基于SNP标记的小麦高通量身份鉴定模式

为探索小麦品种高通量SNP身份鉴定模式,利用 wheat 90K SNP芯片对380份小麦品种进行了全基因组扫描、分析和评价,从中筛选出高质量、高分辨率、单拷贝和均匀分布的候选SNP标记384个,能将除近等基因系以外的所有品种区分开;基于组合最优化算法,获得小麦品种高通量鉴定最少SNP位点组合一套,包含14个SNP标记,区分能力与384个SNP标记相同。将14个SNP位点转化成KASP标记,分析选取的95份样品,结果显示,芯片平台和KASP平台上的基因分型结果一致。考虑品种实际鉴定过程中存在样本量大、高度近似品种少等情况,权衡准确、经济、灵活、快速、通量高等检测需求,建议品种高通量身份鉴定可采取“核心位点+扩展位点”的模式进行。本研究为小麦等农作物品种SNP高通量身份鉴定技术体系的建立和指纹数据库的构建提供了有利的参考。     

Cereal variety identification using MALDI-TOF mass spectrometry SNP genotyping

Correct identification of cereal varieties is important to food quality, safety and authenticity and recently, molecular markers have been applied to cereal varietal identification. Two major factors restricting the wider application of molecular markers in crop plant genetics have been throughput and cost per data point. However, in recent years, novel molecular tools, the re-invention/re-application of mature technologies, and miniaturisation of technology has both increased throughput and significantly reduced these costs. Here we have applied Sequenom^® MassARRAY^® MALDI-TOF mass spectrometry using a collection of recently developed SNPs to facilitate cereal varietal identification. We used a multiplexed Sequenom^® MassARRAY^® mass spectrometry SNP assay targeting 45 loci to genotype a collection of Australian barley varieties. Of the 45 loci screened, 33 were informative and were used to generate a unique barcode of SNPs for each variety tested. Only one variety could not be distinguished from two others due to a high level of varietal heterogeneity. This assay format provided a flexible, cost-effective, robust and moderate throughput SNP genotyping method well suited to varietal identification and purity analysis in cereals.     

Cereal variety identification using MALDI-TOF mass spectrometry SNP genotyping

Correct identification of cereal varieties is important to food quality, safety and authenticity and recently, molecular markers have been applied to cereal varietal identification. Two major factors restricting the wider application of molecular markers in crop plant genetics have been throughput and cost per data point. However, in recent years, novel molecular tools, the re-invention/re-application of mature technologies, and miniaturisation of technology has both increased throughput and significantly reduced these costs. Here we have applied Sequenom^® MassARRAY^® MALDI-TOF mass spectrometry using a collection of recently developed SNPs to facilitate cereal varietal identification. We used a multiplexed Sequenom^® MassARRAY^® mass spectrometry SNP assay targeting 45 loci to genotype a collection of Australian barley varieties. Of the 45 loci screened, 33 were informative and were used to generate a unique barcode of SNPs for each variety tested. Only one variety could not be distinguished from two others due to a high level of varietal heterogeneity. This assay format provided a flexible, cost-effective, robust and moderate throughput SNP genotyping method well suited to varietal identification and purity analysis in cereals.     

一种大豆SNP分型新方法

单核苷酸多态性(SNP)在大豆基因组中分布广泛,SNP分型是大豆SNP遗传作图,关联分析、分子标记辅助选择等研究的重要技术.本文以Rhg4基因开发的5个SNP为例,介绍了一种大豆SNP分型的新方法-片段长度差异等位基因特异性PCR(FLDAS-PCR).采用AS-PCR原理,针对某个SNP位点设计2条相差4-5bp的特异引物和1个公用引物,PCR产物约100bp或150bp,2种等位基因型PCR产物相差4-5bp,在2个特异引物3'端第3和4碱基位置分别人为引入错配碱基来提高PCR特异性,通过6%变性聚丙烯酰胺凝胶电泳可将纯合和杂合基因型检测出来.探讨了特异引物浓度和退火温度对Rhg4-1592扩增效果的影响,研究表明,可通过调整特异引物浓度和退火温度优化扩增效果.FLDAS-PCR对18份种质分型结果与PCR产物克隆测序法一致,表明本研究建立的FLDAS-PCR法是一种简便、快捷、新型的大豆SNP分型方法.     

