太空诱变玉米核不育材料的细胞学观察及不育基因的SSR定位

1、太空诱变玉米核不育材料的细胞学观察及不育基因的SSR定位作物遗传育种：李式昭 指导教师：曹墨菊 教授四川农业大学玉米研究所于1996年利用返回式实验卫星搭载六份优良玉米材料，现已从中获得一份雄性不育突变体。经遗传分析证实，该突变体为一对隐性单基因控制的细胞核雄性不育材料，并已将该不育基因初步定位于玉米第3染色体的长臂上。为探明太空诱变玉米细胞核雄性不育材料花粉败育的细胞学机理及不育基因所在位点，本实验以该不育材料为研究对象，主要研究内容包括：（1）以核不育材料的姊妹交群体作为育性调查和细胞学观察群体，利用整体花药及游离细胞观察和石蜡切片技术对该不育材料的小孢子发育进行了细胞学观察。（2）以核不

2、育材料的F2代群体作为育性调查和SSR定位群体，用玉米第3染色体3.06位点的SSR引物进行差异筛选，进一步缩短现有分子标记与不育基因之间的遗传距离，并以此为基础构建核不育基因区间的分子标记遗传连锁图。主要结果如下：1. 对核不育材料的姊妹交群体进行田间育性鉴定，发现2个小区的姊妹交群体都发生育性分离，且每个果穗均符合可育:不育为1:1的分离比例。姊妹交群体总计295株，其中完全可育127株，完全不育168株。适合性测验结果为：C2=5.424，小于2（0.01，1）=6.63，表明姊妹交群体中可育株和不育株的育性分离符合孟德尔分离比率1:1。对核不育材料的F2群体进行田间育性鉴定，发现10个

3、小区的F2群体都发生育性分离，且每个果穗均符合可育:不育为3:1的分离比例。F2群体总计2002株，其中完全可育1539株，完全不育463株。适合性测验结果为：C2=3.647，小于2（0.01，1）=6.63，表明F2群体中可育株和不育株的育性分离符合孟德尔分离比例3:1。在姊妹交群体和F2群体中均验证出该不育性状确实受一对隐性纯合核不育基因控制。2. 以核不育材料的姊妹交群体为细胞学观察群体，对该不育材料的小孢子发育进行了细胞学观察。结果表明，太空诱变玉米核不育材料的花粉败育主要发生在减数分裂的二分体期间，表现为二分体自溶，形状不规则。同时发现早在减数分裂之前，少数药室的花粉母细胞和绒毡层

4、细胞就出现发育异常，花粉母细胞有解体自溶趋势，不能形成正常的二分体，有些虽可发育至四分体，但不能完成后续的发育；而绒毡层细胞的异常从花粉母细胞时期一直持续到二分体直至四分体。花药发育后期，则观察到绒毡层细胞液泡化并径向膨大，充满整个药室；或绒毡层细胞完全解体，药室变为空腔，结果均表现为无花粉型的败育类型。3. 以核不育材料的F2代群体为SSR定位群体，对该不育基因所在位点进行研究。根据田间育性调查结果随机选取一定数量的不育株和可育株，采用单株法进行SSR引物的筛选，并根据初步定位结果，利用分子标记bnlg197和umc1012进行验证，以确认该核不育基因在玉米第3染色体3.06位点附近。同时合

5、成3.06位点上的37对已知SSR引物进行差异筛选，结果表明分子标记umc1674，umc2076，umc1644，dupssr17，bnlg197，umc2271，umc1951和bnlg1047与不育基因表现为连锁遗传，进一步将不育基因定位在分子标记umc2076和umc1674之间，遗传距离分别为4.4 cM和3.7 cM，并以此为基础构建核不育基因区间的SSR标记遗传连锁图，为下一步克隆该基因奠定基础。关键词：玉米；太空诱变；细胞核雄性不育（GMS）；花粉败育；SSR标记Cytological Observation and SSR Mapping of Genetic Male St

6、erile Mutant Induced By Space Flight in MaizeCrop Genetics and Breeding: Li Shizhao Advisor: Prof. Cao MojuMaize Research Institute of Sichuan Agricultural University (MRI, SAU) sent maize seeds for satellite abroad in 1996. A male sterile mutant was found among six excellent maize materials. The pr

7、imary research indicated that the male sterile trait was governed by a single homozygous recessive gene, and the male sterile gene was located on maize chromosome 3L. In order to determine the cytological mechanism of microspore abortion and the precise location of maize genetic male sterile (GMS) g

8、ene, the maize GMS materials induced by space flight were used as the experiment objects. The researches include two aspects: (1) The sister cross population derived from maize GMS materials were used for sterility analysis and cytological observation. Intact anther observation, isolated cells obser

9、vation and paraffin slice were adopted in this research. (2) The F2 population derived from male sterile materials were used for sterility analysis and simple sequence repeats (SSR) location. Linkage analysis with 37 pairs of SSR primers from maize chromosome 3 bin 3.06 to shorten the genetic distan

10、ce between molecular marker and the GMS gene, then established the genetic linkage map of GMS gene region. The main results concluded as follows:1. Sterility investigation in field showed that each sister cross population populations (295 plants) were segregated in sterility. Among them, 127 were to

11、tally male fertile and 168 were totally male sterile. Chi-square analysis showed that C2=5.424, which was smaller than 2（0.01，1）=6.63. The segregating ratio of male fertile plants to male sterile plants fitted well to a 1:1 Mendelian ratio. Sterility investigation in field showed that each F2 popula

12、tions (2002 plants) were segregated in sterility. Among them, 1539 were totally male fertile and 463 were totally male sterile. Chi-square analysis showed that C2=3.647, which was smaller than 2（0.01，1）=6.63. The segregating ratio of male fertile plants to male sterile plants fitted well to a 3:1 Me

13、ndelian ratio. The results in sister cross population and F2 population indicated that the male sterile trait was governed by a single homozygous recessive GMS gene.2. The sister cross population derived from male sterile materials were used for cytological observation to understand the process of m

14、icrospore abortion of GMS mutant induced by space flight in maize. The results showed that pollen abortion occured mostly in dyad stage of meiosis in GMS mutant. The dyad were degenerated with abnormal shape. The pollen mother cells (PMC) and tapetal cells developed abnormally in a few anther before

15、 the meiosis. The PMCs began to dissolve and degenerate, some PMCs could not become normal dyad cells. Some PMCs entered the tetrad stage, but did not complete the following development. The abnormal behavior of tapetal cells lasted from PMC stage to dyad stage and tetrad stage. At late anther devel

16、oping stage, the tapetal cells became to vacuolated, anther chamber was full of giant tapetum and the microspore degenerated completely. The other phenomenon was that the tapetum degenerated early, the microspore degenerated and disappeared. The result of both two types of abortion was that the empt

17、y anther lacked of pollen. 3. The F2 population derived from male sterile materials were used for SSR location to determine the precise location of maize GMS gene induced by space flight. According to the sterility investigation in field, the randomly selected fertile plants and sterile plants were

18、used for SSR markers selection. On the basis of previous location by Liu, the molecular markers bnlg197 and umc1012 were used to ensure that the GMS gene was on maize chromosome 3 bin 3.06. At the same time, 37 pairs of of the SSR markers from maize chromosome 3 bin 3.06 were obtained, and then began to identify SSR markers. Linkage analysis showed that the SSR markers umc1674, umc2076, umc1644, dupssr17, bnlg197, umc2271, umc1951 and bnlg1047 were linked with the GMS gene. Finally the GMS gene was mapped between mar