基于 RAD-seq 的菜心 InDel 标记开发及应用

【目的】开发多态性丰富的的InDel分子标记,为菜心育种提供研究基础。【方法】以Chiifu-401-42的基因组序列为模板,利用4份菜心材料的RAD-seq重测序数据,在全基因组范围内鉴定InDel位点。利用生物信息学方法筛选4份菜心材料间具有潜在多态性的InDel位点,挑选分布于10条染色体的80个InDel位点设计引物,对55份菜心种质材料进行遗传多样评价。【结果】通过与参考基因组序列比对,在4份菜心材料的重测序数据中共鉴定出84 510个InDel位点,其中插入/缺失长度大于5 bp的3 609个InDel位点在4份菜心材料间具有潜在多态性。挑选80个InDel位点进行PCR扩增验证,58个（72.5%）在4份测序样品中具有多态性,在55份菜心种质材料中检测到133个等位位点,多态性信息含量（PIC）为0.263～0.618,平均值为0.443。基于InDel标记的检测结果可知,55份菜心种质的遗传相似系数在0.366～0.756,平均值为0.570;在遗传相似系数为0.564时55份菜心种质被分为4个类群,但各类群间的耐热性没有明显差异。【结论】本研究开发的InDel标记具有...     

16种观赏石斛兰的遗传关联性分析

石斛兰种质资源的评价与创新是育种工作的基础。为了挖掘石斛兰资源有效的分子标记以及优良目的性状相关基因,本研究主要观测了16份石斛兰种质资源形态学性状以及利用RAD-seq分子标记技术分析这些品种资源之间的遗传进化关系,对16种观赏石斛兰形态学与RAD-seq分子标记进行了关联分析。结果表明,形态学分析显示16个石斛兰品种间遗传变异较大,相似系数在0.776～0.926之间;与形态学结果相比,16个石斛兰品种RAD-seq的测序结果显示种间遗传差异更大,与参考基因组相比相似系数在0.252 2～0.898 2之间。RAD-seq聚类分析表明,聚石斛、霍山石斛和金钗石斛亲缘关系较近;形态学关联分析发现,这3个石斛兰品种都具有花香且花序侧生现象,其他石斛兰品种没有此特征。对这3个石斛兰品种区别于其他品种的SNP标记位点相关序列进行提取和注释,发现了大量的次生代谢途径和光合作用相关基因。16个观赏石斛兰品种间存在较丰富的遗传多样性,其中聚石斛、霍山石斛和金钗石斛亲缘关系较近,并发现分类结果可能与花香和花序侧生现象相关。     

Construction of high-density linkage map and identification of QTLs associated with resistance to black rot in radish (Raphanus sativus) by RAD sequencing

High-density marker-based QTL mapping can serve as an effective strategy to identify novel genomic information to facilitate crop improvement. In this study, we genotyped an F₂ population (KB12-1 × PP12-1) using a RAD-seq approach and constructed a high-density linkage map for radish. After a series of filtering procedures were performed, 17,124 SNPs and 3,336 indels with aa × bb genotyping were retained to obtain bin markers. Then, a linkage map comprising a total of 1,221 bin markers in nine linkage groups spanning 1,467.3 cM with an average marker interval of 1.2 cM was constructed. We evaluated the resistance of the F₂ mapping population to black rot using F₃ progeny, and two major QTLs related to black rot resistance were identified based on this map. Among these QTLs, qBRR2 on Chr.2 explained 26.97% of the phenotypic variation with a LOD score of 11.93, and qBRR7 on Chr.7 accounted for 27.06% of the phenotypic variation with a LOD score of 11.83. The additive effect of qBRR2 was positive (14.97); however, qBRR7 had the opposite effect (−11.99). The high-density linkage map and the major QTLs qBRR2 and qBRR7 provide new important information for disease resistance gene discovery and utilization in genetic improvement.     

Mapping of QTLs controlling epicotyl length in adzuki bean (Vigna angularis)

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F₂ population derived from a cross between the breeding line ‘Tokei1121’ (T1121, long epicotyls) and the cultivar ‘Erimo167’ (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F₅ residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.     

