【作者】 余渝；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 棉花是重要的经济作物,它不仅可以提供天然纤维,而且棉籽也是食用油的重要来源；棉花产业的兴衰对我国农民增收和纺织工业的发展都具有非常重要的意义。新品种在棉花生产中的贡献率达30%以上,长期以来棉花遗传育种研究者主要依靠常规育种技术改良棉花品种性状。由于棉花基因组大且遗传复杂,所以改良的局限性较大；而现代分子生物学的诞生给棉花遗传改良带来了新的空间,分子标记技术与常规育种技术相结合可加快育种进程,缩短育种年限,减少工作量,同时改良较多的不良性状,提高选择效果。本论文运用SSR标记技术,主要开展以下研究工作：(1)来源于草棉EST-SSR标记引物的开发、鉴定和评价；(2)棉花种间群体雌、雄配子重组率差异研究以及对棉花种间群体SSR标记遗传图谱的影响；(3)棉花种间BC1群体偏分离的遗传剖析；(4)棉花种间BC1群体高密度SSR标记遗传连锁图谱构建与SSR-EST功能的初步分析。1.草棉(G herbaceum) EST-SSR的遗传评价根据GenBank中公布的247条草棉EST序列,搜索SSR并进行引物设计其中25条序列含有27个SSR,1-6bp重复类型都存在,2bp和3bp重复的频率较高。为了明确其在A、D和AD基因组中的可转移性,依据25条序列共设计25对EST-SSR引物,其中22对引物对棉属的24份种质资源可扩增出清晰可辨的DNA条带,产生92个多态性片段,平均每对引物产生3.64个多态性片段。引物的多态性信息含量(PIC)在0.49-0.91之间,平均为0.81。6对引物在BC1种间作图群体[(鄂棉22×3-79)×鄂棉22](鄂棉22以下简称Emian22)中表现多态性,产生7个多态性位点,其中5个为共显性,2个为显性。除HAU230b标记在BC1分离群体中表现偏分离外,其余引物符合孟德尔式分离。6个位点被整合到陆地棉和海岛棉种间BC1遗传连锁图谱的6条染色体上；有4个位于A亚基因组的4条染色体上(Chr06、10、11和12),2个位于D亚基因组的2条染色体上(Chr19和20)。来源于草棉EST-SSR标记的开发将有助于四倍体棉花起源、进化、基因组结构和功能的研究。2.雌、雄配子重组对棉花种间(G hirsutum and G barbadense)遗传连锁图谱的遗传距离影响以本实验室构建的SSR标记BC1遗传连锁图谱为基础,通过不同交配方式构建的回交群体B ([Emian22×(Emian22×3-79)和C ([(Emian22×3-79)×3-79])来研究雌、雄配子的重组率差异。用Mapmaker/exp3.0和MAPInspector作图软件,分别以群体B和C构建了反映雌、雄配子重组率的遗传连锁图2张,图谱含有313个标记、30个连锁群,长度分别为4532.9 cM和4464.4 cM,标记间平均距离分别为14.48 cM和14.26 cM。经检验表明,雌、雄配子的重组率对图谱总距离的影响不显著。通过分析雌、雄配子重组率对单条染色体重组率的影响发现,群体B中染色体(连锁群)的图距长于群体C的有21条,其余9条连锁群短于群体C。t测验表明有6个对应连锁群的图距差异达到显著水平,表明雌、雄配子重组对部分染色体的遗传图距有影响。尽管很多标记间的遗传距离存在差异明显,但通过2x2卡方测验表明只有17个标记区间的差异是由雌、雄配子重组导致的,由雄配子引起有4个,由雌配子引起有13个。进一步：分析发现雄配子重组主要引起标记间遗传距离变长,即重组率增加；雌配子重组主要引起标记间遗传距离变短,即重组率减少。本研究还讨论了雌、雄配子重组差异在作物遗传育种中的应用。3.棉花种间回交群体偏分离的遗传剖析偏分离现象在作物种间杂交群体中普遍存在。为了研究棉花种间群体分子标记偏分离的原因,我们采用正反交回交群体来研究雌、雄配子选择所引起的偏分离。在BC1群体[(Emian22x3-79)×Emian22](群体A)中产生的1026个SSR多态性标记位点中有114个SSR标记表现偏分离,其中107个偏分离标记被定位到染色体上。将这114个偏分离的SSR标记分别在群体B和C中进行验证,结果表明,群体A中有61个标记在群体B和C中都表现正常分离,即偏分离是由于杂交群体导致的；36个标记在群体B或C中表现偏分离,即偏分离是由于雌、雄配子的竞争能力不同造成的。由配子竞争能力导致的偏分离标记分布于14条染色体上,D亚基因组上分布多于A亚基因组。偏分离标记在第2、16和18染色体上分布最多。由雌配子竞争能力导致的偏分离标记多数分布于A亚基因组的染色体上,但由雌配子竞争劣势导致偏分离的标记在第18染色体分布最多。由雄配子竞争能力导致的偏分离标记多数分布于D亚基因组的染色体上；但由雄配子竞争优势导致的偏分离标记在第16染色体上分布最多。由配子竞争劣势导致的偏分离标记在第2和7染色体上有标记聚集现象。棉花分子标记偏分离的研究对于标记辅助选择中亲本的选配、杂交方式的确定都具有一定指导意义。4.棉花种间BC1群体高密度SSR标记遗传连锁图谱的构建与SSR-EST功能初步分析本研究以Zhang等(2008)建立的BC1群体和获取的1026个SSR多态性位点数据为基础,利用有关文献公布的SSR和EST-SSR标记引物与本实验室自主设计的3536对EST-SSR引物共12722对SSR引物进行亲本多态性筛选,共有2187对引物可用于本实验设计的BC1群体的多态性研究,在BC1群体中共产生2528个多态性位点。4419对gSSR引物产生1023个多态性位点,8303对EST-SSR引物产生1505个多态性位点,引物的多态性率分别为21.2%和15.6%。用作图软件Joinmap3.0进行连锁遗传分析和图谱构建,2318个标记位点进入棉花基因组26条染色体,图谱全长4418.9 cM；另有56个标记建立了13条短连锁群,但目前还不能确定其位于棉花基因组的哪一条染色体上；此外,还有154个标记没有进入任何连锁群。在定位到棉花基因组26条染色体中多态性位点数最多的Chr19为135个标记,标记数最少的Chr02、Chr04均为53个,平均每条染色体为89个标记,其中定位于棉花A亚基因组的标记为1044个,定位于棉花D亚基因组的标记为1274个。26条染色体中,标记间平均距离最大的Chr02为2.78 cM,标记间平均距离最小的Chr14为1.12 cM,整个图谱标记间平均距离为1.91 cM。所有标记中有425个(占总标记数的16.8%)标记表现偏分离(χ2=3.84,P<0.05)；其中358个(占总标记数的14.2%)偏分离标记被定位到本研究建立的连锁群上,定位到棉花已知染色体上的偏分离标记为323个,定位到未知染色体上的偏分离标记的35个,另有67个(占总标记数的2.6%)偏分离标记未定位到连锁群上。对定位到棉花26条染色体上的2318个SSR标记通过生物分析软件找到相应EST序列,通过Gene Oniology(GO)注释。这些EST序列在分子功能、生物过程、细胞组分三大类功能中共注释上1812个功能；部分EST可以注释上多个功能,另一部分EST在目前棉花EST数据库中还不能注释上任何功能,相信随着棉花功能基因组研究的不断深入,这些EST终究会被注释上相应的功能。在level3水平上,注释到分子功能的SSR-EST共1236条(最大的一类为nucleic acid binding,占13.37%)；注释到生物过程的SSR-EST共2110条(最大的一类为cellular metabolic process,占16.54%)；注释到细胞组分的SSR-EST共2273条(最大的一类为intracellular,占20.50%)。本研究建立的以EST-SSR为主的SSR标记高密度遗传连锁图谱,对于研究棉属的起源与进化、棉花基因组结构与功能、棉花产量与纤维品质相关性状的精细定位、重要农艺性状基因的图位克隆、分子设计育种以及分子标记辅助选择育种都具有十分重要的意义。更多还原

