基因组DNA混合池技术在SSR引物筛选中的可靠性分析

SSR-PCR产物的重测序是SSR标记技术开发和应用的必要步骤。本研究以6个桉树DNA样品为对照组,1个基因组DNA混合池为试验组进行基因组DNA混合池在SSR引物筛选中的可靠性性分析。通过SSR-PCR产物的重测序分析,6个桉树单一DNA样品与基因组DNA混合池的扩增序列比对结果分析显示,两种模板扩增结果差异不显著,即基因组DNA混合池可以替代多个单一DNA样品进行SSR引物的高效筛选。利用20对SSR引物在6种桉树DNA样品中的扩增序列进行多态性分析发现,同一位点不同桉树种间的扩增序列存在长度多态性和核苷酸多态性,从而证实了SSR序列的多态性和复杂性,为SSR标记技术在桉树遗传育种研究中的应用提供了理论依据。     

构建黍稷分子遗传图谱SSR引物的筛选

采用SSR-PCR扩增技术,将实验室利用高通量测序与磁珠富集法结合开发得到的1 210对黍稷SSR引物进行筛选,筛选出在会宁大黄糜、陇糜8号、太原55号和会宁野糜子4个黍稷品种之间表现出条带清晰、多态性明显、重复性较好的引物;并通过提取DNA以及PCR过程中退火温度的简单优化来建立比较适宜的黍稷SSR分子标记反应体系。结果表明,在DNA提取过程中将ddH2O的量减少到100μL,增加65℃的水浴时间并增加4%β-巯基乙醇和DNA浓度等可以获得较高质量的DNA。利用优化好的反应体系从1 210对SSR引物中筛选出116对多态性较好的引物,占总数的9.59%,其中,群体一的2个亲本所特有的引物有36对,群体二的2个亲本中特有的引物有97对,2个群体共有的多态性引物有19对;对引物碱基结构分析发现,选用的1 210对引物中有1 173个双碱基重复SSR和36个三碱基重复SSR;筛选到的多态性引物中双碱基重复SSR有102个。三碱基重复SSR有14个。筛选出的多态性SSR标记能用于黍稷的遗传多样性和群体遗传结构研究。     

构建黍授分子遗传图谱ssR引物的筛选

采用SSR-PCR扩增技术,将实验室利用高通量测序与磁珠富集法结合开发得到的1210对黍援SSR引物进行筛选,筛选出在会宁大黄糜、陇糜8号、太原55号和会宁野糜子4个泰援品种之间表现出条带清晰、多态性明显、重复性较好的引物;并通过提取DNA以及PCR过程中退火温度的简单优化来建立比较适宜的泰援SSR分子标记反应体系。结果表明,在DNA提取过程中将ddH20的量减少到100μL,增加65℃的水浴时间并增加4%β-筑基乙醇和DNA浓度等可以获得较高质量的DNA。利用优化好的反应体系从1210对SSR引物中筛选出116对多态性较好的引物,占总数的9.59%,其中,群体一的2个亲本所特有的引物有36对,群体二的2个亲本中特有的引物有97对,2个群体共有的多态性引物有19对;对引物碱基结构分析发现,选用的1210对引物中有1173个双碱基重复SSR和36个三碱基重复SSR;筛选到的多态性引物中双碱基重复SSR有102个。三碱基重复SSR有14个。筛选出的多态性SSR标记能用于泰援的遗传多样性和群体遗传结构研究。     

燕麦SSR标记开发及其在品种指纹图谱构建中的应用

使用磁珠富集法开发燕麦(Avena sativa L.) SSR分子标记,从中筛选出合适的引物构建燕麦10个国审品种的DNA指纹图谱。结果表明,通过磁珠富集法获得了150对SSR引物,从中筛选出6对多态性好且扩增稳定清晰的引物,共检测到50个多态性片段;使用其中2对引物构建了燕麦10个国审品种的DNA指纹图谱。筛选出的6个SSR引物具有很好的多态性,可应用于更多燕麦品种的DNA指纹图谱构建和遗传多样性分析。每个国审燕麦品种均具有唯一DNA指纹编码,可用于燕麦品种真伪鉴定,为品种权保护提供科学依据。     

应用SSR鉴定化学杀雄杂交油菜秦杂油19种子纯度的研究

为有效建立化杀杂交油菜品种的纯度鉴定方法,本研究利用简单重复序列(SSR)技术,对秦杂油19及其亲本进行了分子鉴定,从800对SSR引物中筛选出了5对带型清晰、扩增效果好且能将亲本与杂交种完全区别的引物。利用其中的SR767和SR790两引物对三者的单株进行了分析验证,三者的扩增图谱与标准图谱对应率极高,分别为97.33%、98.00%、100%和98.67%、96.67%、98.00%;利用两对SSR引物对秦杂油19的栽培品种进行分别鉴定和比较研究,结果显示两对不同引物的SSR分子鉴定结果的单株吻合率96.67%~98.32%之间,结果表明,利用SSR分子标记可以用于杂交油菜纯度鉴定。     