利用SNP标记鉴定青稞种质资源

近年来,青稞(Hordeum vulgare var.nudum Hook. f.)育种速度逐步加快,青稞品种的类别和数量日渐增多,形成了丰富的青稞品种资源。然而,在青稞资源的大量引种和品种资源交换过程中,造成了同名异物、同物异名的现象,因而建立高效、准确的青稞品种鉴定技术体系和数据系统迫在眉睫。为基于青稞品种基本信息实现青稞品种的快速和准确鉴定,本研究利用简化基因组(GBS)测序获得的青稞基因组高通量SNP基因分型数据,对314份青稞种质资源进行群体结构分析;根据SNP注释结果,筛选获得位于外显子区的SNP并计算其杂合率和遗传多态性指数;用Genstat的去冗余(IRREDUNDANT)指令通过顺序算法(sequential algorithm)获得能够区分参试青稞种质资源的核心SNP位点组合,并构建DNA指纹图谱,结合参试材料地理来源等基本信息构建青稞品种的分子身份证。结果表明,群体遗传结构分析可将314份参试青稞材料划分为3个类群,类群划分与其材料类型密切相关。从4 954个位于外显子区域的高质量SNP位点中筛选出14个多态性高且能完全区分青稞种质资源的SNP位点,称其为核心SN...     

玉米穗型和粒型性状的GWAS及其关联位点驯化和改良分析

以32份大刍草、68份玉米地方品种和来源广泛的294份玉米自交系为材料,在对294份玉米自交系进行简化基因组测序的基础上,采用GWAS方法对11个玉米穗型和粒型性状进行关联分析。结果表明,11个性状共检测到44个关联的SNP位点,其中,28个位点与6个穗型性状相关联,16个位点与3个粒型性状相关联。进一步整合32份大刍草和68份地方品种相应位点的序列数据,44个关联位点中共发现29个SNP在3类群体间共有。通过Fisher的精确检验发现,7个SNP的等位基因频率在大刍草到地方品种中发生了显著变化,14个SNP的等位基因频率在地方品种到自交系中发生了显著变化,3个SNP的等位基因频率则在大刍草到地方品种以及地方品种到自交系中均发生了显著变化,表明这些位点可能经历了驯化或/和人工改良。     

利用核心SNP位点鉴别玉米自交系的研究

根据多态性水平、染色体位置等信息从公共数据库上筛选了48个玉米核心SNP位点。利用48重-SNPLex分型系统对105份玉米自交系进行SNP基因分型分析,探索SNP标记在玉米品种鉴定中的应用前景。结果表明,48个SNP位点中有42个位点峰型正常;PIC值在0.019～0.375之间,平均为0.242;任何两份自交系间的遗传距离均在0.015以上,即42个SNP位点的基因分型数据信息可以将105份自交系材料区分开。     

基于全基因组SNP构建甘蓝型油菜指纹图谱

甘蓝型油菜是世界上重要的油料作物,可用作油料、饲料以及食品等,具有较高的经济价值。为了高效快速地鉴定并区分油菜品种,加强油菜品种管理,本试验利用505份油菜种质的重测序数据进行SNP鉴定,根据杂合率、位点缺失率以及多态性等指标对SNP集合进行筛选,得到了能够高效鉴定油菜种质的核心SNP位点组合,构建了DNA指纹图谱。该套核心位点组合包括897个位点,其MAF、PIC的平均值分别为0.41、0.474。使用该套SNP位点组合进行品种区分,每两个材料之间的差异位点数目90%为357～508。对核心SNP位点进行精简,最少用17个SNP标记可完全鉴定该套种质。本研究使用高质量的油菜重测序数据,筛选出了897个核心SNP位点,并利用该套核心SNP组合构建了油菜的特征指纹图谱,为油菜遗传多样性分析、品种鉴定以及种质管理提供数据参考。     

