梨遗传连锁图谱的构建与比较分析

【目的】利用已公开发表的梨和苹果的SSR（Simple Sequence Repeat）引物以及从梨转录组开发的SSR引物构建本研究作图群体的遗传连锁图谱,为后期梨重要性状QTL定位和分子标记辅助选择等奠定基础。【方法】以西洋梨品种‘红茄’（Red Clapp Favorite）为母本,东方梨品种‘晚秀’（Mansoo）为父本,构建F1代作图群体。将所选用的SSR引物在亲本和4个子代个体进行PCR扩增,初步筛选出扩增结果符合JoinMap 4.0软件中“CP”作图模式要求的引物,随后在F1群体中检测,选用JoinMap 4.0软件对分离数据进行连锁分析,分别构建亲本的连锁图谱。以双亲图谱在各连锁群上的同源标记作为锚定位点,对双亲图谱进行整合。【结果】利用PCR技术对不同来源的共909对SSR引物（526对梨和283对苹果公开发表的SSR引物,从梨转录组开发的100对SSR引物）进行初步筛选后,发现来自苹果的SSR引物有效扩增片段的比例和多态性均较低,而来自梨和梨转录组开发的SSR引物相对较高。筛选出207对符合作图要求的SSR引物在群体中扩增,构建亲本的连锁图谱。母本图谱中的141个标记分布在17个连锁群上,总长度757.34 cM,标记间平均5.37 cM;父本图谱中的153个标记分布在19个连锁群上,总长度1 149.43 cM,标记间平均7.51 cM。【结论】对不同来源的SSR引物构建的双亲连锁图谱进行整合,最终得到一张由186个SSR标记,覆盖基因组长度1 125.33 cM的整合图谱。     

马铃薯基因组计划研究进展

马铃薯是重要的茄科作物,在自然界中存在二倍体、三倍体、四倍体、五倍体和六倍体,同时是块茎形成、淀粉合成、对病原物应答研究的模式植物。由于茄科植物基因组存在高度的共线性,为此,开展番茄基因组计划及功能基因组学研究可以为其他蔬菜、水果等重要经济作物的研究提供理论和方法上的直接借鉴。从马铃薯的遗传图谱、物理图谱、序列图谱和转录图谱,以及已从马铃薯中克隆的重要基因等方面详细阐述了马铃薯基因组研究的最新进展。     

应用Charleston×东农594重组自交系群体构建SSR大豆遗传图谱

 以美国半矮杆大豆品种Charleston为母本，东北农业大学高蛋白大豆品系东农594为父本及其F2:10代重组自交系的154个株系为试验材料，利用164个在亲本之间表现多态的SSR引物对群体进行了分析，构建了一张大豆遗传图谱。该大豆遗传图谱总长度1 913.5 cM，标记间平均距离为11.89 cM。每个连锁群长度变动在0.4～309.5 cM之间，连锁群上的标记数在2～28个之间。各连锁群上的SSR标记并不是均匀分布的，其中A1、C2、D1a三个连锁群存在标记密集区。与国内外已构建完成的5张大豆遗传图谱比较表明，该图谱与国外的大豆公共遗传图谱对应性较好。     

Construction of Soybean Genetic Map with RIL Population by Charleston ×Dongnong 594

F2:10 RIL population with 154 lines, crossed by Charleston as female parent and Dongnong 594 as male parent were used.164 SSR primers were screened with the two parents and amplified on the 154 lines. A new soybean molecular genetics map, named NEAUSRI-GMS, was constructed by Mapmaker. The total length of the soybean genetic map is 1 913.5 cM,and the average distance among markers is 11.89 cM. The length of linkage group varied from 0.4 to 309.5 cM, and the markers on the linkage group varied from 2 to 28. The distribution of SSR markers on every linkage group is not even. High density region of markers existed on linkage group Al, C2, and Dla. Compared with 5 soybean genetic maps constructed at home and abroad, NEAUSRI-GMS has high homologous with the public genetic map abroad.     

Construction of Genetic Linkage Map Based on SSR Markers in Peanut(Arachis hypogaea L.)

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci (QTLs) for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines (RILs) derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut (Arachis hypogaea L.). A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut (Arachis hypogaea L.). The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.     

Construction of Genetic Linkage Map Based on SSR Markers in Peanut （Arachis hypogaea L.）

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci （QTLs） for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines （RILs） derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut （Arachis hypogaea L.）. A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut （Arachis hypogaea L.）. The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.     