基于梨EST和基因组序列SSR引物的批量开发与应用

目前基于梨属植物基因组开发的SSR引物太少,远不能满足其品种鉴别、遗传多样性分析和分子标记辅助育种等研究与应用的需要。为进一步开发更多的梨SSR引物,本研究从NCBI公共数据库检索到梨的4 338条ESTs和478条基因组序列。通过前处理和聚类去冗余后得到全长为343.81 kb的无冗余的686条ESTs,搜索获得725个SSRs,其平均长度为17.34 bp。在一至九个核苷酸的重复基元中,其主要类型是单核苷酸重复和二核苷酸重复,两者所占的比例分别为84.44%和10.34%。BLASTx同源性比较分析发现,上述686条ESTs中305条在数据库中可以找到同源序列,占ESTs总数的44.46%,功能已知蛋白质中碧桃来源的ESTs所占比例最大,为41.37%。GOA功能分类结果表明,293条ESTs可划分为生物过程、分子功能和细胞成分3大功能类,其中与细胞过程相关的ESTs数量最多,为103条。序列分析结果显示,EST-SSRs与基因组SSRs的平均检出频率分别为2 108.74 loci/Mb和91.11 loci/Mb,两者具有显著的差异。本研究基于梨的EST和基因组序列,应用MISA批量开发技术分别获得了111对EST-SSR引物和291对基因组SSR引物,随机抽取及合成11对引物的验证结果表明,开发得到的SSR引物在梨属植物中具有较好的通用性。     

利用SSR荧光标记构建20个高粱品种指纹图谱

为建立高粱种子纯度及真实性鉴定技术体系,构建高粱栽培品种DNA指纹图谱数据库是必要的。利用SSR荧光标记技术筛选出36对SSR荧光标记引物,构建我国高粱杂种优势利用以来中晚熟区主推的20个品种的SSR指纹图谱。36对SSR引物共扩增出235个等位基因,平均每个等位基因检测到多态性位点7个,多态性位点变化范围为2~12个。20个高粱品种36个SSR位点的遗传多样性指数和多态性信息量的变化范围分别为0.3490~0.8813和0.3144~0.8696,平均值分别为0.6976和0.6571。从36对SSR引物中筛选到多态性丰富的2对引物作为高粱品种鉴定的特征引物,2对SSR特征引物组合可区分所有供试品种。20个高粱品种的SSR指纹图谱互不相同,可以作为各品种特定的图谱,为品种鉴别提供依据。     

水稻中以遗传多样性分析为基础的分子标记

一系列分子标记系统的有效性允许我们比较其中两种标记系统在估计不同分类单位相互关系时的效率。本研究的目的是用简单序列重复(SSR)标记和随机扩增多态DNA(RAID)标记来评价40个水稻(Oryza sativa L)栽培品种和5个野生近缘种的遗传多样性。用36个十体引物和38个SSR引物对扩增后评价了这些试样的多态性。在40个栽培品种和5个野生近缘种中产生了总共499个RAPD标记，具有90．0％多态性。在所使用的38个SSR引物对中，仅有1个位点即RM115是多态的。     

玉米杂交种DNA指纹图谱及其在亲子鉴定中的应用

采用79对SSR引物分析了86个玉米杂交种的72份亲本自交系的遗传多态性,从中筛选出扩增带型清晰、稳定的50对引物用于构建86个杂交种的DNA指纹图谱数据库;进而确定10对核心引物和判别标准用于亲子鉴定研究。结合玉米SSR分子标记检测技术和单籽粒DNA快速提取方法,DNA指纹数据库和判别标准可有效地用于玉米杂交种的亲子鉴定。     

基于热胁迫甜玉米雌穗发育差异表达基因的SSR标记开发

为了开发适用于甜玉米抗性遗传的SSR标记,通过对高通量测序基因表达谱技术获取的热胁迫下粤甜13号雌穗发育差异表达基因进行分析,应用生物信息学方法从玉米DNA数据库中开发SSR标记。结果表明,以玉米参考基因数据库获取的58个基因相应序列中,发现53个基因序列存在252个SSR位点,SSR检出率为4.8%,从中开发了251对SSR引物。合成了其中100对SSR引物经PCR及电泳检测,12对引物能在3个甜玉米材料中检测出较好的多态性,占被检测引物的12.0%,扩增带型清晰、稳定性好,这些SSR引物可应用于今后甜玉米抗逆研究中。     