基于RAD-seq简化基因组测序的19个地方鸡种遗传进化研究

旨在基因组水平上揭示地方鸡种的遗传进化,发掘重要种质特性基因。本研究利用简化基因组RAD-seq测序鉴定19个地方鸡种（每个品种按照家系选取30个个体,10公、20母）基因组SNP标记,计算观察杂合度（Ho）、核苷酸多样度（Pi）、近交系数（Fis）、遗传分化系数（Fst）和基因流（Nm）等遗传统计量指标,分析地方鸡种的遗传多样性和遗传结构,并通过选择信号检测鉴定基因组受选择基因。结果表明,在19个地方鸡种中鉴定出400 562个SNPs标记。瓢鸡（PJ）和文昌鸡（WC）的遗传多样性最为丰富,观察杂合度（Ho）分别为0.246 8、0.243 0,核苷酸多样度（Pi）分别为0.278 1、0.265 5;河南斗鸡（DJ）的遗传多样性相对匮乏,Ho为0.156 0,Pi为0.175 2;东乡绿壳蛋鸡（DX）和边鸡（BJ）的近交系数最高（Fis>0.160 0）。瓢鸡与文昌鸡、惠阳胡须鸡（HX）、藏鸡（ZZ）、大围山微型鸡（WX）,惠阳胡须鸡与文昌鸡,藏鸡与茶花鸡（CH）间的遗传分化最低（Fst<0.100 0）,对应的基因流最高（Nm>0.240 0）。河南斗鸡与其它品种间的遗传分化均处于较高水平,与其中15个品种间的Fst>0.200 0、Nm<1.000 0。遗传聚类分析（2个引入品种做外群）将地方鸡种总体上分为5类,与品种形成历史和地理分布基本吻合。通过选择信号分析,在19个地方鸡种合并群体中检测出9个常染色体上的26个区域受到选择作用,包含31个受选择基因。这些受选择基因广泛参与免疫系统调节、生殖机能调控、应激响应、代谢等生物学过程。利用基因组SNP标记能更全面准确地揭示地方鸡种的遗传多样性和遗传结构,选择作用主要体现在对地方鸡种抗逆抗病特性、配子活力及行为等方面的塑造。     

基于RAD-seq的荸荠SSR标记开发

荸荠(Eleocharis dulcis)是一种重要的特色水生蔬菜,为开发用于荸荠遗传学研究的简单重复序列(SSR)分子标记,本研究基于RAD-seq技术对荸荠进行简化基因组测序,并进行SSR标记开发与引物设计。结果显示,共检测到5039个SSR位点,其中4127个SSR位点可利用并成功设计引物。在可利用的SSR位点中,三碱基重复基序类型所占比例最高,数量为1894个,占位点总数的45.89%;其次是二碱基重复基序类型,数量为1406个,占位点总数的34.07%。随机选取了100对引物进行有效性验证,扩增成功率为93%,从扩增成功的引物中选取83对引物对两个品种进行多态性检测,83对引物共得到232条条带,其中多态性条带为128条,具有多态性的引物为60对,多态性比率为72.28%。通过RAD-seq开发的荸荠SSR标记有效性和多态性较高,为后期荸荠种质资源的高效利用、品种的遗传改良及分子育种提供了基础。     

基于简化基因组的甜叶菊分子标记开发

本研究旨在筛选和开发具有多态性的甜叶菊基因组分子标记。选取4个甜叶菊品种为材料,利用简化基因组测序技术(RAD-seq)开发SSR和SNP标记。开发并验证了149对多态性丰富、重复性好、特异性高的简单重复序列(SSR)和4组功能性的SNP标记,开发的多态性基因组分子标记中包含了甜叶菊糖苷合成途径关键基因的新标记StKASPSNP-7。验证了基于KASP技术开发甜叶菊功能SNP标记的可行性,为甜叶菊遗传图谱构建、分子鉴定奠定了基础。本研究开发的SSR和SNP标记可应用于甜叶菊的遗传图谱分析、品种真实性的高效快速鉴定以及高糖苷含量新品种的开发。     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.     

印尼虎鱼RAD-seq数据中微卫星标记的初步筛选及特征分析

采用RAD-seq(Restriction-site associated DNA sequencing)对印尼虎鱼(Datnioides pulcher)进行简化基因组测序,获得13308 806个高质量干净读长(HQ clean reads),利用SSR搜索软件在所得序列中共检测到26359个SSR位点,由496种重复基序组成,主要分布在三、四、五碱基重复类型中,单碱基重复序列中最多的类型为A/T,二碱基重复序列中以AC/GT和AG/GT重复单元为主,三碱基重复序列中以AGG/CCT、AGC/CTG、AGG/CCT为优势类型;在数量上,二碱基重复类型SSR位点最多(19492个),占总SSR位点的73.95%;除了单核苷酸类型外,SSR基序的重复次数主要集中在4-9之间,多态性SSR数目为2 783个。根据SSR位点两翼序列,利用Primer5.0成功设计出20 066对SSR引物,引物设计成功率为76.13%,依据测序数据中的重复类型和重复数,随机选择100个二至五碱基重复类型SSR在6个虎鱼样本中验证其可用性和多态性,有73对引物通过扩增可获得有效目的片段,其中20个SSR验证为具有多态性。开发所得SSR序列可用于印尼虎鱼及其近缘物种遗传分析、遗传图谱构建以及分子标记辅助育种等工作,同时也证实了利用简化基因组测序技术开发SSR标记和发现样品间具有多态性的SSR标记的可能性。