An Integrated QTL Map of Fungal Disease Resistance in Soybean(Glycine max L.Merr):A Method of Meta-Analysis for Mining R Genes

Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world.Conflicting results of fungal disease resistance QTLs from different populations often occurred.The objectives of this study were to:(i)evaluate evidence for reported fungal disease resistance QTLs associations in soybean and(ii)extract relatively reliable and useful information from the "real" QTLs and mine putative genes in soybean.An integrated map of fungal disease resistance QTLs in soybean was established with soymap 2 published in 2004 as a reference map.QTLs of fungal disease resistance developed from each of separate populations in recent 10 years were integrated into a combinative map for gene cloning and marker assisted selection in soybean.107 QTLs from different maps were integrated and projected to the reference map with the software BioMercator 2.1.A method of meta-analysis was used to narrow down the confidence interval,and 23 "real" QTLs and their corresponding markers were obtained from 12 linkage groups(LG),respectively.Two published R genes were found in these "real" QTLs intervals.Sequences in the "real" QTLs intervals were predicted by GENSCAN,and these predicted genes were annotated in Goblet.228 resistance gene analogs(RGAs)in 12 different terms were mined.The results will lay the foundation for a bioinformatics platform combining abundant QTLs,and offer the basis for marker assisted selection and gene cloning in soybean.     

SNPS在大豆等作物遗传及改良中的应用

单核苷酸多态性(singlenucleotidepolymorphism,SNP)是指DNA序列上的单个碱基变异,它具有分布广、多态信息量大、易于检测和统计分析等优点,被称为继RFLP和微卫星标记后的第三代基因遗传标记。单核苷酸多态性是等位基因间序列差异最为普遍的类型,可作为一种高通量的遗传标记。已建立PCR扩增目标序列及其产物测序和电子SNP(eSNP)等多种发现和检测SNP的方法。大豆等作物也已开展了SNP分析。一些栽培作物种质的多样件不断减少,其结果连锁不平衡(linkagedise鄄quilibrium,LD)增加,这有利于目的基因座上SNP单元型(haplotype)与表型的相关性分析。SNP已在作物基因作图及其整合、分子标记辅助育种和功能基因组学等领域展示了广泛的应用价值。     

禾谷类作物的比较基因组研究

 水稻是基因组最小的禾谷类作物,饱和遗传连锁图谱的构建,以及在此基础上开展的标记辅助选择和抗病基因克隆,表明水稻基因组研究已经领先于其他禾谷类作物。比较基因组研究表明：小麦、玉米、高粱、谷子和甘蔗的基因组均可由水稻染色体区段重新排列而成，这些区段上DNA标记的排列顺序在各个种之间保留。各种作物基因组大小的差异可能由于各个区段内基因间重复顺序扩增的程度不同所致。根据这些区段在各种作物染色体的排列顺序，有人提出根据水稻染色体区段排列单个原始禾谷类染色体的设想，为深入研究禾谷类作物的进化遗传提出了全新的思路。禾谷类作物基因组之间的共线性有利于在小基因组内克隆大基因组作物的同源基因，使生物技术在作物育种中发挥更大的作用。     

Intron-Targeted Intron-Exon Splice Conjunction （IT-ISJ） Marker and Its Application in Construction of Upland Cotton Linkage Map

To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.     

An Integrated QTL Map of Fungal Disease Resistance in Soybean (Glycine max L. Merr):A Method of Meta-Analysis for Mining R Genes

Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world. Conflicting results of fungal disease resistance QTLs from different populations often occurred. The objectives of this study were to: (i) evaluate evidence for reported fungal disease resistance QTLs associations in soybean and (ii) extract relatively reliable and useful information from the "real" QTLs and mine putative genes in soybean. An integrated map of fungal disease resistance QTLs in soybean was established with soymap 2 published in 2004 as a reference map. QTLs of fungal disease resistance developed from each of separate populations in recent 10 years were integrated into a combinative map for gene cloning and marker assisted selection in soybean. 107 QTLs from different maps were integrated and projected to the reference map with the software BioMercator 2.1. A method of meta-analysis was used to narrow down the confidence interval, and 23 "real" QTLs and their corresponding markers were obtained from 12 linkage groups (LG), respectively. Two published R genes were found in these "real" QTLs intervals. Sequences in the "real" QTLs intervals were predicted by GENSCAN, and these predicted genes were annotated in Goblet. 228 resistance gene analogs (RGAs) in 12 different terms were mined. The results will lay the foundation for a bioinformatics platform combining abundant QTLs, and offer the basis for marker assisted selection and gene cloning in soybean.     

A physical map of the 1-gigabase bread wheat chromosome 3B

As the staple food for 35% of the world's population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17-billion-base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)-based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.     

