用复合区间作图法在YM型温敏雄性不育系的1Bs染色体上检测到不育基因的1个主效QTLrfv1-1和1个微效QTLrfv1-2。rfv1-1位于SSR标记Xgwm18和Xwmc406之间, 与两标记的遗传距离分别为6.0 cM和4.6 cM, LOD值为8.80, 加性效应23.87, 显性效应10.44, 可解释表型变异的23.91%; rfv1-2位于Xwmc406和Xbarc8之间, 与两标记的遗传距离分别为4.0 cM和3.4 cM, LOD值为3.10, 加性效应17.59, 显性效应5.99, 可解释表型变异的7.78%。本研究初步定位了YM型小麦温敏雄性不育系1Bs染色体片段上不育基因的QTL, 为进一步准确定位该基因奠定了基础。     

谷子6号染色体雄性不育基因的克隆

克隆谷子雄性不育材料1066A的不育基因,分析不育基因与可育基因存在的突变位点,为揭示谷子雄性不育分子机制、利用分子标记辅助选择方法选育多用途的不育材料奠定基础。利用谷子全基因组测序数据及前人不育基因定位结果克隆谷子雄性不育材料1066A的雄性不育基因,发掘导致不育的突变位点,旨在为从分子水平揭示谷子不育机制、利用分子标记辅助选择方法选育多用途的不育材料奠定基础。首先利用生物信息学方法从豫谷1号6号染色体找到1个雄性不育基因位点(Si015780m.g),该基因全长5 027个碱基,编码479个氨基酸,且位于前人用分子标记定位的基因组区间内。根据豫谷1号不育基因序列设计2对特异引物在雄性不育材料1066A的基因组DNA进行PCR扩增。将扩增产生的2个基因组片段进行拼接后在谷子不育材料1066A中获得2 561 bp的基因序列,包含了下游部分编码区。通过对豫谷1号、张谷、1066A的不育基因部分编码序列及推定的蛋白质序列进行比对分析,结果发现谷子不育材料1066A的不育基因编码序列存在3处突变:2处单碱基替换和1处单碱基插入,这3处突变导致谷子不育材料1066A的不育基因蛋白的第402,403个氨基酸由异亮氨酸和亮氨酸替换成缬氨酸和异亮氨酸,同时导致其不育基因编码的蛋白在第466个氨基酸处发生提前终止。3处突变中2处氨基酸替换对编码蛋白的功能影响不大,因此,认为谷子不育材料1066A的不育基因蛋白翻译提前终止可能是导致其产生不育的原因。     

谷子显性雄性不育基因Msch的AFLP标记

利用雄性不育是实现谷子杂种优势利用最经济、有效的途径之一.为了寻找与不育基因Msch紧密连锁的分子标记,提高不育系的选育效率,本研究构建了Msch不育/可育近等基因系(NILs),通过对400对AFLP引物组合进行筛选,找到了与不育基因紧密连锁的两个AFLP标记(P17/M37224和P35/M52208),与不育基因的遗传距离分别是2.1 cM和1.4 cM,而且位于不育基因的同一侧,标记间相距0.7 cM.这两个AFLP标记可有效用于分子标记辅助选择育种.     

甘蓝型油菜显性细胞核雄性不育基因的AFLP标记

用甘蓝型油菜双基因显性细胞核雄性不育系Rs1046A和欧洲油菜品种Samourai构建了一个回交分离群体。在群分法（BSA）构建的不育池和可育池中共筛选了256对AFLP引物组合,找到了与不育基因紧密连锁的两个AFLP标记(EA03MC1599和EA07MC01235), 它们与不育基因的遗传图距分别是3.5 cM和5.5 cM,而且位于不育基因的同一侧,标记间相     

基于AFLP和SSR标记的高粱分子遗传连锁图构建

以茎秆糖份含量高的高粱自交系1095和低糖高粱自交系N3杂交获得的F2分离群体(205个个体)为材料,采用AFLP(amplified fragment length polymorphism)和SSR(simple sequence repeat)两种分子标记,构建了包含273个(232AFLP,41 SSR)标记,覆盖基因组长度为978.1cM的高粱分子标记连锁遗传图.以SSR标记为锚标记,19个连锁群中,18个连锁群各自被归并于高粱的10个连锁群(A-J)中.该连锁图平均图距和最大图距分别为3.6 cM和19.4 cM,未出现大的空隙(gap＞25 cM),归并后的10个连锁群(A-J)分别对应于高粱染色体SBI-01、SBI-02、SBI-03、SBI-04、SBI-07、SBI-09、SBI-10、SBI-08、SBI-06、SBI-05.     

