【作者】 蔡彩平；

【导师】 郭旺珍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 高密度遗传图谱的构建是植物基因组研究的基础。富含功能标记的高密度遗传图谱为准确认识基因组结构,发掘功能基因,定位和利用重要经济性状相关QTLs奠定了基础,同时也为进一步开展基因组进化研究提供了前提条件。在已有研究基础上,本研究开展了棉花新型分子标记的开发和高密度遗传图谱的构建研究。本研究利用新开发的源于三个棉种(雷蒙德氏棉、陆地棉及海岛棉)的eSSRs、陆地棉Maxxa的gSSRs、基因的RT引物,网上公开释放的部分STV及Gh编号的引物,共计3592对,以及组配的部分REMAP标记,在作图亲本TM-1及Hai7124之间筛选多态性,进而完成图谱的加密工作。整合后的图谱包含2577个位点,覆盖了3591.0 cM的遗传距离,标记间的平均遗传距离为1.39 cM,全部分布于26条染色体上。图谱有四大特点：：(1)富含功能标记。图谱中功能标记占总位点数的57.04%,eSSR位点占功能标记的78.44%；(2)明确四倍体栽培棉种中A亚组存在两个相互易位,涉及A2／A3和A4／A5四条染色体。在棉花基因组的13个部分同源群上共检测到180对SSR引物扩增产生的重复位点,在A2／D3,A3／D2,及A4／D5,A5／D4上共检测到28对重复位点；(3)存在四个偏分富集区。图谱中含偏分离位点220个,占总位点数的8.54%,在A2、A3、A7和D7上存在4个大的偏分富集区,且每一染色体上的偏分离富集区其偏分离方向一致；(4)是一张基于四倍体栽培棉种的高密度遗传图谱,可直接用于QTL定位及相关农艺性状分析等后续研究。通过比较四个棉种中所有随机设计的eSSR引物及源于陆地棉Maxxa的gSSR引物的重复基元类型、分布及其多态性之间的关系,发现在eSSRs中,重复基元最多的为三核苷酸重复；在gSSRs中,重复基元最多的为六核苷酸重复。四核苷酸重复在不同来源的SSR中所占的比例均较少；在源于四个棉种的eSSRs中,复合型及四、六核苷酸重复基元的多态率比较高；在多态引物中,所占比例最多的重复基元本身的多态率并不是很高,二核苷酸重复基元中,AC／TG的多态率较高,四核苷酸重复基元中,ATAC／TATG的多态率较高。比较了四个棉种中eSSR位点在图谱中的分布情况。发现：源于亚洲棉的eSSR.位点,At：Dt=1.5：1；源于雷蒙德氏棉的eSSR位点,At：Dt=1：1.5；源于陆地棉及海岛棉的eSSR位点,At：Dt=1：1。也就是说,源于A基因组棉种亚洲棉的eSSR位点在At亚组中检测到的要多,源于D基因组棉种雷蒙德氏棉的eSSR位点在Dt亚组中检测到的要多,源于四倍体棉种陆地棉及海岛棉的eSSR位点均匀分布在At、Dt亚组中。通过比较同一引物在TM-1、Hai7124及Maxxa中扩增产物分子量的大小,参照其在二倍体祖先棉种阿非利加棉及雷蒙德氏棉中扩增条带的特点,并结合该引物在图谱中的定位结果,将37个BACs准确定位到相应的亚组中。其中At亚组中有12个,Dt亚组中有25个。根据陆地棉Maxxa 97个BAC克隆中的344组LTR保守区域设计了14个单引物,与同一来源的60对SSR引物随机组配为REMAP标记,共产生188个多态位点,检出率高达11.19%,这些位点遍布在四倍体棉种整个基因组中。初步建立了棉花REMAP分子标记技术体系。通过共有的SSR标记将本实验室18个陆地棉图谱整合为一张高密度的陆地棉种内遗传图谱。该图谱包含67个连锁群,694个位点,覆盖了3552.8 cM的遗传距离,标记间的平均遗传距离为5.12 cM,并将67个连锁群全部定位到相应的26条染色体上,几乎覆盖了整个四倍体棉种基因组。研究了源于5个高强纤维供体(7235、HS427、PD6992、渝棉1号及J415)的95个优质纤维QTL的分布特点,结果显示：Dt亚组上比At亚组上检测到的优质纤维QTL要多,且更为集中。说明尽管At亚组的祖先产生可纺织的纤维,而Dt亚组的祖先不产生有价值的纤维,但Dt亚组的祖先在纤维生长发育及多倍体棉种进化过程中扮演着非常重要的角色。这是迄今为止拥有多个遗传背景且最为高密度的陆地棉整合遗传图谱,该图谱为棉花产量、棉纤维品质及抗性相关QTL的定位奠定了基础,并将进一步应用到陆地棉的分子设计育种中。更多还原

【Abstract】 A high-density molecular map is the basement of genome research in plant. A saturated genetic map shares new light to our understanding of structural genomics, and is useful in the novel gene discovery, economically important QTLs analysis, et al. In this paper, we were mining DNA molecular markers and constructing high density genetic mapping in cotton.Total 3592 primer pairs were developed, including eSSRs from G. raimondii, G. hirsutum, G. barbadense-derived ESTs, gSSRs from 70 BAC clones of G. hirsutum cv. Maxxa and RT primers, REMAP maker, STV, Gh primers. They were used to screen polymorphisms in order to enhance our backbone genetic map in allotetraploid cotton. After integrating these new loci, our enhanced genetic map consists of 2577 loci covering 3591.0 cM, at 1.39 cM intervals in 26 linkage groups. This microsatellite-based, gene-rich linkage map contains 57.04% functional marker loci, of which 78.44% are eSSR loci. There were 208 duplicated loci, bridging 13 homoeologous At/Dt chromosome pairs. Two reciprocal translocations after polyploidization were further confirmed between A2 and A3, and between A4 and A5 chromosomes. Four big distorted intervals were found in the A2, A3, A7 and D7 chromosome. All distorted loci in the same chromosome were skewed towards the heterozygotes or homozygotes. This high-density cultivated cotton species genetic map will be used for QTL tagging and agronomic traits analysis.At the same