QTL analysis for plant height and fine mapping of two environmentally stable QTLs with major effects in soybean

Plant height is an important agronomic trait, which is governed by multiple genes with major or minor effects. Of numerous QTLs for plant height reported in soybean, most are in large genomic regions, which results in a still unknown molecular mechanism for plant height. Increasing the density of molecular markers in genetic maps will significantly improve the efficiency and accuracy of QTL mapping. This study constructed a high-density genetic map using 4 011 recombination bin markers developed from whole genome re-sequencing of 241 recombinant inbred lines (RILs) and their bi-parents, Zhonghuang 13 (ZH) and Zhongpin 03-5373 (ZP). The total genetic distance of this bin map was 3 139.15 cM, with an average interval of 0.78 cM between adjacent bin markers. Comparative genomic analysis indicated that this genetic map showed a high collinearity with the soybean reference genome. Based on this bin map, nine QTLs for plant height were detected across six environments, including three novel loci (qPH-b_11, qPH-b_17 and qPH-b_18). Of them, two environmentally stable QTLs qPH-b_13 and qPH-b_19-1 played a major role in plant height, which explained 10.56–32.7% of the phenotypic variance. They were fine-mapped to 440.12 and 237.06 kb region, covering 54 and 28 annotated genes, respectively. Via the function of homologous genes in Arabidopsis and expression analysis, two genes of them were preferentially predicted as candidate genes for further study.     

夏大豆重组自交系群体遗传图谱构建及开花期QTL分析

【背景】开花期是大豆重要的生育期性状,不仅决定了大豆品种的适种范围,而且对大豆的产量和品质有重要影响。江淮地区是中国重要的大豆产区,目前对该地区夏大豆开花期性状遗传基础研究相对较少。【目的】利用2个夏大豆材料杂交衍生的重组自交系群体对开花期进行QTL定位,为分子标记辅助选择育种和基因克隆提供依据。【方法】以科丰35(KF35)和南农1138-2(NN1138-2)为亲本,构建了含91个家系(F2:8)的重组自交系群体(NJK3N-RIL),在6个环境下调查开花期性状数据。利用限制位点相关DNA测序(restriction-site associated DNA sequencing,RAD-seq)技术对群体亲本及家系材料进行SNP标记分型,并利用窗口滑动法进行bin标记划分。利用bin标记构建该群体的遗传图谱,结合多年多点的表型数据,使用QTL Network 2.2软件中的基于混合线性模型的复合区间作图法(mixed-model based composite interval mapping,MCIM)和Windows QTL Cartographer V2.5₀     

西瓜遗传图谱构建及果实相关性状QTL分析

【目的】利用CAPS及SSR标记构建西瓜遗传图谱,对西瓜果实相关性状进行QTL分析,为西瓜果实性状改良、主效基因精细定位及克隆奠定基础。【方法】授粉后40 d对母本PI186490、父本LSW-177以及两者杂交获得的F2群体的果实进行采摘,对每个果实的果形指数、中心和边缘可溶性固形物、中心和边缘果肉硬度、果皮硬度、种子长度、种子宽度、种子厚度以及种子百粒重进行调查,将所得数据用软件SPSS19进行统计分析。通过Illumina HiSeq 2000高通量测序平台对两亲本材料进行基因组重测序,每样品产出10 G数据量,覆盖西瓜基因组20×以上,所得数据以已经发布的基因组数据为参考基因组,用bwa软件进行基因组组装,组装后利用Samtools软件进行SNP发掘,利用perl语言自编脚本提取SNP位点前后1 000 bp的序列,将SNP及其侧翼序列输入软件SNP2CAPS以转化为CAPS标记。在每条染色体上平均选取20个CAPS酶切位点,利用Primer Premier 5软件在突变位点上下游100-500 bp左右设计CAPS引物,进行PCR扩增和酶切检验,酶切产物用1%琼脂糖凝胶电泳检测。SSR引物来源于前人发表文献,PCR扩增产物用聚丙烯酰胺凝胶电泳检测。对所有分子数据进行卡方检验,在其中选择符合1﹕2﹕1比例的标记用于构建遗传连锁图谱。利用Mapmaker/Exp version 3.0软件构建遗传连锁图谱,用Group命令对标记进行连锁分组,标记数目少于8的连锁群用Compare命令进行排序优化,标记数多于8的连锁群用Try命令排序。绘制遗传图谱使用Map Chart 2.1软件。QTL分析运用QTL Network 2.0软件,利用置换测验做1 000次重复,临界阈值为P=0.005,采用复合区间作图法,在每条染色体上以1.0 cM步行速度在全基因组范围内扫描,分析QTL加性效应和上位效应。【结果】本遗传连锁图谱共包含16个连锁群,涉及CAPS标记87个,SSR标记9个,覆盖基因组1 484.3 cM,平均图距15.46 cM。利用QTL Network 2.0分析,检测到6个西瓜果实相关性状的8个QTL位点和1对上位效应位点,其中包括果形指数QFSI 1、中心可溶性固形物QCBR、中心果肉硬度QCFF、边缘果肉硬度QEFF、种子长度QSL各1个,种子宽度QSWD 1、QSWD 2、QSWD 3 3个;上位效应位点包括果形指数FSI 2、FSI 3。表型贡献率大于等于10%的QTL有6个,可解释11.7%-18.8%的遗传变异。【结论】以CAPS标记为主要标记构建西瓜遗传图谱,并且定位了控制西瓜果实相关性状的8个加性QTL与1对上位性QTL,可用于进一步精细定位与克隆西瓜果实优良性状基因。     

