栽培花生中基因表达序列标记的产生和标记开发

本试验用抗叶片斑点病和番茄斑枯病毒的花生品系C34—24的叶片以及耐干旱胁迫和收获前黄曲霉素污染的花生品系A13的未成熟荚果制备成mRNA，然后用其mRNA构成两种cDNA文库，最后用这两种cDNA文库来开发出了栽培花生（Arachishy pogaea L．）中的表达序列标记（Expressed Sequcncetag，Esr）文库。对随机选择的cDNA克隆进行部分地排序，以便产生总共1825个ESTs，     

分离植物目的基因全长cDNA和启动子的新方法--快速定位转录起始位点(RITIS)

启动子是调控外源基因在植物体内表达的“开关”。随着植物转基因技术的广泛应用,无论是基础研究还是应用研究,人们希望能够充分利用启动子来准确控制外源基因在植物体内的表达,使目的基因的“开”和“关”、表达的“多”和“少”、在“何地”和“何时”表达等,能够听从人的指挥,以实现植物育种的分子设计。因此,快速分离和鉴定植物体内各种特异启动子已经成为植物基因工程研究的热点和难点。本文在互补末端连接反向PCR(CELI-PCR)技术基础上建立起一种快速分离目的基因全长cDNA和启动子序列的新方法。该方法利用CELI-PCR进行染色体连续步移,获取足够长的目的基因及其上游基因组DNA序列,再根据转录起始位点是目的基因转录本和启动子的分界点,其下游转录本中的外显子可通过RT-PCR扩增,而上游启动子序列则不能被RT-PCR扩增这一特点,借助RT-PCR进行cDNA连续步移,直到获得全长cDNA,确定启动子基因5'非翻译区的位置,进而精确定位转录起始位点。从而获取目的基因准确的启动子序列和全长cDNA序列。因此,建立在CELI-PCR基础上的RITIS技术,可绕过繁琐的构建cDNA库和5'-RACE等方法快速分离目的基因全长cDNA和启动子序列。     

EST技术及其应用

EST (Expressed Sequence Tag,EST)指的是一组cDNA的部分序列,一般长度为150~500bp,是由大规模随机挑取的cDNA克隆测序得到的组织或细胞基因组的表达序列标签。一个EST代表生物某一时期的某种组织或细胞的一个表达基因。主要综述了EST技术的原理方法以及EST技术在构建遗传学图谱、分离与鉴定新基因、基因表达谱的研究、比较基因组学的研究和制备DNA芯片等方面的应用。     

EST技术及其应用综述

EST(Expressed Sequence Tag,EST)指的是一组cDNA的部分序列,一般长度为150~500bp,是由大规模随机挑取的cDNA克隆测序得到的组织或细胞基因组的表达序列标签。一个EST代表生物某一时期的某种组织或细胞的一个表达基因。主要综述了EST技术的原理方法以及EST技术在构建遗传学图谱、分离与鉴定新基因、基因表达谱的研究、比较基因组学的研究和制备DNA芯片等方面的应用。     

黄瓜作图亲本间分子标记的多态性分析

利用AFLP,SRAP和SSR3种不同类型的分子标记技术,研究了作图亲本F-3与HZL04-1间的多态性。结果表明,这3种标记技术多态性检测效率不同,其中,AFLP揭示作图亲本间多态性效率最高,SRAP次之,SSR多态性检测效率最低。选择高效揭示多态性的分子标记技术是提高黄瓜遗传图谱构建效率的关键。分析探讨了几种分子标记技术的优缺点,认为应用AFLP,SRAP构建黄瓜遗传图谱为最佳,SSR可以作为补充的标记。     

梨AFLP分子连锁图谱的构建与分析

遗传图谱是数量性状基因定位、基因图位克隆及分子辅助育种等研究的基础,而目前国内在梨树上这方面的研究尚属空白.试验以早美酥×八月红的杂交F1 92株实生苗为作图群体,采用了扩增稳定、多态性丰富的16对Mse I/EcoR I引物组合,对分离群体进行AFLP多态性检测,共获得208条多态性带,其中偏离孟德尔遗传的比率29.8%(P<0.01).应用Joinmap 3.0软件且结合"双假测交"策略,对208个AFLP分子标记进行了遗传连锁分析,构建了一张包含145个AFLP标记,分属20个连锁群的梨分子遗传连锁图谱,每个连锁群包含4～20个标记,平均为9.06个标记,图谱总长度覆盖梨基因组618.7 cM,标记间平均图距为6.58 cM.此图谱为梨树基因定位、比较基因组学和重要农艺性状的QTL定位等研究奠定了基础.     