Development of new markers to genotype the functional SNPs of SSIIa, a gene responsible for gelatinization temperature of rice starch

Gelatinization temperature (GT) is an important quality predictor that determines the cooking quality of rice. GT is genetically controlled by the starch synthase IIa (SSIIa) gene. Two functional single nucleotide polymorphisms (SNPs) inside the SSIIa have already been found to be responsible for the variation of GT. One of these, GC/TT SNP at 4329/4330 bp, could be genotyped by four primers in a single PCR ( Bao et al., 2006a), but another one, G/A SNP at 4198 bp, has not been detected by a PCR-based marker. Here, we developed cleaved amplified polymorphic sequence (CAPS) and derived cleaved amplified polymorphic sequence (dCAPS) markers to detect these SNPs. A dCAPS marker that the PCR products were cleaved by the BseR I restriction endonuclease was designed to detect GC/TT SNP. Both CAPS and dCAPS markers were designed to detect G/A SNP using the restriction endonuclease Nla III and Tsp45 I, respectively. All the markers developed were co-dominant. It was known that the A allele of G/A SNP was rare among rice germplasm, but it was still in use by rice breeders. 11 rice accessions including landrace and breeding lines with A allele of G/A SNP were detected. The F₂ individuals from two crosses were used to analyze the co-segregation between the SNP alleles and the GT. The segregation ratio of two SNPs did not conform to the expected Mendelian ratio of 1:2:1, but the SNPs were co-segregated with GT. The markers developed in the present study would be useful in molecular breeding for the improvement of the quality of rice grain.     

小麦周8425B及其衍生品种与黄淮麦区主栽品种的遗传解析

为了解小麦骨干亲本周8425B及其衍生品种与黄淮麦区主栽品种的遗传结构和遗传多样性,利用Illumina 90KiSelect SNP标记技术对周8425B及其16份衍生品种和23份黄淮麦区主栽品种进行全基因组扫描。结果显示,在40份小麦材料中,有22 466个多态性SNP位点被定位在21条染色体上,不同基因组间多态性SNP标记的分布依次为BAD。周8425B及其衍生品种的遗传相似系数变化范围为0.640 5~0.926 4,平均值为0.739 8,与黄淮麦区主栽品种间遗传差异较小。供试材料的遗传相似系数变化范围为0.530 1~0.963 4,平均值为0.672 1,并被划分为4个类群,聚类分析结果与系谱较为吻合。周8425B对其衍生一代、二代、三代的平均贡献率为79.48%、76.73%和74.24%,随世代的增加而不断降低,且在不同基因组间的遗传贡献率表现为DAB。全基因组扫描结果显示,周8425B衍生品种共有6 789个SNP位点保留了周8425B的遗传基因,不同基因组继承的SNP位点数不同,依次为BAD,这些选择位点可能与重要基因的遗传传递有关,可能是周8425B成为骨干亲本的主要遗传特征。     

Identification of New Resistance Loci Against Sheath Blight Disease in Rice Through Genome-Wide Association Study

Sheath blight (SB) caused by the soil borne pathogen Rhizoctonia solani is one of the most serious global rice diseases. Breeding resistant cultivar is the most economical and effective strategy to control the disease. However, no rice varieties are completely resistant to SB, and only a few reliable quantitative trait loci (QTLs) linked with SB resistance have been identified to date. In this study, we conducted a genome-wide association study (GWAS) of SB resistance using 299 varieties from the rice diversity panel 1 (RDP1) that were genotyped using 44 000 high-density single nucleotide polymorphism (SNP) markers. Through artificial inoculation, we found that only 36.5% of the tested varieties displayed resistance or moderate resistance to SB. In particular, the aromatic and aus sub-populations displayed higher SB resistance than the tropical japonica (TRJ), indica and temperate japonica sub-populations. Seven varieties showed similar resistance levels to the resistant control YSBR1. GWAS identified at least 11 SNP loci significantly associated with SB resistance in the three independent trials, leading to the identification of two reliable QTLs, qSB-3 and qSB-6, on chromosomes 3 and 6. Using favorable alleles or haplotypes of significantly associated SNP loci, we estimated that both QTLs had obvious effects on reducing SB disease severity and can be used for enhancing SB resistance, especially in improving SB resistance of TRJ sub-population rice varieties. These results provided important information and genetic materials for developing SB resistant varieties through breeding.     

