【作者】 王沛政；

【导师】 张天真；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 新疆具有适宜棉花种植得天独厚的自然生态条件。新疆“矮密早”栽培技术,促进了新疆棉花生产的迅速发展。近十年来,新疆棉花种植面积、单产、总产已位居全国各产棉省之首,目前已成为国家重要的棉花产区。棉花枯萎病（Fusarium oxysporum）和黄萎病（Verticillium dahliae kleb）是世界性的主要棉花病害。新疆棉花枯黄萎病发生已久,严重威胁了新疆棉花生产,而其自育的大部分品种又不抗枯黄萎病,如何提高新疆棉花抗病性乃当务之急。传统育种多依赖于形态学标记（如株高、穗形、粒色、芽黄和花色素等）,随着科学的发展,育种家开始在细胞学、生理生化和DNA等水平上寻找与育种目标性状紧密连锁的遗传标记,以便对目标性状进行追踪选择。本研究选用目前由新疆广泛种植的陆地棉品种构建的F₂群体进行QTL定位研究,以期在新疆“矮密早”栽培技术条件下筛选出一些与棉花抗病、产量构成及株型相关QTL,有助于今后新疆棉花分子标记辅助育种效率的提高。一、基于EST-SSR标记的新疆陆地棉遗传多样性研究利用EST-SSR标记对新疆近30年来种植的陆地棉品种进行了遗传多样性研究。42个EST-SSRs对50个陆地棉的进行了基因型鉴定,共产生了91个多态性位点;50个陆地棉间的相关系数在0.11-0.83之间,研究表明50个陆地棉间有较高的遗传多样性,但新疆本地自育陆地棉品种的遗传多样性比较窄。聚类分析发现新疆自育品种大都包含前苏联棉花种质血统,并且与其系谱基本一致。结果证明来由EST开发的SSR标记可以用来估计陆地棉间的遗传多样性。二、新疆陆地棉黄萎病抗性QTL筛选及其定位高抗非落叶型黄萎病陆地棉品种“新陆中10”与高感非落叶型黄萎病陆地棉品种“军棉1号”、“新陆早7号”分别配制两个F₂作图群体;新陆中10号×军棉1号群体图谱含62个标记位点,覆盖棉花基因组593.6CM;新陆中10号×新陆早7号群体图谱,含有78个标记位点,连锁群总的长度830.2cM。两个作图群体F_2:3家系在黄萎病发病高峰期铃期和吐絮期共检测到8个黄萎病抗性QTLs,LOD均大于3。其中4个QTLs的抗性基因位点来自抗性母本,分别位于4个不同的染色体（Chr.5、Chr.15、Chr.20、Chr.25）,另外4个QTLs的抗性基因位点来自感病亲本,位于Chr.9、Chr.13上。由此认为陆地棉对非落叶性黄萎病的抗性是多基因控制的。其中染色体13上共检测到3个QTLs,由于这3个QTLs均在标记NAU1211附近,它们可能为同一QTL。我们与已有报道均在Chr.5、Chr.15、Chr.9、Chr.13、Chr.20或同源区域定位到抗性相关QTLs。三、新疆主栽陆地棉枯萎病抗性QTL筛选与定位以高抗枯萎病品种与高感枯萎病品种构建了2个F₂陆地棉种内作图群体。利用新疆棉花枯萎病菌进行群体抗病鉴定,以F_2:3家系对枯萎病抗性来估计F₂单株抗病性。鉴定结果表明中棉所35号和军棉一号群体的F_2:3家系对枯萎病抗性鉴定表明抗感符合3:1分离,把棉株对枯萎病抗性做为显性标记进行连锁作图分析发现,枯萎病抗性基因与标记JESPR304的紧密连锁,距离为5.3 cM,利用本实验室饱和作图群体,将标记JESPR304（特征条带140bp）定位到D3（Chr.17）上。该群体F_2:3家系枯萎病抗性病指经转化成正态分布后,复合区间作图共测到与枯萎病抗性相关5个QTLs,分别位于4个不同的连锁群上（Chr.7、Chr.15、Chr.23、Chr.17）。其中两年均在Chr.17检测到枯萎病主效抗性QTL,与单侧标记JESPR304的距离为0.06-0.2cM,解释表型变异均大于50.0%。苏棉10号和长绒棉作图群体的84个SSR标记位点的单标记分析筛选到1个标记（JESPR304）和枯萎病抗性基因相关,该标记距离枯萎病抗性基因8.00 cM,解释表性变异19.9%。选用内地抗病品种和新疆本地感病品种进行验证JESPR304标记与抗性的相关性,96.6%内地抗病品种扩增出标记JESPR304抗性位点,而27%的新疆感病品种扩增出标记JESPR304抗性位点,表明标记JESPR304与品种抗性的相关性密切。初步确定JESPR304标记与抗枯萎病抗性基因紧密连锁。四、新疆陆地棉主栽品种产量性状QTL的标记与定位在新疆“矮密早”栽培技术体系下,上述3个作图群体的F_2:3家系共鉴定、筛选出皮棉产量以及单铃重、衣分、籽指、结铃数等5个产量构成因子QTLs 16个:其中与籽指有关的QTL共5个,分别位于Chr.1、Chr.5和Chr.7上;与衣分有关的QTL5个,分别位于Chr.7、Chr.13和连锁群6上;与铃重有关的QTL5个,在Chr.7上有4个、Chr.17上1个;与皮棉产量有关的QTL1个,位与Chr.17上。在Chr.7上共定位了9个包括单铃重、衣分和籽指在内的QTLs,由于单铃重、衣分和籽指等性状有明显的相关性,可以看出,它们的QTL在同一区域分布的现象很明显,这些同一区域分布的QTL可能会在今后新疆陆地棉标记辅助育种中起到一定作用。同时对没有分配到连锁群上的标记位点进行单标记分析,共检测出8个与产量性状相关的标记,解释的表型变异在5%-11%之间。我们在Chr.1、Chr.5染色体水平上的产量相关QTLs定位与前人研究相同,其余QTLs是在“矮密早”栽培条件下检测出的新QTLs。五、新疆陆地棉主栽品种形态性状QTL的标记与定位前述3个作图群体共鉴定、筛选出果枝始节、株高、叶主脉、叶次脉等15个稳定的QTLs:其中2个果枝始节QTL位于Chr.5和Chr.7上;3个株高QTL分别位于Chr.13、Chr.25和Chr.17上;筛选出叶主脉及叶次脉的QTL共10个,位于Chr.7、Chr.15、Chr.17、Chr.19、Chr.21、Chr.23和连锁群17、6上,解释表型变异在6.8%-24.4%之间。对没有分配到连锁群上的标记位点的单标记分析,在LOD值大于2的水平下,检测出9个与棉株形态性状相关的标记,其中与株高相关标记3个,另外6个标记与叶主脉及叶次脉相关。本研究定位在Chr.15、Chr.21、Chr.23、Chr.25上的棉株形态性状QTLs,在染色体水平上的定位与前人报道相同,其它QTL在染色体水平上（Chr.7、Chr.17、Chr.19）定位与前人研究不同,可能是新检测出的QTL。更多还原

