【作者】 张艳欣；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 棉花（Gossypium L）是世界上最重要的经济作物之一,为我们提供了天然的纺织纤维和食用油,随着高效快速的纺纱技术的引进与普及,以及人民群众对衣着要求的普遍提高,对我国棉花品种的总体纤维品质提出了更高的要求,但是常规育种方法要求育种群体大,时间长,难度也较大,很难打破强度与产量之间的负相关,取得突破性进展。随着基因组研究的飞速发展,分子育种成为棉花纤维品质遗传改良的重要途径。新的分子标记不断被开发并用于棉花高度整合的遗传图谱的构建,棉纤维基因结构和功能的研究,棉纤维品质数量性状位点的定位及分子标记辅助选择（MAS）。1.海岛棉EST-SSR引物的开发与应用研究植物EST（expressed sequence tags）计划作为植物基因组计划的一个重要组成部分,已经在多种物种中开展起来,EST资源库的不断扩充为采集SSR提供了海量数据。现有的棉花EST-SSR引物大都开发于中棉、雷蒙德氏棉和陆地棉,尚没有海岛棉的EST-SSR开发研究。众所周知,海岛棉是最重要的棉种之一,从中可以发掘新的遗传材料用于陆地棉的纤维品质改良。本研究基于实验室已构建的海岛棉Pima3-79（遗传标准系）纤维发育的cDNA文库中的98条EST序列设计了119对EST-SSR引物。详细地分析了这批新的EST-SSR引物的特征,而且评价了其在物种间的可转移性,在遗传多样性分析及遗传连锁图构建方面的应用价值。在这些SSR中,三核苷酸重复基序AAG含量最为丰富,达11.76%。用36份材料（13份A基因组二倍体材料,11份D基因组二倍体材料和12份AD基因组异源四倍体材料）来评价新EST-SSR引物,119对引物中有76对成功扩增,共产生313条多态性条带,平均每对引物产生4.11条。在二倍体棉种中的扩增效率从78.95%（瑟伯氏棉）到94.74%（中棉和草棉）,在四倍体棉种中的扩增效率从93.42%（黄褐棉）到100%（达尔文棉）。在二倍体棉种中棉的扩增多态性率为11.84%,与在四倍体棉种陆地棉的扩增多态性率相同。引物的多态性信息含量（PIC）变化于0.17-0.95之间,平均达0.53。根据Jaccard’s遗传相似系数对所用36份材料进行了聚类分析,聚类结果为三大类,分别为A基因组材料,D基因组材料和AD基因组材料。此外,有21对引物在本实验室的种间回交群体[（鄂棉22×Pima3-79）×鄂棉22]DNA中表现多态性扩增,产生24个多态性位点,其中22个被锚定于该遗传连锁图的12条染色体。这批新的EST-SSR引物在遗传多样性分析,比较作图和分子标记辅助选择（MAS）育种的研究中具有较高的应用价值。2.海陆棉种间BC₁群体的高密度遗传连锁图谱构建与QTL定位对棉花来说,以分子标记为铺垫的基础研究和应用研究才刚刚处于起步阶段,关于棉花分子标记遗传连锁图谱和数量性状位点定位的研究仍需大量积累。以简单、可靠、适于高通量分析的分子标记构建高密度的棉花遗传连锁图谱,将不断增多的基因组信息剖析到相应的染色体片段上,才能使分子标记辅助选择育种、重要数量性状基因的图位克隆等后续的研究得到顺利开展。在众多的分子标记中,SSR标记具有分布广泛、位点特异性、共显性、信息量高等优点,是构建棉花遗传连锁图谱最有优势的标记。本研究采用SSR分子标记为基础,以海陆棉种间[（鄂棉22×Pima3-79）×鄂棉22]的141个BC₁单株为作图群体,构建了一张完全基于SSR标记的高密度的遗传连锁图谱,并且以此图谱为基础,运用复合区间作图法进行纤维品质的QTL检测。用6310对SSR引物（3714对gSSR,2596对EST-SSR,共13个系列）进行鄂棉22和Pima3-79亲本间多态性的检测,共筛选出在双亲间表现多态性的SSR引物1026对（占16.3%）,用于进行BC₁群体扩增,产生了1040个多态性SSR位点,用MapMaker/EXP.3.0软件,以LOD值≥8.0,标记间最大间距≤40cM为阈值构建了遗传连锁图谱,917个标记位点进入44个连锁群（26条染色体）,图谱全长5452.2cM,标记间的平均距离为5.9cM。位于连锁图上的偏分离标记共有107个,有72.9%标记偏向于杂合子,基因组SSR的偏分离标记比例（11.7%）与EST-SSR的偏分离标记比例（11.5%）基本相当,偏分离热点区（SDR）共有11个,分布于At染色体组上的偏分离标记数（35）和SDR数（2）明显少于Dt染色体组上的偏分离标记数（72）和SDR数（9）,而染色体chr02、chr16、chr18上分布的偏分离标记数量分别达到了11个、20个、23个。对本研究所用2596对EST-SSR引物的重复基序信息及其多态性表现进行统计分析。结果表明:四核苷酸基序的多态性率（11.33%）高于二核苷酸基序的多态性率（7.90%）及三核苷酸基序的多态性率（6.72%）,五核苷酸与六核苷酸的多态性率更低（4.00%）;ACAT,AC和ACT的多态性率较高（分别达到17.24%,16.67%和12.99%）,而基序ACG和AGG的多态性率最低（3.33%）;SSR位点长度为27bp,33bp和24bp的引物与其他引物相比,有着更大的产生多态性的潜力。以上结果将对今后EST-SSR引物的设计提供参考价值。以BC₁单株和BC₁F₂家系的纤维品质性状值用复合区间作图法进行QTL检测（LOD≥3.0）,检测出纤维长度（2个）、整齐度（1个）、马克隆值（1个）、伸长率（3个）、强度（4个）等性状的QTL共11个,其中有6个来源于At染色体亚组,5个来源于Dt染色体亚组,解析9.80%～16.82%的表型变异。来自Pima3-79的位点起增效作用的QTL有3个,加性效应值在0.33到1.10之间。更多还原

【Abstract】 Cotton（Gossypium L.） is one of the most important economic crops that provide natural textile fiber and edible oil throughout the world.In the past few years, improvement in the quality of cotton fiber has become more important because of profound innovations in spinning technology.While cotton yield traits are negatively related with fiber quality traits which make it difficult to improve yield and quality at the same time by traditional breeding ways.With the rapid development of genomic technologies,molecular breeding has become an important method for