小麦高温敏感雄性不育基因htms1的精细定位及候选基因分析

Xinong 291S is a high-temperature-sensitive male sterile mutant, which derived from a spontaneous mutation of common wheat cultivar Xinong 291. It has a great potential for establishing an efficient two-line hybrid wheat breeding system, which propagate male sterile line in winter wheat regions and with the hybrids being produced in spring wheat regions of China. Our previous studies suggested that the male sterility of Xinong291S was controlled by two recessive major genes, which designated as htms1 and htms2 respectively. The htms1 gene was located on chromosome 5B by aneuploid analysis. The objectives of the present research are as follows: (1)Construct F2 populations of cross Xinong 291S/Zhoumai 18, and select closely linked SSR and SRAP markers of the two male sterile genes by using methods of bulked segregant analysis (BSA) and recessive class analysis (RCA). (2)Construct large-scale F2 populations of cross Xinong 291S/Chinese Spring, and select recombinants of extremely sterile plants by using the molecular markers closely linked to the htms1 gene. Fine mapping of the htms1 gene with EST (expressed sequence tags) markers, which development based on collinearity of wheat with Brachypodium distachyon and rice. (3)Predict candidate genes of the htms1 by bioinformatics analysis, and perform sequence analysis of these genes in Xinong 291S, Xinong 291, Chinese spring and Zhoumai 18 by cloning and sequencing. Analyze temporal and spatial expression patterns of the selected candidate genes in different organs. The results of the present study will lay a good foundation for marker-assisted selection and revealing the molecular mechanism of photoperiod thermo-sensitive genic male sterility in wheat.

西农291S是从普通小麦西农291中发现的一种高温敏感雄性不育突变体，在两系杂交小麦育种上具有巨大应用潜力。课题前期研究表明，西农291S的育性主要受2对隐性核不育基因（htms1、htms2）控制，其中htms1基因位于5B染色体上。本项目拟开展研究内容：①利用西农291S/周麦18的F2分离群体，结合集群分离分析法与隐性群体分析法，筛选与2对不育基因紧密连锁的SSR、SRAP等分子标记；②在西农291S/中国春的F2大规模分离群体中，筛选极端不育重组单株，借助比较基因组学开发EST标记，逐步缩小目标区域，实现对htms1的精细定位；③利用生物信息学方法预测htms1的候选基因，并进行候选基因克隆、测序，检测西农291S与西农291等品种间存在的DNA序列差异，分析候选基因的时空表达模式。研究结果将为后续的分子辅助选择奠定良好基础，对于揭示小麦光温敏雄性不育的分子机制具有重要意义。

小麦光温敏两系法具有制种程序简便、恢复源广、易选配出强优势组合等优点，是小麦杂种优势利用的一条重要途径。光温敏不育系是两系法利用小麦杂种优势的基础，发掘和研究小麦光温敏不育基因及其雄性不育的分子机理，对实现快速培育小麦光温敏不育系具有重要的理论意义和应用价值。西农291S是从普通小麦西农291中发现的一种高温敏感雄性不育突变体，其育性转换明显，不育性易于恢复，在两系杂交小麦育种上具有巨大应用潜力。本研究对西农291S的光温敏不育基因htms1、htms2进行分子标记定位，以西农291S/中国春F2代作为定位群体，采用集群分离分析法与隐性群体分析法策略，利用小麦全基因组分布的SSR标记进行多态性筛选，再通过部分单株验证，筛选出染色体6BS上5个SSR标记与隐性核不育基因htms1连锁。对F2群体中极端不育株和极端可育单株进行分子检测和连锁分析，结果表明该连锁群最优排序为Xcfd13、htms1、Xgdm113、Xwmc104、Xbarc1169、Xwmc398，且两两标记间的遗传距离分别为4.9cM、3.8 cM、3.0 cM、11.2 cM、8.2 cM。由于htms1基因两侧连锁的SSR标记距离较远，难以按原计划借助比较基因组学开发新标记，最终导致无法精细定位htms1基因。西农291S/矮抗58 F2群体的亲本间多态性较差，始终未筛选出与htms2基因连锁的SSR标记。本研究已利用Affymetrix小麦660K高密度SNP芯片技术，对西农291和西农291S、西农291S分别与中国春和矮抗58构建的2对混合池的DNA样品进行全基因组扫描，SNP分型结果的后续分析工作正在进行中。差异SNPs可用于开发新的分子标记，实现htms2基因定位，尤其是htms1基因精细定位，进而通过生物信息学方法预测分析htms1候选基因。