【Abstract】 As a major source of fibers,cotton is an important economic crop and plays an important role in the global economy.The technology of "short,dense and early"（short plant height,dense populations and earlier maturity） accelerated the development of Xinjiang cotton production.The cotton in Xinjiang covered about 20%of the total cotton area in China and contributes nearly about 33%to the national cotton yeild.Fusarium wilts and Verticillium wilts are the main diseases of cotton in the world.The incidence area of Fusarium wilt reached to 46 thousand hectare in 1995 according to the survey of the plant protection station in Xinjiang.At present,the majority of the varieties from nurtures in Xinjiang were not resistance to Fusarium wilt and Verticillium wilts.How to enhance the resistance of variety to disease has become the important factor for breeder.Selection breeding and planting cultivars of resistance to the disease were the most economical effective solution to the problem.As breeding a new cultivar through the traditional ways needed a long period,which didn’t meet the stringent demand of production.Traditional selection breeding is more dependent on the phenotypic of crop.Phenotypic traits marker --plant height,grain color,shoot yellow,ears--have been taken important action in tradition breeding.In this study,the objective of this research is to screen the stable QTL related resistance and agronomic traits basing on different populations under the technology with "short,dense and early" and these QTLs will be used effectively for MAS during the process of breeding in the future.1、Genetic Diversity among Xinjiang Upland Cotton Cultivars Based on EST-SSR MarkersThe genetic diversity among Xinjiang 50 upland cotton cultivars was investigated based on EST-SSR.A total of 91 bands with polymorphism had been detected in these cultivars by screening the 42 EST-SSRs loci.Based on EST-SSR data,the coefficient of similarity among 50 upland cotton cultivars was calculated varying from 0.11 to 0.83.It was indicated that the more genetic diversity among those cultivars was presented,while genetic diversity in same origin in Xinjiang had been narrowed.The 50 cultivars can be divided into two subgroups based on the coefficient of similarity,and most of the tested cultivars were consistent with their pedigree.It was testified that EST-SSR markers derived directly in transcribed:regions of the genome,provide a more-direct estimate of genetic diversity of cotton cultivars.2、QTL Mapping of Genes Resistant to Verticillium Wilt In Xinjiang Upland CottonTwo molecular mapping F₂ populations were derived from the intraspecific cross of the highly tolerant G.hirsutum cv.（Xinluzhong10） and the susceptible G.hirsutum cv. （Junmianland Xinluzhao7）.A total of 5400 simple sequence repeat（SSR） primer pairs were screened over resistant and susceptible parents.The first map comprises 62 loci mapped to 15 linkage groups covering 593.6 cM of the genome.The second genetic map comprises 78 loci mapped to 20 linkage groups covering 830.2 cM of the cotton genome. The two mapping F₂ population were used to analyze quantitative disease-related trait loci （QTLs）.The two mapping F_2:3 families were phenotyped with a non-defoliating V.dahliae. A total of 8 QTLs