Development of novel microsatellite markers for effective applications in Anthurium cultivar identification

Anthurium andraeanum is one of the most economically important floral crops and potted flowers marketed worldwide. Microsatellite markers are currently the preferred molecular marker owing to the many desirable attributes, including hypervariability, codominance, and amenability to high-throughput genotyping; however, there are few polymorphic molecular markers available for Anthurium. The object of this study was to develop and characterize novel microsatellite markers using the Araceae sequences in GenBank of the National Center for Biotechnology Information (NCBI) to contribute to molecular identification for cultivar protection. Using 1,579 Araceae expressed sequence tags (ESTs) and the related nucleotide sequences, 100 candidates contained simple sequence repeat (SSR) motifs that were suitable for primer design. Furthermore, 100 pairs of SSR primers were screened against a set of 28 diverse genotypes representing 24 cultivars that included four registration cultivars which were bred from the Taiwan Agricultural Research Institute (TARI) and 20 commercial cultivars, appended with three hybrid progeny and a mutant line. From the selected six polymorphic SSR loci, 52 alleles were amplified and 27 distinct genotypes were found, except for ‘Tropical’ and its mutant, with a mean number of eight alleles per locus. The polymorphism information content (PIC) ranged from 0.86 to 0.93. Based on these results, we proposed a key identification set using four microsatellite markers that is sufficient to discriminate among 24 cultivars. Because the Anthurium microsatellite markers developed in this study are primarily from expressed sequence tags or related genomic sequences, they can be used for cultivar identification and, accordingly, contribute to genetic evaluations in breeding programs.     

新疆彩色棉23个品种指纹图谱的构建及遗传多样性分析

以新疆截止2012年审定的23份新彩棉品种为材料,利用SSR标记进行DNA指纹图谱的构建和遗传多样性分析。从5000对SSR引物中,挑选出多态性高、稳定性好、均匀分布在棉花26条染色体上的52对引物,在23份新彩棉品种中筛选出核心引物47对,SSR扩增检测到多态性基因型位点数共计162个,每个标记检测到的基因型位点数在2~7之间,平均为3.45个;引物多态信息量(PIC)值介于0.4537~0.8686之间,平均值为0.7096。结果显示:在23份新彩棉品种中,14份品种采用特异或特征引物可以一次性区分开,其余9份品种需要采用引物组合来实现区别该品种与其他品种。最少选用18对特异引物及组合引物就可以完全区分开新彩棉1~23号品种。利用18对SSR标记构建了新彩棉1号至23号品种的指纹图谱。利用NTSYS-pcV2.10软件聚类分析表明:23个新彩棉品种遗传相似系数变化范围是0.3781~0.9298,平均为0.5511,表明新彩棉品种之间存在着丰富的遗传多样性。     

Genetic diversity,structure and fruit trait associations in Greek sweet cherry cultivars using microsatellite based (SSR/ISSR) and morpho-physiological markers

It is important to couple phenotypic analysis with genetic diversity for germplasm conservation in gene bank collections. The use of molecular markers supports the study of genetic marker-trait associations of biological and agronomic interest on diverse genetic material. In this report, 19 Greek traditional sweet cherry cultivars and two international cultivars, which were used as controls, were grown in Greece and characterized for 17 morpho-physiological traits, 15 simple sequence repeat (SSR) loci and 10 inter simple sequence repeat (ISSR) markers. To our knowledge, this is the first report on molecular genetic diversity studies in sweet cherry in Greece. Principal component analysis (PCA) of nine qualitative and eight quantitative morphological parameters explain over 77.33% of total variability in the first five axes. The SSR markers yielded a combined matching probability ratio (MPR) of 9.569 × e−12. The 15 SSR loci produced a total of 92 alleles. Ten ISSR primers generated 91 bands, with an average of 9.1 bands per primer. Expected heterozygosity (gene diversity) values of 15 SSR loci and 10 ISSR markers averaged at 0.683 and 0.369, respectively. Based on stepwise multiple regression analysis (MRA), SSR alleles were found associated with harvest time and fruit polar diameter. Furthermore, three ISSR markers were correlated with fruit harvest and soluble solids and four ISSR markers were correlated with fruit skin color. Stepwise MRA identified six SSR alleles associated with harvest time with a high correlation (P < 0.001), with linear associations with high F values. Hence, data analyzed by the use of MRA could be useful in marker-assisted breeding programs when no other genetic information is available.     