【Abstract】 Cotton (Gossypium spp.) is an important cash crop in China and many other countries. It is the second largest source of textile fiber and edible oil throughout the world. The prosperity or decline of cotton yield is very important to the income of farmers and the development of textile industries. The new cultivars of cotton contribute up to 30% to cotton industries. For a long time, researchers have been involved in improving yield traits mainly by employing conventional breeding techniques. Tetraploid cotton has a larger and complicated genome, which is the limiting factor of cotton improvement. Modern molecular biological techniques have brought new ways for cotton improvement. With the combination of marker assisted and conventional breeding, cotton breeding process has been accelerated. It has helped cotton breeders to increase yield and quality by improving the efficiency of selection.This study was planned to reveal the following aspects by SSR technology:(1) Genetic evaluation of EST-SSRs derived from Gossypium herbaceum, (2) The difference between male and female gametes recombination rates by interspecific backcross of cotton (3) Analysis of genetic segregation distortion of SSR molecular markers in cotton interspecific population (4) Construction of a high density genetic linkage map from interspecific backcross population of cotton.1. Genetic evaluation of EST-SSR derived from Gossypium herbaceumEST-SSRs were isolated from 247 EST sequences of G. herbaceum documented in GenBank. Twenty-seven perfect SSRs were identified from twenty-five unique ESTs. These SSRs contained 1-6bp nucleotide motifs with high frequency for 2bp and 3bp nucleotide motifs. In order to clarify the transferability of A, D and AD sub-genomes, SSRs were designed from 25 pairs of EST-SSR primers. Twenty-two of them could amplify 24 cotton accessions and produced 92 polymorphic fragments. The PIC (Polymorphism information content) values ranged from 0.49 to 0.91 with an average of 0.81. Among the 25 pairs EST-SSR primers, six pairs of them revealed polymorphism between Emian22 and 3-79 and yielded seven polymorphic loci (five were co-dominant and two dominant) in the BC1 [(Emian22×3-79)×Emian22] population. Only HAU230b showed distorted segregation in the BC1 population. Six polymorphic loci were integrated into six chromosomes of our interspecific BC1 backbone genetic linkage map among which, four loci were mapped on four chromosomes of A sub-genome (Chr.6,10,11,12), and two loci on two chromosomes of D sub-genome (Chr.19 and 20). The development of EST-SSRs derived from Gossypium herbaceum will contribute to the origin, evolution and the genomic structure of the tetraploid cotton.2. Research on the difference between male and female gametes recombination rates by interspecific backcross of cottonTwo linkage maps covered with 313 markers have been established by populations B and C of BC1 which was formed by male and female gametes recombination, based on the BC1 genetic linkage map created by our laboratory. The lengths of B and C linkage maps were 4532.9 cM and 4464.4 cM respectively and the mean distances of markers in the linkage maps were 14.48 cM and 14.26 cM respectively.By analyzing the influence of male and female gametes recombination rates to the whole chromosome, there were no