基于全基因组SNP高效鉴定燕麦种质资源

为探究鉴别燕麦品种特异性所需SNP标记的最少数量与鉴别效果,以25个生产上大面积推广的栽培燕麦品种为材料,利用燕麦Illumina Inc.iSelect 6K微珠芯片进行基因分型,按照具有多态性、最小等位基因频率（MAF）大于0.05、缺失率小于0.50的条件进行筛选,获得2 214个高质量的SNP标记。所有SNP标记的平均MAF为0.75,平均多态性信息含量（PIC）为0.28。群体遗传结构分析将25个燕麦品种划分为5个类群。通过去冗余分析,获得8个能够高效鉴别燕麦参试品种的SNP标记。这8个SNP标记的平均MAF为0.62,平均PIC为0.35,表现出丰富的遗传多样性。测序结果显示,所筛选的8个SNP标记具有较好的稳定性和重现性。进一步利用这8个SNP标记构建了25个燕麦栽培品种的SNP指纹图谱,为燕麦栽培品种真实性鉴定和纯度检测提供了可用的标记组合。     

Haplotype Diversity at Sub1 Locus and Allelic Distribution Among Rice Varieties of Tide and Flood Prone Areas of South-East Asia

Single nucleotide polymorphisms and restriction digestion-based haplotype variations among 160 flood prone rice varieties were analyzed with enzymes Alu I and Cac8 I to generate polymorphisms at Sub1A and Sub1C loci(conferring submergence tolerance), respectively. Haplotype associated with phenotype was used to study the haplotype variations at Sub1A and Sub1C loci and to determine their functional influence on submergence tolerance and stem elongation. Three patterns at Sub1A locus, Sub1A0(null allele), Sub1A1(does not cut) and Sub1A2(one SNP), and four patterns at Sub1C locus, Sub1C1, Sub1C2,Sub1C3 and Sub1C4, were generated. Both tolerant Sub1A1 and intolerant Sub1A2 had the same length, but the difference was presence of a restriction site in the Sub1A2, but absent at the Sub1A1. Further, two types of polymorphism were detected at the Sub1C, one included major length polymorphisms(165, 170 and 175 bp) and the other was a single restriction site at different position. Eight haplotypes(different combinations of the two loci), A1C1, A1C2, A1C4, A2C2, A2C4, A0C2, A0C3 and A0C4, were detected among 160 varieties. Haplotype A1C1 was comparatively more related to haplotypes A1C2 and A1C4, having the same Sub1A allele, and these haplotypes were found only in Bangladeshi, Sri Lankan and Indian varieties. Most tolerant varieties in A1C1 haplotype showed slow elongation, having tolerant specific Sub1A1 and Sub1C1 alleles. Further, the varieties Madabaru and Kottamali(A2C2) also showed moderate level of tolerance without Sub1A1 allele. These varieties were different with FR13 A and also suspected to carry different novel tolerant genes at other loci. These materials could be used for hybridization with Sub1varieties for pyramiding additional tolerant specific alleles into a single genotype for improving submergence tolerance in rice.     

An evaluation of gains in breeding for resistance to the cocoa swollen shoot virus disease in Ghana

