【作者】 谢文刚；

【导师】 张新全； B.Shaun Bushman；

【作者基本信息】 四川农业大学 ， 草业科学， 2013， 博士

【摘要】 鸭茅(Dactyl is glomerata L.)起源于欧洲和北非及亚洲温带地区,是世界著名的多年生牧草,具有草质柔嫩、高产、优质、耐荫且适应性强等优点,蕴藏着丰富而优良的基因资源,开发利用潜力大。但该物种分子遗传育种基础研究滞后,育种效率低,急需加快分子育种进程。开展鸭茅资源分子遗传多样性评价、遗传图谱构建及重要农艺性状基因定位对于鸭茅基因资源保护、综合利用、重要农艺性状遗传改良及提高鸭茅育种水平和育种效率有重要意义。因此本研究利用鸭茅SSR分子标记分析了亚洲地区二倍体和四倍体鸭茅种质内和种质间遗传变异；同时综合利用SSR和SRAP两种分子标记构建了首张二倍体鸭茅遗传连锁图谱；在此基础上利用鸭茅EST-SSR分子标记和AFLP标记构建了四倍体鸭茅遗传图谱并首次定位鸭茅抽穗期QTL；通过454高通量测序平台,获得大量鸭茅开花基因序列,并成功定位了Vrn3春化基因。主要研究结果如下：1、二倍体与四倍体鸭茅遗传多样性及亲缘关系的SSR分析用SSR标记对来自亚洲地区的16份鸭茅(Dactylis glomerata L.)种质的160个单株进行遗传多样性研究。获得下述结果：(1)21对引物共扩增出多态性条带143条,平均每对引物扩增出6.8条,多态性条带比率(P)达90.7%；多态性信息含量(PIC)范围在0.17(A01F24)到0.45(A01E02)之间,平均为0.33；Shannon多样性指数(Ⅰ)范围在0.0463到0.3547之间,表明供试鸭茅种质具有丰富的遗传多样性。(2) AMOVA分析表明65.75%的遗传变异存在于鸭茅种质内部,75.58%的遗传变异存在于地理区域内,二倍体与四倍体鸭茅群体间遗传差异不显著,其遗传变异仅为2.62%。(3)对各地理类群基于Nei氏无偏估计的遗传一致度(GI=0.876)可将16份鸭茅分为两大类：所有的四倍体聚为同一大类,二倍体在两个大类中都有分布。聚类结果总体表明：来自相同或相似生态地理环境、相同倍型的材料基本聚为一类,呈现出一定的相关性。2、二倍体鸭茅遗传连锁图谱构建以遗传和表型差异大的鸭茅亲本01996和YA02-103杂交获得的111个F1单株为作图群体。利用164个SSR标记和108个SRAP标记构建二倍体鸭茅遗传连锁图谱。父本遗传图谱涉及9个连锁群,包含90个标记(57个SSR和33个SRAP),图谱总长为866.7cM,标记间平均图距为9.6cM,基因组覆盖率为81%。母本遗传图谱涉及10个连锁群,包含87个标记(54个SSR和33个SRAP),图谱总长为772.0cM,标记间平均图距为8.9cM,基因组覆盖率为75%。标记偏分离比率为15%,基于10个“桥标记”共检测到5个同源连锁群。研究结果可为鸭茅重要农艺性状QTL定位奠定基础,为鸭茅分子育种研究提供了基础信息。3、四倍体鸭茅遗传连锁图谱构建及抽穗期QTL定位本研究以鸭茅抽穗期存在显著差异的鸭茅亚种him271(晚花型)和asch621(早—中等开花型)为杂交亲本,获得了包含284个单株的F1作图群体,综合利用鸭茅EST-SSR和AFLP分子标记构建了四倍体鸭茅高密度遗传连锁图谱。亲本图谱分别包含了28个共分离连锁群和7个整合连锁群。利用亲本图谱间共有的38个“桥标记”,构建了7个同源连锁群,并基于鸭茅EST-SSR标记与水稻基因组的线性关系,确定了7个同源连锁群与鸭茅染色体间的对应关系。Him271图谱总长947cM,包含183个标记位点,标记平均密度为5.2cM。而asch621图谱包含193个标记,图谱全长1038cM,标记平均密度为5.5cM。亲本连锁群长度在98到187cM之间。利用区间作图法在连锁群2、5和6上共检测到7个影响开花性状的QTL,解释的表型变异在7.85%到24.19%之间。该研究结果为鸭茅分子标记辅助育种,重要农艺性状遗传改良,混播草地晚熟鸭茅品种选育及近一步开展功能基因和比较基因组研究奠定了重要基础。4、鸭茅开花基因发掘与定位基于水稻、小麦、黑麦草、高羊茅等物种的开花基因(如：FT、Hd、VRN基因等)保守序列,设计基因探针28条。并利用开花基因探针对鸭茅基因组开花基因序列进行捕获。通过454高通量测序平台,共获得读长(reads)约21000条,叠连群(Contig)序列2094条,序列长度在105bp到1975bp之间。BLASTn序列搜索表明：Contig40、Contig441和Contig558与VRN、Hd和FT基因具有较高同源性。根据同源序列设计了20对候选开花基因引物,并对F1作图群体扩增以检测其分离比例。最终Vrn3基因引物在F1作图群体中具有1：1的分离比例,成功定位在Him271的第3个连锁群上。更多还原