the genetic improvement of cotton fiber.New sets of molecular markers should be developed to facilitate the development of a high-resolution integrated genetic map for structural and functional studies of cotton fiber genes and marker assisted selection（MAS） of genes that could enhance cotton fiber quality.1.Studies of new EST-SSRs derived from Gossypium barbadenseThe plant EST（expressed sequence tags） program is one of the most important parts which compose the plant genome program.The expansion of EST.data base is a foundation of development of EST markers.EST-SSRs can be used in studies of plant genetics and evolution;they also have higher levels of transferability to related species than genomic SSR markers.Furthermore,the functions of EST-SSRs are usually presumable.However,existing cotton EST-SSR markers are mostly derived from G. arboreum and G.hirsutum,while G.barbadense derived EST-SSRs are scarce.It is well-known that G.barbadens is one of the most valuable cotton species and it is an ideal candidate for providing new genetic material for improving fiber quality in G.hirsutum.In this paper,one hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G.barbadense cv.Pima3-79.The characteristics of these EST-SSRs were defined, and their transferability,applications in genetic diversity analysis and linkage mapping were evaluated. Among the SSRs,trinucleotide AAG appeared at a high frequency of 11.76%. Thirty-six accessions（consisting of 13 diploids of the A genome,11 dipioids of the D genome and 12 allotetraploids of the AD genome） were employed to test new EST-SSRs. Seventy-six EST-SSRs were successfully amplified,and yielded 313 polymorphic fragments,with an average of 4.11 fragments per primer pair.Cross-species transferability of the 76 EST-SSRs in diploid Gossypium species ranged from 78.95%（in G.thurberi） to 94.74%（in G.arboreum and G.herbaceum）,whereas in tetraploid species from 93.42%（in G.mustelinum） to 100%（in G.darwinii）.Polymorphism in diploid species G.arboreum was the same as in tetraploid species G.hirsutum,both were 11.84%. The PIC ranged from 0.17 to 0.95 with an average of 0.53.Based on Jaccard’s genetic similarity coefficient,these 36 accessions were clustered into three groups.Twenty-one EST-SSRs exhibited polymorphisms in BC₁ population[（Emian22×Pima3-79）×Emian22],24 polymorphic loci were generated,while 22 of the 24 polymorphic loci were integrated with our interspecific BC₁ backbone genetic linkage map,and anchored in 12 chromosomes.This study effectively proved that EST-SSRs from G.barbadense are valuable for genetic diversity analysis and genetic mapping.2.Construction of dense genetic linkage map by interspecific population for QTL mappingIn the past decade,several successive cotton molecular maps have been constructed using diverse DNA molecular markers and mapping populations.A detailed genetic linkage map covering a large region of the cotton genome based on some informative, portable and practical markers will provide a platform for evolutionary