芒属植物叶绿体InDel标记的开发与应用

研究利用芒属植物(芒02381和荻04005)叶绿体全基因组测序的结果,开发12对InDel标记。采用正交设计法优化cpInDelPCR体系,得到模板DNA、dNTPs、Mg2+、引物、TaqDNA聚合酶5个关键因素在体系中的最佳组合。12对InDel标记对43份芒属和2份甘蔗材料进行扩增,共扩增出538条条带,其中3对引物扩增出13条多态性条带,占总数的2.4%。将所有位点读带转化为数值矩阵并用UPGMA法聚类,在遗传相似系数1.4的水平上,和南荻聚为一类,而芒和与五节芒、双药芒、红山茅、尼泊尔芒、甘蔗聚为一类,4个具有杂交种特征的材料单独聚为一类。试验开发的芒属植物cpInDel标记可用于芒与荻的区分,指导芒属植物的杂交育种。     

Development and Application of Cotton InDel Markers Based on High Throughput Sequencing

[Objective] Dimorphic InDel markers can be used for cotton variety identification and purity detection, to improve the accuracy and efficiency of cotton seed testing, and to play a role in molecular breeding of cotton. [Method] Based on the whole genome sequencing of 121 cotton varieties from different sources, the InDel markers with high polymorphism were developed according to polymorphism information content(PIC) and were applied in the genetic distance analysis and cluster analysis by using 66 cotton varieties in China. [Result] Totally 10 967 InDel were identified based on the next generation sequencing data of 121 cotton varieties. Among the 85 pairs of InDel primers synthesized, 64 were selected including 35 from At group and 29 from Dt group. The minimum average allelic frequency(MAF) of At and Dt chromosomes were 0.45 and 0.32, respectively, while the PIC were 0.49 and 0.40, respectively. The genetic distances of the 66 cotton varieties ranged from 0.04 to 0.65 centimorgan (cM), with an average of 0.39 cM. The two varieties with the largest genetic distance were Simian 3 and CCRI 36, and the two varieties with the smallest genetic distance were Xumian 18 and Xuza 3. [Conclusion] The 64 cotton dimorphic InDel markers can effectively reveal the relationships among varieties based on the genetic distance, and distinguish cotton varieties from different sources, which has certain theoretical significance and application value.     

白菜基因组导入芥菜的远缘杂交及后代分离偏向性

【目的】研究远缘杂交将结球白菜的A基因组导入包心芥菜,利用高分辨率熔解曲线(high-resolution melting, HRM)结合InDel标记辅助选择技术,筛选出保留白菜基因较多的回交后代,改良包心芥的结球性,丰富包心芥菜的种质资源。【方法】利用幼胚培养技术辅助6份包心芥菜与2份结球白菜进行远缘杂交和回交,选取20个InDel标记追踪白菜A基因组全部染色体,从192的BC₁单株中筛选出白菜A基因组标记占比较高的单株。田间调查筛选结球性近似结球白菜的单株。【结果】远缘杂交结合幼胚培养获得了9个种间杂种组合和14个BC₁群体。筛选并获得203个在全基因组分布均匀的可区分白菜与芥菜的HRM-InDel标记;利用20个InDel标记追踪192株BC₁单株的白菜基因,分离群体偏向传递白菜基因组,有54棵单株标记含量超过90%。【结论】幼胚培养技术可提高白菜与芥菜远缘杂交后回交的结实率,并可加快育种进程;筛选出了白菜基因组占比较高的组合和单株;分离群体明显偏向于传递白菜基因。     

樱桃番茄种质资源亲缘关系的InDel和SSR标记分析

选用18对InDel引物和15对SSR引物对30份樱桃番茄高代自交系育种材料进行遗传多样性分析。多态性分析结果表明,InDel标记比SSR标记的多态性低;遗传距离分析结果表明,30份樱桃番茄种质材料两两间的遗传距离(GD)在0.052~0.993之间,平均遗传距离仅为0.391,遗传距离小于0.5的种质材料占到了77.24%。在相似系数为0.65的水平上,可将30份樱桃番茄材料分为3类。     

Development ofNew InDel Marker to Detect Genotypes of Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility inRice简

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility (CMS) in japonica rice, a functional marker InDel-Rf-1a based on the difference of nucleotide sequence in Rf-1a locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-1arf-1a showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-1a locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F₂ segregation population, and three genotypes in Rf-1a locus could be distinguished distinctly. Therefore, the marker InDel-Rf-1a could be widely used for genetic identification and marker-assisted selection (MAS) in breeding japonica restorer lines.     