SSR方法标记谷子光敏雄性不育基因

为加快谷子杂种优势利用的研究进展、寻找谷子光敏雄性不育基因,利用SSR方法对谷子光敏雄性不育基因进行了分子标记。首先用166对引物在谷子光敏不育系GM与恢复系恢东1号两亲本间进行了筛选,其中有61对引物在亲本间存在差异;经F2群体153株单株验证后,仅有一对引物b159和目标基因连锁;通过Kosambi函数计算,其连锁距离为13.5 cM,位于第6条染色体。     

甘蓝型油菜细胞质雄性不育恢复基因的SSR标记及初步定位

以甘蓝型油菜恢复系Q 16C和不育系2116A为亲本构建F2群体,根据BSA法的构建原则,使用F2群体分别构建了可育基因池与不育基因池。运用98对SSR引物对油菜亲本及基因池进行了多态性分析,结果表明,有1对引物(OS 31)能在亲本和基因池间表现出多态性,使用该引物继续对F2群体的230个单株进行分析,表明标记(OS 31)与恢复基因Rfp的遗传距离为0.08cM,可作为恢复系辅助育种的候选标记。     

水稻特异亲和基因S-e的分子定位

水稻籼粳亚种间杂种具有强大的优势，但亚种间杂种的不育性限制了这一优势的利用。开展杂种不育基因的定位工作，对于进一步了解杂种不育性的遗传基础，克服亚种间杂种的不育性具有重要的意义。本研究选用粳型品种台中65的近等基因系E47-1和籼型品种广陆矮4号为材料，利用74个SSR标记对杂种F2群体进行偏态分离标记的筛选，同时根据F2和F3群体花粉育性和具有偏态分离的SSR标记之间的连锁关系，对特异亲和基因(F1花粉不育基因)S-e座位进行了分子定位，取得了以下主要结果：1、利用116个均匀分布在水稻12条染色体上的SSR标记对籼粳两亲本进行多态性筛选。结果有101个SSR标记在亲本间具有多态性，15个SSR标记在亲本间无多态性，SSR标记在亲本间的多态率高达87．07％。2、选用74个亲本间具有多态性的SSR标记对E47-1／广陆矮4号组合F2群体的偏态分离进行了初步的筛选和分析。发现有6个染色体区段的9个SSR标记在F2群体中存在偏态分离，它们分别位于第3、第6、第7、第10、第11和第12染色体上，卡方值均达到显著或极显著水平。6个染色体区段中有2个严重偏态分离区段，分别位于第6和第12染色体。3、通过对F2群体的花粉育性和偏态分离区段的SSR标记基因型的相关关系分析，表明位于第12染色体上的SSR标记RMl9附近存在一个F1花粉不育基因。继而在该标记附近设计位置特异性微卫星标记PSM401、PSMl80、PSMl82，利用F3作图群体，将特异亲和基因S-e座位定位在分子标记PSM401、PSMl80和PSMl82、RMl9之间，该基因与各标记的遗传距离分别为2．3cM、1．3cM、3．7cM和4．3cM。4、选取在S-a、s-b、S-c、S-d、S-e五个座位均纯合、花粉表现为部分不育的F2单株，发展了另一R群体，表明该群体存在另一特异亲和基因座s-f。本研究利用SSR标记，对特异亲和基因S-e进行了分子定位。S-e座位的分子定位，进一步丰富和完善了特异亲和性的学术观点，并为分子标记辅助选育水稻的粳型亲籼系奠定了基础。     

小麦花粉致死基因Ki的SSR标记分析

小麦雄性不育主要是通过花粉的败育表现,其不育材料对小麦杂种优势的利用研究具有重要意义和价值,国外研究表明,某些特定普通小麦品种间杂交F₁表现的花粉部分不育现象,受控于核基因组花粉致死基因Ki,为了筛选小麦花粉致死基因Ki的连锁标记,利用现代分子生物学技术通过定位该基因,克隆出花粉致死基因连锁标记片段,为小麦雄性不育种质材料的转育提供有效的选择标记。对小麦花粉致死基因Ki进行了分子标记定位,以‘中国春’和澳大利亚春小麦品种的BC₁F₁代作为定位群体,利用分离群体分组分析法(BSA)对位于小麦6B染色体上85对SSR引物进行多态性筛选,具有多态性的引物再通过BC₁F₁定位群体进行验证,从中筛选出与目的基因连锁的2个SSR标记Xgwm626和Xgpw4138。运用Mapmaker 3.0软件进行连锁分析。结果表明,Xgwm626和Xgpw4138与Ki基因的遗传距离分别为9.2 cM和6.9 cM,且2个SSR标记位于目的基因两侧,并将Ki定位于小麦6BL染色体上。研究结果为Ki基因的分子标记辅助选择和进一步精细定位奠定了基础。     

Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean

In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F₂ population derived from a single F₁ plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.     

Molecular mapping of Cg1, a gene for resistance to anthracnose (Colletotrichum sublineolum) in sorghum

Anthracnose, caused by the fungus Colletotrichum sublineolum is one of the most destructive diseases of sorghum and has been reported in most areas where the crop is grown. Several control strategies have been developed but host plant resistance has been regarded as the most effective strategy for disease control. Here, we describe the search for molecular markers that co-segregate with Cg1, a dominant gene for resistance originally identified in cultivar SC748-5. To identify molecular markers linked with the Cg1 locus, F_2:3 plants derived from a cross to susceptible cultivar BTx623 were analyzed with 98 AFLP primer combinations. BTx623 was chosen as the susceptible parent because it is also one on the parents used in creating RFLP and AFLP maps and BAC libraries for sorghum. Four AFLP markers that cosegregate with disease resistance were identified, of which Xtxa6227 mapped within 1.8 cM of the anthracnose resistance locus and all four AFLP markers have been previously mapped to the end of sorghum linkage group LG-05. Sequence scanning of BAC clones spanning this chromosome led to the discovery that Xtxp549, a polymorphic simple sequence repeat (SSR) marker, mapped within 3.6 cM of the anthracnose resistance locus. To examine the efficacy of Xtxa6227 and Xtxp549 for marker-assisted selection, 13 breeding lines derived from crosses with sorghum line SC748-5 were genotyped. In 12 of the 13 lines the Xtxa6227 and Xtxp549 polymorphism associated with the Cg1 locus was still present, suggesting that Xtxp549 and Xtxa6227 could be useful for marker-assisted selection and for pyramiding of Cg1 with other genes conferring resistance to C. sublineolum in sorghum.     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.     

大豆质核互作雄性不育系NJCMS3A双亲雄性育性基因的SSR标记

利用大豆质核互作雄性不育系NJMCS3A的质、核供体亲本N21566和N21249构建F₂和BC₁F₁育性分离群体进行雄性育性的遗传分析与基因定位。结果表明, F₁正反交可育,F₂和BC₁F₁的可育株与不育株分离比例经χ²测验分别符合3∶1和1∶1,表明NJCMS3A供体亲本雄性育性由一对基因控制,可育等位基因为显性。该基因可能是NJCMS3A的一个恢复基因。选用793对SSR引物对F₂和BC₁F₁群体分别进行育性基因定位,发现该育性基因位于O连锁群上,在Satt331和Satt477标记之间,与Satt331、CSSR133和Satt477标记距离的次序一致,分别为8.1~10.4 cM、11.4~16.4 cM、13.3~19.2 cM。     

Fine mapping of Rf2, a major locus controlling pollen fertility restoration in sorghum A1 cytoplasm,encodes a PPR gene and its validation through expression analysis

Sorghum is one of the pioneering cereal crops where cytoplasmic male sterility (CMS) was successfully exploited for mass production of F₁ hybrid seed. Mapping genes for fertility restoration (Rf) is an important aspect of understanding the molecular basis of fertility restoration in crop plants. In this study, we fine‐mapped a fertility restoration locus, Rf2 of sorghum reported earlier (Jordan, Mace, Henzell, Klein, & Klein, 2010 ), involving two F₂ populations (296A × RS29 and 296A × DSV1) and newly developed SSR markers delimited Rf2 locus to 10.32‐kb region on chromosome 2. The Rf2 locus was tightly linked with two new SSRs, MS‐SB02‐3460 (0.14 cM) and MS‐SB02‐3466 (0.75 cM) on both sides, and hosted only one gene (Sobic.002G057050) of PPR gene family. Another new SSR marker developed in the study, MS‐SB02‐37912, forms the part of PPR gene and could act as a perfect marker in marker‐assisted breeding for fertility restoration involving Rf2 in sorghum breeding. The strong involvement of Sobic.002G057050 gene in fertility restoration was supported through RNA expression analysis.     