【Abstract】 Orchardgrass (Dactylis glomerata L.) is indigenous to Eurasia and northern Africa, has been naturalized on nearly every continent, and is one of the top perennial forage grasses grown worldwide, owing to its good nutrition, high yield and good adaptability. Orchargrass gene resources are diverse and rich and have a great potential for breeding use. But few tools and information are available for molecular breeding, gene mapping and genetic improvement of the species. Accordingly, this research aimed to offer an important information and basis for further molecular assisted selection (MAS) breeding, improve economically important traits, enhance breeding efficiency and shorten breeding process of the speices.In this study, SSR molecular markers were used to detect the genetic diversity and variation between and within diploid and tetraploid orchardgrass accessions collected in Asian. A combination of simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular markers was used to construct the first diploid orchardgrass map. Orchardgrass EST-SSR markers and AFLP markers were employed to construct a high-density tetraploid orchardgrass maps. QTLs associated with heading date in this species were reported first. Based on454high-throughput sequencing, we obtained amounts of sequences of flowering candidate genes in orchardgrass. Finally, Vrn3gene was successfully mapped on previous maps. These results were as follows:1. Diversity comparison and phylogenetic relationships of diploid and tetraploid orchardgrass(Dactylis glomerata L.) germplasm as revealed by SSR markersPhylogenetic relationships among or within16accessions collected from Asia were investigated using SSR markers. The21SSR primer pairs generated a total of143polymorphic alleles, with an average allele per locus of6.8. The average polymorphic rate (P) for this species was90.7%. The polymorphic information content (PIC) ranged from0.17(A01F24) to0.45(A01E02) with an average of0.33. And the Shannon’s information index of diversity (Ⅰ) ranged from0.0463to0.3547, suggesting a high degree of genetic diversity. Analysis of molecular variation (AMOVA) revealed larger genetic variation within accessions (65.75%) and geographical regions (75.58%) rather than between them, while the ploidy level variance among the accessions contributed only2.62%. The16accessions were grouped into two major clusters (GI=0.876). Specifically, all tetraploid accessions originating from different regions were grouped into the same cluster whereas the diploid accessions were grouped into the two major clusters. The dendrogram was concordant with the morphological variability, agronomic traits and karyotype.2. Genetic linkage maps of diploid orchardgrass (Dactylis glomerata L.)The objective of this study was to construct a diploid (2n=2x=14) orchardgrass genetic linkage map useful as a framework for basic genetic studies and plant breeding. A combination of simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular markers were used for map construction. The linkage relationships among164simple sequence repeats (SSR) and108sequence-related amplified polymorphism (SRAP), assayed in a pseudo-testcross F1segregating population generated from a cross between two diploid parents were used to construct male (01996) and female (YA02-103) parental genetic maps. The paternal genetic map contained90markers (57SSR markers and33SRAP) over9linkage groups and the maternal genetic map was comprised of87markers (54SSR and33SRAP) assembled over10linkage groups. The total map distance of the male map was866.7cM representing81%genome coverage, whereas the female map spaned772.0cM, representing75%coverage. The average map distance between markers was9.6cM in the male map and8.9cM in the female map. The level of segregation distortion observed in this cross was15%. Homology between the two maps was established between five linkage groups of the male maps and five of the female map using10bridging markers. The information presented in this study establishes a foundation for extending genetic mapping in this species and will serve as a framework for mapping QTLs and provide basic information for further molecular breeding studies.3. A genetic linkage map of tetraploid orchardgrass (Dactylis glomerata L.) and QTL analysis for heading dateA combination of EST-based SSR markers and AFLP markers were used to genotype an F1population of284individuals derived from a very late heading D. glomerata ssp. himalayensis parent (him271) and an early to mid-heading D. glomerata ssp. aschersoniana parent (asch621). Two parental maps were constructed with28cosegregation groups and seven consensus linkage groups each, and homologous linkage groups were tied together by38bridging markers. The him271map consisted of183markers with a total length of974cM, the mean density between markers was5.2cM. The asch621map consisted of193markers and spanned a total length of1038cM with mean density between markers of5.5cM. Parental linkage group lengths varied from98to187cM. All but two mapped SSR markers had homologies to physically mapped rice (Oryza sativa L.) genes, and seven orchardgrass linkage groups were assigned based on this putative synteny with rice.7QTLs were detected for heading date on linkage groups2,5, and6in both parental maps, explaining between7.85%and24.19%of the penotypic variation. These results laid a theoretical foundation for further molecular assisted selection (MAS) breeding, improvement of important agronomic traits, later-mature cultivar release for mixture pasture and functional and comparative genetic analysis.4. Flowering gene identification and mapping in orchardgrassThe conserved sequences of flowering candidate genes (VRN, Hd, FT) in rice, wheat, ryegrass and tall fescue were used to design28baits. Solution capture method was employed to capture flowering genes in orchardgrass. Approximately21,000reads were generated from454sequencing, which were assembled into2,094contigs varying in size from105bp to1975bp. After BLASTn searches, contig404, contig441and contig558had hits to these bait sequences, including the Vrn, Hd, and FT candidate genes.20pairs of candidate gene primers were designed and amplified in F1mapping population to test segregation ratio. Finally, Vrn3with1:1segregation ratio was successfully mapped on LG3of the him271parental map.更多还原