time, relationship was observed between the level of polymorphism, motif type of the randomly developed eSSRs and gSSR from BAC clones of G. hirsutum cv. Maxxa. Among the eSSRs, trinucleotide are the most abundant motif, tetranucleotide are the least abundant motif. For the gSSRs, hexanucleotide are the most abundant motif, tetranucleotide are the least abundant motif. Their eSSRs polymorphic rates were higher for compound motif, tetranucleotide, hexanucleotide repeats. We also observed the polymorphic rate which the most abundant motif type was not high. AC/TG was the higher polymorphic rate in dinucleotide repeat, ATAC/TATG was the higher polymorphic rate in tetranucleotide repeat.A comparison of the eSSRs tagging information derived different genome showed that eSSR derived from G. arboretum were tagged At:Dt=1.5:1, derived from G raimondii were tagged At:Dt=1:1.5, derived from G. hirsutum and G. barbadense were tagged At:Dt =1:1. The tagging results showed that eSSRs derived from A genome species were preferentially tagged in the A-subgenome, and eSSRs from D genome species were preferentially tagged in the D-subgenome in the tetraploid linkage map. However, eSSRs derived from AD genome species were equally tagged in the A-and D-subgenomes of the tetraploid linkage map.By comprehensive analyses of the amplified product molecular size among tetraploid G. hirsutum cv. Maxxa, acc. TM-1, and G. barbadense cv. Hai7124, and diploid G. herbaceum var. africanum and G. raimondii,37 BACs were further anchored to their corresponding subgenome chromosomes, showing 12 BACs from the A-and 25 from the D-subgenome.14 primers were developed from 344 LTRs of 97 BAC clones in G hirsutum cv. Maxxa.60 gSSRs developed from 70 BAC clones were randomly assembled with 14 primers. Their REMAP maker produced 188 polymorphic loci, evenly distributing in the whole genome. REMAP technique was preliminarily constituted in cotton.Using SSR as bridge markers, a saturated intraspecific genetic linkage joinmap was constructed from 18 different mapping populations of Upland cotton (Gossypium hirsutum L.). The new integrated Upland cotton intraspecific genetic linkage joinmap comparises 694 loci, that mapped to 67 linkage groups assigned perfectly to their corresponding 26 chromosomes with the average distance between markers of 5.12 cM, covering 3553 cM or nearly 100% of the total recombinational length of the tetraploid cotton genome. Further, elite fiber QTL from five super fiber quality donors,7235, HS427, PD6992, Yumian 1 and J415, were aligned on the joinmap and showed that more elite QTL were biased non-randomly on Dt subgenome than on At subgenome, even if At progenitor produces spinnable fiber but Dt progenitor does not. The result suggested that the Dt subgenome from the non-fiber-production ancestor plays an important role in the genetic control of fiber growth and development in polyploidy cotton. So far, this is the most saturated genetic linkage joinmap assembled from different genetic backgrounds of cotton populations in G hirsutum. The research is ongoing for the identification of QTL allele and homoelogous relationship for breeding target traits, such as yield, fiber quality and resistance etc, and further provides the foundation for molecular breeding by design in G hirsutum.更多还原