The present study examines the gains in resistance to cocoa swollen shoot virus (CSSV) infection from investments in breeding over the past seven decades. The general susceptibility to CSSV infection of the West African Amelonado that dominated plantings prior to the start of formal research in 1938 necessitated the introduction of germplasm of Upper Amazon origin to better contain the disease spread. Included in this study are findings of two recent experiments. In the first, the genetic basis for resistance in the clone mvT85, developed from gamma radiation of clone T85/799 and putatively resistant to CSSV disease was investigated. In the second experiment, the comparative levels of resistance in sets of old, current and new cocoa varieties were tested following inoculations with the severe CSSV strain 1A. Absence of nucleotide differences at 29 single nucleotide polymorphism (SNP) loci between mvT85 and T85/799, and lack of segregation for resistance in the full-sib and backcross populations derived from mvT85 indicated that mvT85 did not carry novel genes for improving cocoa for CSSV disease resistance. Moreover, there were no differences in resistance to CSSV disease between mvT85 and T85/799. These observations conflict with the previous report that mvT85 is immune to CSSV disease, and distinct from T85/799. Between variety groups, disease severity scores based on three successive leaf flushes after inoculation were not effective in discriminating among them. Disease severity assessed eight months after inoculation was the most important criterion for separating varieties for resistance to CSSV disease. As expected, the older varieties were the most sensitive to infection. No differences were found between current varieties derived exclusively from Upper Amazon clones and new varieties. Contrary to the generally held opinion of a higher level of resistance in existing inter Upper Amazon cultivars, varieties derived from crosses using Catongo, RB 49 and C-SUL 7 (all of Lower Amazon origin) as males with specific Upper Amazon varieties were among the most resistant. A re-appraisal of variety recommendations for areas of mass infection and for less affected areas is advocated.     

几个马铃薯品种产量及品质形成的差异

以3个马铃薯品种为供试材料,在块茎增长期进行分期取样,对不同品种的产量和品质形成状况进行比较,以摸清不同品种的块茎形成特性,为特定品种配套适宜栽培技术的实施提供参考。结果表明：供试3个品种的单株产量存在显著差异,延薯4号的产量最高,薯块膨大早,商品薯率高;东农309的产量其次,薯块膨大较早,商品薯率较高;克新13号的产量最低,薯块膨大偏晚,商品薯率中等;但3个品种的单株结薯数量和干物质含量相近,均为结薯数量适中、干物质含量中等的中晚熟鲜食型品种。在生产上应根据品种特性配套栽培技术,以保证各品种获得较好的产量和品质。     

Genetic Fingerprinting of Potato Varieties from the Northwest Potato Variety Development Program

The Northwest Potato Variety Development (NWPVD) Program has released 45 improved potato varieties since 1985. Thirty-four potato varieties, four breeding clones and two advanced selections from NWPVD Program, and six commonly-grown potato varieties were fingerprinted using 32 simple sequence repeat (SSR) markers and 12,808 single nucleotide polymorphism (SNP) markers. Of 32 SSR markers, 29 exhibited significant polymorphism across all the 46 potato clones studied. A total of 143 alleles were observed with an average of 4.6 alleles per SSR marker. These markers span all 12 chromosomes of potato, with a maximum of five markers from chromosome VIII and minimum of one marker from chromosome VI. The polymorphic information content (PIC) and expected heterozygosity (H_e) of the SSR markers ranges between 0.18 to 0.75 and 0.20 to 0.78, respectively. Based on PIC, H_e, and ease of scoring, we recommend a set of eight SSR markers: STG0016, STI0004, STI0012, STI0023, STI0030, STI0033, STM1016 and STM1104 for fingerprinting NWPVD varieties. Out of 12,808 SNPs, 88.8% resulted in reliable three cluster diploid calling of which 87.8% were polymorphic. Tetraploid calling resulted in 44.2% of SNPs of which 94.5% were polymorphic. Our study provided fingerprinting resources for the NWPVD varieties and can be used in issues related to intellectual property rights, ownership, trademark and diversity analysis.     