comparisons with other species,comparative mapping,QTL mapping and MAS,and structural and functional studies of the cotton genome.Simple sequence repeats（SSRs）,alternatively called microsatellites,are regarded as useful and efficient markers for map construction of cotton,which are abundant,locus-specific,often codominant and multiallelic.In this study,an interspecific linkage map of allotetraploid cotton was developed in a BC₁ population[（Emian22×Pima3-79）×Emian22].This map was based entirely on genome-wide simple sequence repeat（SSR） markers.Totally 6310 SSR markers （including 13 sets of markers） were employed to screen polymorphisms between ’Emian22’ and ’Pima3-79’,approximately 16%（1026/6310） of all SSR markers revealed polymorphic bands,then yielded 1040 polymorphic loci in BC₁ population.The molecular map was generated using MAPMAKER version 3.0b program（LOD scores≥8.0,recombination frequency≤0.40）,with 44 linkage groups assigned to 26 chromosomes,and 917 loci spanning 5452.2 cM of the genome.The average distance between loci was 5.9 cM,covering the A-subgenome and D-subgenome uniformly. Significant segregation distortion Was observed for 107 loci（11.7%）,of which 29（27.1%） loci skewed toward the ’Emian22’ parent,while 78（72.9%） loci skewed toward the heterozygote.The frequency of distorted loci of genomic SSRs（11.7%） was coincident with EST-SSRs（11.5%）.In total,11 SDRs were detected in seven linkage groups,2 SDRs（35 loci） appeared in the A-subgenome,and 9 SDRs（72 loci） appeared in the D-subgenome.Many of the distorted loci concentrated in Chr02,Chr16 and Chr18.This map was characterized and analyzed in detail,including the distributions of genomic SSRs,expressed sequence tag（EST）-SSRs,and distorted markers.Furthermore, the relationship between motif characteristics（sizes,types,lengths） and the level of polymorphism in EST-SSRs was also surveyed.The results showed that polymorphism rates of tetranucleotide motifs（11.33%） was higher than dinucleotide motifs（7.90%） and trinucleotide motifs（6.72%）,the percentages of polymorphic hexanucleotide and pentanucleotide were both only 4.00%;ACAT,AC,and ACT motif types had the highest level of polymorphism（17.24%,16.67%and 12.99%,respectively）,while ACG and AGG were the lowest（both 3.33%）;loci with lengths of 27 bp,33 bp,and 24 bp were more likely to be polymorphic.These results will provide information to assist in designing future EST-SSRs.The method of composite interval mapping was applied to search for QTL（LOD≥3.0） by the phenotypes of BC₁ plants and corresponding BC₁F₂ families.On the basis of this analysis yielded 11 QTL:2 QTL controlling fiber length,1 QTL controlling fiber uniformity,1 QTL controlling micronaire,3 QTL controlling fiber elongation,4 QTL controlling fiber strength.Among them,6 were mapped on At chromosome and 5 were mapped on Dt chromosome,explained 9.80%～16.82%of phenotypic variance.In three of the QTL,Pima3-79 alleles had positive additive genetic effect,the additive genetic effect values ranged from 0.33 to 1.10.更多还原