significant effects to the genetic linkage maps caused by the recombination rates of male and female gametes. There 21 genetic linkage groups in population B were much longer than those in population C, and 9 genetic linkage maps were shorter. The T test revealed that there were 6 linkage maps and 2 linkage maps showed significant differences at 0.05 level and high significant differences at 0.01 levels of significance.Then analyzing the recombination rates of SSRs derived from male and female gametes recombination rates, we found there were 17 markers comprised of 4 male gametes and 13 female gametes showed significant differences at 0.05 levels using 2×2 contingency Chi-square test. Further study showed that the male gametes mainly lead to a longer distance of markers in the linkage map of cotton, i.e. increase the recombination rates. Meanwhile a shorter distance of markers in the linkage map of cotton was caused by the female recombination rates, i.e. decrease the recombination rates. There will be different effects with different recombination rates, so we can chose many kinds of combination modes according to the breeding objectives in crop genetics and breeding, as well as molecular marker-assisted selection.3. Analysis of genetic segregation distortion of SSR markers in the interspecific population of cottonInterspecific cross population between Upland cotton and Sea-island cotton is very common in cotton genetic linkage map. Segregation distortion was ubiquitous among interspecific backcross population. In order to study the reasons for segregation distortion, populations of positive and negative crosses were used. A total of 114 SSR markers showed segregation distortion among 1026 marker in BC1 mapping population [(Emian22×3-79)×Emian22] (Pop A), of which 107 segregation distortion markers was located on chromosome. These 97 SSR markers were validated in population B [Emian22×(Emian22×3-79)] (Pop B) and population C [(Emian22×3-79)×3-79] (Pop C). In Pop A, segregation distortion of 61 markers were caused by cross mode and segregation distortion of 36 markers were caused by competitive competence of male or female gametes. Segregation distortion markers caused by competitive competence of male and female gamete were distributed on chromosome 14. These markers were found to be more frequent on "D" sub-genome than "A" sub-genome. Most of segregation distortion markers were distributed on chromosome 2,16 and 18. Segregation distortion markers caused by competitive competence of female gamete were distributed on chromosomes of "A" sub-genome. Most of segregation distortion markers caused by competitive disadvantage of female gamete were distributed on chromosome 18. Most of segregation distortion markers caused by competitive competence of male gamete were distributed on chromosomes of "D" sub-genome. Clusters of segregation distortion markers caused by competitive disadvantage of gamete were found to locate on chromosome 2 and 7. Segregation distortion markers caused by competitive disadvantage of male gametes were distributed on chromosome 16. The research of segregation distortion will be important for parent selection and the hybrid approach in marker-assisted selection.4. Construction of high density genetic linkage map by interspecific backcross population of cottonWith the improvement of molecular