across two populations and two disease development periods were detected by Composite Interval Mapping（CIM）.Four QTLs of resistance to V.wilt were detected on four linkage groups and explained 59.1%,63.5%,13.6%and 26.9%of the phenotypic variance with LOD＞3 respectively.The other four QTLs were detected on two linkage groups,with LOD peaks ranging from 2.6 to 21 and explained 7.3%,56.9%,32.7%, and 7.18%of the phenotypic variance.This might mean that the resistance of V.wilts was controlled by polygene.Three QTLs located on the position of chromosomes 13,which is nearer the marker NAU1211,had large effect,it might be the same QTLs.In comparison with previously published data,some QTLs related to V.wilt localized on the five chromosomes（Chr.5,Chr.9,Chr.13,Chr.15 and Chr.20） were localized in similar regions or homeologous regions as previously reported QTLs.Those QTLs can be used effectively for MAS during the process of breeding in the future.3、Mapping of Genes Resistant to Fusarium Wilt of Upland Cotton in XinjiangFusarium wilt is a serious disease in most cotton growing areas of the world.The identification of resistance genes is critical for developing sound breeding strategies.In this study a genetic linkage map was constructed using 173 F₂ individuals developed by acrossing two elite lines of Upland cotton（Zhongmiansuo35 and Junmian1） as parents.The maps with 75 loci were covered 743.1cM length of the cotton genome.Phenotype of F₂ population to disease-related trait was evaluated by Phenotype of F_2:3 families.In the assessment of individual phenotype of disease resistance as dominant marker,the gene of resistance to Fusarium wilt were located on Chr.17 groups by linkage map analysis using bridge markers.In 2004 a new genetic linkage map was constructed by acrossing the same two elite lines of Upland cotton.The result still suggested that one major QTL with the resistance to Fusarium wilt were tightly linked to JESPR304.Another genetic linkage map was constructed using 160 F₂ individuals developed by acrossing two elite cultivars of Upland cotton,"Changrong",highly sensitive to wilt and "Sumian10",highly resistant to wilt.One major gene detected with resistance to Fusarium wilt was tightly linked to JESPR304 with 0.80cM genetic interval distance and explained the phenotypic variance of 19.9%.In order to detect the degree of linkage between the resistance gene and JESPR304, 40 susceptible Upland cotton cultivars and 86 resistant Upland cotton cultivars were screened using JESPR304.The results showed there is intensely linkage between the resistance gene and JESPR304.Using composite Interval Mapping method a total of five QTLs resistant to Fusarium wilt were detected on four linkage groups（Chr.7、Chr.15、Chr.23、Chr.17） in Zhongmiansuo35 and Junmian1 population.One major QTL with resistance to Fusarium wilt showed to be tightly linked to JESPR304 with 0.06 to 0.2cM genetic interval distance,and it explained over 50%of the phenotypic variance.The gene /QTL will be of