Identification of powdery mildew resistance loci in wheat by integrating genome-wide association study(GWAS) and linkage mapping

Wheat powdery mildew(Blumeria graminis f.sp.tritici, Bgt) is a disease of increasing importance globally due to the adoption of high yielding varieties and modern sustainable farming technologies.Growing resistant cultivars is a preferred approach to managing this disease, and novel powdery mildew resistance genes are urgently needed for new cultivar development.A genome-wide association study was performed on a panel of 1292 wheat landraces and historical cultivars using 5011 single nucleotide polymorphism(SNP)markers.The association panel was evaluated for reactions to three Bgt inoculants, OKS(14)-B-3-1, OKS(14)-C-2-1, and Bgt15.Linkage disequilibrum(LD) analysis indicated that genome-wide LD decayed to 0.1 at 23 Mb, and population structure analysis revealed seven subgroups in the panel.Association analysis using a mixed linear model(MLM) identified three loci for powdery mildew resistance on chromosome 2 B, designated QPm.stars-2BL1,QPm.stars-2BL2, and QPm.stars-2BL3.To evaluate the efficacy of GWAS in gene discovery,QPm.stars-2BL2 was validated using F2 and F2:3 populations derived from PI420646 × OK1059060-126135-3.Linkage analysis delimited the powdery mildew resistance gene in PI 420646 to an interval where QPm.stars-2BL2 was located, lending credence to the GWAS results.QPm.stars-2BL1 and QPm.stars-2BL3, which were associated with four SNPs located at 457.7–461.7 Mb and two SNPs located at 696.6–715.9 Mb in the Chinese Spring reference IWGSC RefSeq v1.0, respectively, are likely novel loci for powdery mildew resistance and can be used in wheat breeding to improve powdery mildew resistance.     

Integration of Genomics into Rice Breeding

One of the major challenges in genetics has been to identify the nucleotide polymorphisms responsible for phenotypic variation. Through intensive analysis, several major quantitative trait loci (QTLs) for agronomic traits in rice have been identified and the underlying candidate genes have been delimited. Advanced mapping populations, including chromosome segment substitution lines, have enhanced the power of genetic analysis to detect QTL alleles, even those with minor effects. Recent examples of marker-assisted selection have proven the potential of this strategy for crop improvement. The genome-wide discovery of single nucleotide polymorphisms (SNPs), even among closely related cultivars, has enhanced the power of allele mining in a wide range of rice breeding materials. An array-based SNP genotyping system can be used to visualize pedigree haplotypes in breeding materials, including landraces and modern cultivars. All of these technologies are accelerating the genetic dissection of complex agronomic traits and further improvement of rice.     

抗SCN位点rhg1与Rhg4在种质资源中的单倍型分析及分子标记开发

rhg1和Rhg4是抗大豆胞囊线虫病种质所具有的主要的抗性基因,利用与rhg1和Rhg4位点紧密连锁的SSR标记和SNP标记对15份抗感病大豆种质进行基因分型。结果表明:rhg1位点连锁SSR标记Satt309对抗病种质的检出率为55.56%,Rhg4位点连锁SSR标记Sat_162对抗病种质的检出率为66.67%;rhg1位点序列引物PCR产物630bp位置存在腺嘌呤与胞嘧啶(A/C)突变,感病等位为A碱基,抗病等位为C碱基,在Rhg4位点标记引物SHMT的PCR产物上2 749 bp位置存在胞嘧啶和鸟嘌呤(C/G)突变,感病等位为C碱基,抗病等位为G碱基。rhg1和Rhg4位点内SNP对抗病种质的检出率分别为77.78%和100%。利用高分辨率溶解曲线法设计rhg1和Rhg4位点SNP标记,在扫描温度为65℃起始,60℃保持,94℃终止时可得到理想的分型结果。     

南方稻区国家水稻区域试验品种的微卫星标记分析

利用前期研究筛选的12个微卫星标记（SSR）首选标记对2005年南方稻区国家水稻区域试验199个水稻品种进行了DNA指纹鉴定,构建了199个水稻品种×12个首选标记的南方稻区国家水稻区域试验品种DNA指纹库。在首选标记座位上,常规稻和杂交稻的主导等位基因基本相同,籼稻和粳稻的主导等位基因差异较大;常规稻品种的纯合度高,多数杂交稻品种的杂合度较高、杂合座位数呈正态分布。比较相同母本的系列杂交稻组合,表明主导不育系系列组合具有较高的组内品种间母本一致性。此外,对南方稻区国家水稻区域试验品种特异性鉴定、DNA指纹库的扩充和鉴定标记数的确定进行了讨论。关键词