用于黄瓜白粉病抗性鉴定的InDel标记

根据黄瓜白粉病抗性主效基因/QTL的插入/缺失突变,将其设计成共显性的In Del分子标记。通过群体连锁分析,发现In Del-MLO1标记与黄瓜白粉病抗性主效基因共分离。应用该标记对24份来自世界各地且白粉病抗性已知的黄瓜材料进行抗性验证,21份材料的抗性表型与标记的带型一致,说明在黄瓜进化和驯化过程中,造成抗病表型的该插入/缺失突变发生普遍。因此,该In Del标记适合于大多数黄瓜材料的白粉病抗性鉴定和分子标记辅助育种,从而加快黄瓜白粉病抗病育种的进程。     

基于SNP和InDel标记的巴西木薯遗传多样性与群体遗传结构分析

As a typical tropical crop, cassava (Manihot esculenta Crantz) has the characteristics of drought resistance, barren resistance, high biomass and so on. In addition to being used for food and forage, it can also be used for production, processing and starch extraction. Due to highly heterozygous cassava genome, breeding is more difficult. Enriching the genetic diversity of cassava germplasm, comprehensively evaluating its genetic background and traits, and discovering superior alleles that control excellent traits are of great significance for cassava breeding in the future. In order to analyze the genetic diversity, genetic relationship and population structure of cassava germplasm in Brazil, 7946 SNPs and 1997 InDels molecular markers were used. Population structure analysis was performed by ADMIXTURE software, and principal component analysis was performed by GCTA software. Brazilian cassava was divided into nine subgroups, and was roughly consistent with the results of cluster analysis using PHYLIP. Among them, subgroup 1, subgroup 2, subgroup 4, subgroup 6, and subgroup 8 could be clustered together respectively, while the samples of other subgroups could be roughly clustered, and there was a certain cross between the samples. The genetic diversity of cassava germplasm in Brazil (0.274) was higher than the genetic diversity level of cassava germplasm in China and Nigeria. Subgroup 5 of Brazil cassava had a relatively high genetic diversity (0.29). The genetic differentiation of subgroups was low (the genetic differentiation vary from 0.03 to 0.15), but higher than domestic cassava germplasm. The genetic distance between cassava accessions varied from 0.084 to 0.297, with the average of 0.228. The results of this study can provide a basis for subsequent association analysis to identify great alleles and introduction.     

中国樱桃InDel标记开发及其在蔷薇科果树中通用性评价

对栽培和野生中国樱桃进行De novo基因组测序和比对，在全基因组范围内检测筛选出50 617个InDel位点，并挑选均匀分布的200个标记进行验证，其中，27个标记在中国樱桃种内表现出多态性。利用多态性标记，对不同来源的192份中国樱桃（168份栽培和24份野生）种质进行基因型分型，共检测到60个等位基因（N_a），平均每个标记2.2个；基因多样性指数（H_s）为0.010 ~ 0.500，平均0.239；多态性信息含量（PIC）为0.010 ~ 0.375，平均0.198，表明中国樱桃遗传基础相对狭窄。根据聚类结果和地理分布将192份中国樱桃种质大致划分为5个群体。筛选出在蔷薇科李亚科樱属、李属、桃属、杏属、苹果亚科梨属、苹果属和蔷薇亚科草莓属、悬钩子属等重要果树共8个属156个个体中表现出较好通用性标记34个。物种与中国樱桃亲缘关系越近，InDel标记的多态性越高。     

利用InDel技术评价甜菜品种的一致性

为了探索InDel分子标记技术在鉴定甜菜品种一致性中的可行性，本研究以8个甜菜品种为试材，每个品种取24个单株，分别提取基因组DNA，利用9对InDel引物对每个甜菜品种的24个单株分别进行InDel-PCR扩增，根据扩增条带分析每个甜菜品种的一致性分布特征。结果表明8个甜菜品种所有位点的平均一致性比率在67.7%~88.1%之间。单一引物在不同品种中表现出的一致性比率具有一定差异。例如，利用ND286引物在品种985和品种1123中检测出的一致性比率为100%，但是在其他品种中检测出的一致性比率分布在46%~63%之间。不同引物在同一品种中表现出的一致性比率可能相同，但其区分出的不一致植株可能相同也可能不同。比如，在平均一致性最好的品种1015中，ND31和ND71引物在24个植株中检测出的一致性比率相同，但ND31引物区分出的差异植株为13、14和15号植株，而ND71引物区分出的差异植株为8、15和21号植株。因此利用单个位点引物对甜菜品种一致性进行鉴定不具有普遍性和科学性，应该综合多个位点的表现进行一致性鉴定，且现阶段甜菜品种一致性鉴定的标准不宜设置过高。     

Localization of a Dwarfing Mutation in Upland Cotton Using InDel Markers Based on Genome Re-Sequencing Data

[Objective] InDel markers that were developed by comparing whole genome re-sequencing data were used to map the AS98 gene of a dwarf mutant of upland cotton (Gossypium hirsutum L.). [Method] An F2 segregation population was constructed from an upland-cotton dwarf mutant (AS98) and its wild type (LHF10), and InDel markers were developed based on genome re-sequencing data from the two parents. The InDel markers and the F2 generation were then used to map the AS98 gene of the dwarf mutant. [Result] A total of 114 InDel markers located in a previous mapping region were screened and used to genotype 223 F2 individuals. Ultimately, phenotypic data and 20 polymorphic primer pairs were used to construct a genetic linkage map. Molecular marker analysis mapped AS98 within a 6.9 cM distance of the marker InDel-50 on chromosome D12. [Conclusion] This study has demonstrated the feasibility of using InDel markers developed from re-sequencing data for gene localization in isolated populations derived from upland cotton dwarf materials. The resulting gene map provides a basis for future fine mapping and molecular-assisted selection breeding.