Marker-assisted breeding for rf1, a nuclear gene controlling A1 CMS in sorghum (Sorghum bicolor L. Moench)

A study on marker-assisted selection (MAS) for the rf1 gene, which controls pollen sterility in the sorghum A1 cytoplasm, was conducted on the offspring population of two crosses between a maintainer line, BTx-622, and two sweet sorghum lines, BJ-299 and Lunen-2, to test the effectiveness of the MAS method and develop maintainer lines with sweet and juicy stalks and corresponding cytoplasmic-nuclear male sterility (CMS) lines. The simple sequence repeat marker Xtxp18 exhibited a high accuracy (95.098 %) for selecting recessive homozygotes for the rf1 gene. The segregation ratio matched the expected ratio calculated according to the reported genetic distance in the F₂ population of the two crosses used. Finally, four excellent maintainer lines/CMS line pairs (F₅/BC₃) with high stalk juice and stalk juice sugar contents were developed. The MAS method based on Xtxp18 for the sorghum rf1 gene could be used for hybrid breeding programs at a low cost in the future.     

Molecular mapping of the fertility restoration locus Rf1 in sunflower and development of diagnostic markers for the restorer gene

Fertility restoration by dominant nuclear genes is essential for hybrid breeding based on cytoplasmic male sterility (CMS) to obtain heterotic effects and high seed yields. In sunflower, only the PET1 sterility inducing cytoplasm has been used in commercial hybrid breeding until now. This particular male sterility was derived from an interspecific hybrid Helianthus petiolaris × H. annuus. For the recent work we used the segregating population RHA325(CMS) × HA342, based on the PET1 cytoplasm. Molecular markers were mapped within 1.1 cM around the restoration locus Rf1. At the distal side, the marker OP-K13_454 mapped at a distance of 0.9 cM and E32M36-155R at 0.7 cM from Rf1. At the proximal side the markers E44M70-275A, E42M76-125A and E33M61-136R were mapped at 0.1, 0.2, and 0.3 cM from the restorer locus, respectively. These markers provide an excellent basis for a map based cloning approach and for marker-assisted sunflower breeding.     

油菜野芥NSa细胞质雄性不育系的特异性分子鉴定

常规细胞质类型鉴定方法花费时间长、工作量大、鉴定效率低、应用性较差。基于基因组重复序列的PCR技术(rep-PCR)在重复序列丰富的线粒体和原核生物的基因组鉴定中效果较好, 本研究对8份油菜资源的细胞质进行分子鉴定,结果表明rep-PCR可以明确区分甘蓝型油菜中常见的6种雄性不育细胞质。同时对甘蓝型油菜和野芥(Sinapis arvensis)体细胞杂交创建的、育性较为稳定的NSa野芥雄性不育胞质进行了分子特异性鉴定,获得了两条NSa不育胞质的特征DNA条带。但波里马和萝卜质不育细胞质的特异性引物在NSa中不能扩增出特异性片段,说明NSa不属于这两种细胞质类型。本研究结果为该不育胞质系统的利用和知识产权保护提供了理论依据和技术支撑。     

Mapping of male sterility gene ms10 in chilli pepper (Capsicum annuum L.)

Most of the hybrid seed in chilli are produced manually, but the use of male sterility (MS) can reduce the cost of hybrid seed production. MS‐12, a nuclear male‐sterile (NMS) line developed at Punjab Agricultural University, Ludhiana (India), has been utilized to develop commercial F₁ hybrids. A recessive gene, designated as ms10, governs MS in MS‐12. Due to recessive gene control, development of new NMS lines incorporating ms10 gene is tedious and time‐consuming. We identified SSR markers AVRDC‐PP12 and AVRDC_MD997* linked to the ms10 gene. A total of 558 primer pairs were screened following bulked segregant analysis (BSA). Linkage analysis in 210 F₂ plants indicated that the two SSR markers were linked to the ms10 gene and the marker AVRDC‐PP12 was closest to the gene at 7.2 cM distance. The marker was mapped to chromosome 1 at genome position 175 694 513 to 175 694 644. Until more closely linked markers are developed, the marker AVRDC‐PP12 would facilitate transfer of ms10 gene through marker‐assisted selection (MAS). Fine mapping would lead to cloning of the ms10 gene.