marker techniques and the improvement of cotton DNA extraction, researches on the use of molecular markers of genetic linkage maps of cotton have achieved rapid development. Until now, more than one molecular marker genetic linkage map of cotton have been established including inter-specific and intra-specific populations. Advanced hybrid approaches including F2, BC1, DH, natural and RIL populations, et al., more molecular markers, such as RFLP, RAPD, AFLP, SCAR, SSR, SRAP and SNP. And more genetic mapping software: Mapmaker/exp3.0 and Joinmap3.0.Based on the BC1 population established by Zhang (2008) and 1026 polymorphic loci of SSRs. 12722 pairs of primers including published SSRs and EST-SSRs as well as designed in our laboratory have been used to scan the polymorphisms between two parents. Among of these markers,2187 primers showed polymorphism between parents while 2528 primers were confirmed to be polymorphic in BC1 population. There were 1023 and 1505 polymorphic loci generated by 4419 SSRs and 8303 EST-SSRs respectively and polymorphic percentages were 21.2% and 15.6%.Map analysis and construction were performed in Joinmap3.0 software, with supposed LOD value (>5.0). The maximum distance of markers was calculated to be<40 cM. Overall,2318 marker loci had been anchored onto 26 linkage groups of cotton genome and length of map was 4418.9 cM. There were 13 short linkage groups established by 56 markers, yet we were unable to locate them on the cotton chromosomes. Another 154 markers did not anchor on any linkage groups.There were 135 markers on Chr.19, whose polymorphic loci were the most one in 26 linkage groups of cotton genome. Chromosomes with the least markers were found on Chr.02 and Chr04 with an average of 89 markers per chromosome. There were 1044 and 1274 markers located on A and D sub-genomes, respectively. The maximum distance of markers was 2.78 cM on Chr.02, compared to the minimum of 1.12 cM on Chr.14. The mean distance of markers in the map was 1.91 cM.There were 425 markers (16.8%) showed segregation distortion (χ2=3.84, P<0.05), of which 358 markers (14.2%) located in the linkage groups.323 markers located in cotton genome,35 markers located in 13 short linkage groups, and 67 markers (2.6%) unallocated in any linkage groups.The 2318 markers located in 26 chromosomes were analyzed by correspondening EST sequences marked by 1812 molecular functions, biological process and cellular element. Some EST sequences were noted on multi-function; some were not noted on any functions in cotton EST database for the present; all of these EST sequences will be noted on homologous function with further research on cotton function genome. In level 3,1236 SSR-EST sequences were noted molecular function, nucleic acid binding is the maximum class(13.37%).2110 SSR-EST sequences were noted biological process, cellular metabolic process is the maximum class (16.54%).2273 SSR-EST sequences were noted cellular element, intracellular is the maximum class (20.50%)With the high density SSRs linkage maps mainly established by EST-SSR in the research, it will be helpful with the evolutionary studies of Gossypium, and cotton genome structure and function as well as cotton yield and fiber development-related genes. It also has great significance for molecular assisted selection and molecular designed breeding.更多还原