鹅掌楸EST-SSR引物开发及通用性分析

通过对6520条鹅掌楸EST序列进行检索,在364条ESTs中发现394个SSRs,鹅掌楸EST-SSRs平均密度为每8.5kb含有1个SSR;在检索出的SSRs中,二核苷酸重复单元的SSRs类型最多,占总数的61.9%。利用SSR-ESTs序列共设计176对EST-SSR引物,其中132对在鹅掌楸上有扩增产物,66对扩增出多态,多态性引物占所设计引物的36.9%。这批EST-SSR引物在物种之间具有较高的通用性。研究表明在鹅掌楸中表现多态的66对EST-SSR引物,85%在中国马褂木中有扩增,54%在白玉兰中有扩增。     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Development of PCR-based markers for a major locus conferring powdery mildew resistance in mungbean (Vigna radiata)

Powdery mildew (PM) can cause significant yield loss in mungbean and several loci conferring resistance to this disease have been identified. A restriction fragment length polymorphism (RFLP) marker (VrCS65) linked closely to one of these loci was used to screen a mungbean bacterial artificial chromosome (BAC) library and positive BAC clones identified were used to develop simple sequence repeat (SSR or microsatellite) and sequence tagged site (STS) markers. Four of the new PCR markers (including two SSRs and two STSs) co-segregated with the original RFLP marker VrCS65, and another SSR marker (VrCS SSR2) was located 0.5 cM away from it. These PCR-based and locus-specific markers could be useful in breeding cultivars with enhanced resistance to PM and in the further characterization of the locus including the isolation of gene(s) responsible for the resistance.     

青海省马铃薯主要栽培品种的SSR遗传多样性

利用SSR标记分析了12份马铃薯栽培品种的遗传多样性及其亲缘关系,结果表明:(1)30对SSR引物共扩增出388条带,其中多态性条带为359条.(2)12份材料的遗传距离介于0.238 5～0.4801 cM之间,平均值为0.397 6 cM.(3)利用由非加权类平均法聚类分析表明,在遗传相似系数0.63处将12份材料被划分为五类:两类为晚熟(其中从国际马铃薯中心引进的青薯9号品种为一类;青薯168,2号,3号,4号品种为另一类),两类为早熟(其中青薯5号品种为一类;青薯8号品种和02-1-1品系为另一类),一类为中熟(因育成的地域不同而分为两个亚类).结果表明,青海省现有主要栽培的马铃薯品种遗传多样性水平较低.为拓宽育成品种的遗传基础,应充分发掘野生二倍体的遗传潜力.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Mapping of quantitative trait loci controlling cotton leaf curl disease resistance in upland cotton

Cotton leaf curl disease (CLCuD) is a major threat to cotton production in Asia and Africa. Using marker-assisted breeding can be the best sustainable approach to tackle CLCuD. Identification of new QTLs in the indigenous cotton germplasm is necessary to combat CLCuD. The current study was designed to construct a genetic linkage map of bi-parental F2:F3 populations developed from highly tolerant MNH-886 and highly susceptible S-12 cotton cultivars. One hundred seven CLCuD-associated simple sequence repeat (SSR) marker alleles were identified as polymorphic and three new QTLs were found on chromosomes C11, C19 and C21. Two QTLs on chromosomes C11 and C19 were detected in both F2 and F3 populations in the region flanked by SSR markers CIR316 and BNL4094, and BNL285 and BNL3348, respectively. Whereas, one QTL on chromosome C21 was detected in the region flanked by SSR markers JESPR158 and JESPR135 in both F2 and F3 generations. The CLCuD-associated QTLs identified in this study can help fine-tune the molecular mapping of the QTLs on the cotton genome against CLCuD.     

Genetic diversity in soybean genotypes from north‐eastern China and identification of candidate markers associated with maturity rating

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to estimate the genetic relationships among 101 soybean cultivars developed in north‐eastern China. Fifty‐three fragments of the 100 RAPD markers and 35 SSR markers tested were polymorphic across the 101 soybean cultivars. Similarity values among these soybean cultivars ranged from 45.2% to 100% for RAPD data, and ranged from 36.1% to 100% for SSR data. The similarity matrices for SSR data and RAPD data were moderately correlated (r = 0.31, P < 0.05). Cluster analyses indicated that the cultivars released from the same seed company were mostly grouped together. A principal component analysis, based on the combined RAPD and SSR data, yielded a good separation of soybean varieties with different maturity ratings [represented by soybean Heat Unit (HU)]. The varieties with HU < 2200 were well separated from those with HU > 2200. Four RAPD markers and eight SSR markers were significantly associated with the maturity ratings of soybean.