great significance in breeding and molecular marker-assisted selection.4、QTL Mapping of Yield Components for Main Cultivars Planted of Upland Cotton in XinJiangWe used three above-mentioned molecular linkage maps for characterizing quantitative trait loci（QTLs） determining yields related traits of cotton which can provide DNA molecular markers to be used in cotton improvement.The three molecular linkage maps shared same 22 pairs of SSR primers,covered all of the cotton genome except chromosome 2 and chromosome 26.Under Xinjiang "short,dense and early" planted condition,16 possible QTLs of yield related traits（Lint yield,Lint percentage,Boll size, Seed index） were suggested with LOD＞2.0.Five seed index QTLs were located on chromosome1,5 and 7;Five lint percentage QTLs were located on chromosome7,13 and LG6;Five boll size QTLs were located on chromosome7,17;One lint yield QTL were located on chromosome17.As nine correlated QTLs of boll size,seed index and lint percentage were located on chromosome7,it was clearly that these QTLs were distributed on same or neighbor position and would play important significances on breeding.All of unlinkaged markers of the three maps were surveyed using single marker analysis method and 8 marker related to yield traits were suggested with LOD＞2.0.In comparison with previously published data,the seed index QTL were assigned to the chromosomes Chr.1 andCh r.5in this paper were the same as previously published reports.The rest QTL on the level chromosomes（Chr.7,Chr.13） were different from previously published reports.5、QTL Mapping of some Plant Morphological Traits for Main Cultivars of Upland Cotton Planted in XinJiangWe used the above-mentioned molecular linkage maps for characterizing quantitative trait loci（QTLs） determining cotton morphological traits.Under Xinjiang "short,dense and early" experiment condition,four morphological traits—plant height,the first Fruit nodes, main-lobe length（L1）,second-lobe length（L2）—were studied,15 possible QTLs/loci which had LOD＞2.5 were suggested.Two first Fruit nodes QTLs were identified on Chr.5 and Chr.7.Three plato height QTLs were located on Chr.13、Chr.25 and Chr.17,and two of its alleles were originated from the male parent.Ten QTLs of main-lobe length（L1） and second-lobe length（L2） were located on Chr.7,Chr.15,Chr.19,Chr.21,Chr.23,LG6, LG17 and Chr.17.All of unlinked markers of the three maping population were surveyed using single marker analysis method and 9 markers related to morphological traits were detected with LOD＞2.0.Three markers were related to yield traits and 6 markers to main-lobe length（L1） and second-lobe length（L2） explained about 5.2-8.0%of the phenotypic variance.In comparison with previously published data,the QTLs assigned to specific chromosomes（Chr.15、Chr.21、Chr.23、Chr.25） in this paper were the same as previously published reports.In addition,we also found the rest QTLs were different from previously published reports on the chromosomes level（Chr.7、Chr.17、Chr.19）.更多还原