A genetic linkage map with 178 SSR and 1901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci(QTLs) controlling agronomically important traits. In this study, simple sequence repeat(SSR) markers and Illumina 9K i Select single nucleotide polymorphism(SNP) genechip were employed to construct one genetic linkage map of common wheat(Triticum aestivum L.) using 191 recombinant inbred lines(RILs) derived from cross Yu 8679×Jing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 c M and 1 000 marker bins, with an average interval distance of 1.66 c M. A, B and D genomes covered 719.1, 703.5 and 237.3 c M, with an average interval distance of 1.66, 1.45 and 2.9 c M, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754(92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184(97.4%) were located on o ne single chromosome, and the rest 31(2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags(ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it will be a useful tool for comparative genomics analysis, fine mapping of QTL/gene controlling agronomically important traits and marker-assisted selection breeding in wheat.     

单核苷酸多态性在大豆育种中的应用

单核苷酸多态性 (singlenucleotidepolymorphism ,SNP)是指DNA序列上的单个碱基变异 ,它具有分布广、多态信息量大、易于检测和统计分析等优点 ,被称为继RFLP和微卫星标记后的第三代基因遗传标记。单核苷酸多态性是等位基因间序列差异最为普遍的类型 ,可作为一种高通量的遗传标记。已建立PCR扩增目标序列及其产物测序和电子SNP(eSNP)等多种发现和检测SNP的方法。大豆等作物也已开展了SNP分析。     

Constructing the wolfberry (Lycium spp.) genetic linkage map using AFLP and SSR markers

Genetic linkage maps are important for quantitative trait locus (QTL) and marker-assisted selection breeding. The wolfberry (Lycium spp.) is an important food and traditional medicine in China. However, few construction genetic linkage maps have been reported because of the lack of genomic and genetic resources. In this study, a population of 89 F₁ seedings was derived from a cross between two heterozygous parents, L. chinense var. potaninii ‘BF-01’ (female) and L. barbarum var. auranticarpum ‘NH-01’ (male), in order to construct a genetic linkage map using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers based on the double pseudo-test cross mapping strategy. The resulting genetic map consisted of 165 markers (74 AFLPs and 91 SSRs) distributed across 12 linkage groups and spanned a total length of 557.6 cM with an average distance of 3.38 cM between adjacent markers. The 12 linkage groups contained 3 to 21 markers and ranged in length from 8.6 to 58.3 cM. Twenty-nine segregated markers distributed in the map were mainly located on LG4 and LG9 linkage groups at P<0.05. This is the first linkage map of Lycium species using SSR and AFLP markers, which can serve as basis for improving genes and selective breeding of the genome assembly.     

Meta-Analysis of 100-Seed Weight QTLs in Soybean

100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, the public map should be chosen which was suitable for the published results integrated, and to improve yield. In this research, an integrated map of 100-seed weight QTLs in soybean had been established with soymap2 published in 2004 as a reference map. QTLs of 100-seed weight in soybean were collected in recent 20 yr. With the software BioMercator 2.1, QTLs from their own maps were projected to the reference map. From published papers, 65 QTLs of 100-seed weight were collected and 53 QTLs were integrated, including 17 reductive effect QTLs and 36 additive effect QTLs. 12 clusters of QTLs were found in the integrated map. A method of meta-analysis was used to narrow down the confidence interval, and 6 additive QTLs and 6 reductive QTLs and their corresponding markers were obtained respectively. The minimum confidence interval (C.I.) was shrunk to 1.52 cM.These results would lay the foundation for marker-assisted selection and mapping QTL precisely, as well as QTL gene cloning in soybean.     

A BAC-based physical map of the major autosomes of Drosophila melanogaster

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.