滇杨优树基因组及其无性系落叶期侧芽cDNA的AFLP分析

本研究采用AFLP分子标记技术,对采集于四川省和云南省的56株滇杨(Populus yunnanensis Dode)优树基因组遗传变异、54个滇杨优树无性系3a苗木落叶期侧芽的基因表达谱进行了分析。DNA-AFLP分析结果表明,筛选出的8对AFLP引物组合共扩增出508条带,平均多态性条带比率为59.25%,观测等位基因数为1.5925,有效等位基因数为1.1581,Nei’s基因多样性指数为0.1129,Shannon’s信息指数为0.1914;除有效等位基因数相等外,四川滇杨优树群体的4项遗传多样性指标均略高于云南滇杨优树群体,但2个优树群体之间存在着较高的基因流(Nm=21.64)。cDNA-AFLP分析结果显示,筛选出的8对AFLP引物组合共扩增出526条带,其中平均差异性条带百分率为82.70%,观测等位基因数和有效等位基因数分别为1.8270和1.2417,Nei’s基因多样性指数为0.1675,Shannon’s信息指数为0.2809;四川滇杨优树群体的基因表达差异性指标均略高于云南滇杨优树群体,且二者之间的基因流值较大(Nm=27.00)。分子方差分析(AMOVA)结果表明,滇杨2个优树群体在DNA水平和cDNA水平的遗传差异均极显著。     

花生DNA分子标记的研究 III 不同限制酶组合AFLP分析效果比较

采用了28对EcoR I/Mse I AFLP选择性扩增引物、64对Pst I /Taq I AFLP选择性扩增引物，对作图亲本24-3、B4及其杂种F1进行了分析，统计了两种限制酶组合的扩增总带数、多态性带数及多态性率。秩和检验结果说明，两种限制酶组合AFLP每对引物扩增出的总带数无显著差异，而多态性条带数目以及多态性率均有极显著的差异。证明尝试不同的限制酶组合可望揭示出花生品系材料间更为丰富的遗传变异性。     

猪HK2基因的克隆及序列分析

为克隆猪己糖激酶2基因,分析其生物功能,采用已报道的人及小鼠HK2的cDNA序列为依据,利用电脑克隆策略获得的ESTs设计引物,扩增新的猪HK2基因cDNA序列。将PCR产物克隆测序,分析获得的核苷酸序列及其编码蛋白的特性,分离的开放阅读框全长2754bp,编码917个氨基酸,与人和小鼠的核苷酸序列同源率分别为90%和87%,与人和小鼠的氨基酸序列同源率分别为96%和94%,利用生物信息学软件分析出此蛋白的理化特性并预测了其蛋白结构,为进一步开展猪HK2基因的结构功能、表达调控的相关研究奠定了基础。     

小麦新种质YW243抗条锈病新基因的AFLP标记

小麦新种质YW243抗条锈病新基因对条锈菌具有较宽抗谱，对26个不同毒性谱的条锈菌菌系免疫到近免疫，与已知基因系抗病谱不同，可能为新的基因或基因组合。遗传分析表明，YW243对条锈菌条中31号小种的抗性由显性单基因YrX控制。通过BSA法对1056对MseI/EcoRI AFLP引物进行筛选，结果表明7对引物可扩增出多态性片段，通过对F2群体单株遗传连锁分析和作图软件分析，结果表明，2个AFLP 标记M54E63-700、M54E64-699与YrX连锁较紧密、遗传距离为9.27 cM，为该基因的精细作图和分子标记辅助育种打下了一定基础。     

AFLP技术及其在干果遗传育种研究中的应用

AFLP（扩增片段长度多态性）是检测DNA多态性的一种高效的分子标记技术,该技术的运用不需要预知基因组的序列特征,可适用于任何来源和各种复杂度的DNA,兼具RFLP技术的可靠性和RAPD技术的方便性。近年来,AFLP技术已在干果遗传育种研究中得到广泛应用。本文对AFLP技术的基本原理和反应程序进行了介绍,综述了其在干果种质资源研究、遗传图谱构建、基因标记、分子辅助选择、基因表达等方面的应用,并对AFLP技术存在的问题及其在干果遗传育种研究上的应用前景进行了探讨。     

Clustering of amplified fragment length polymorphism markers in a linkage map of rye

Amplified fragment length polymorphisms (AFLPs) are now widely used in DNA fingerprinting and genetic diversity studies, the construction of dense genetic maps and in fine mapping of agronomically important traits. The AFLP markers have been chosen as a source to extend and saturate a linkage map of rye, which has previously been generated by means of restriction fragment length polymorphism, random amplified polymorphic DNA, simple sequence repeat and isozyme markers. Gaps between linkage groups, which were known to be part of chromosome 2R, have been closed, thus allowing the determination of their correct order. Eighteen EcoRI‐MseI primer combinations were screened for polymorphism and yielded 148 polymorphic bands out of a total of 1180. The level of polymorphism among the different primer combinations varied from 5.7% to 33.3%. Eight primer combinations, which revealed most polymorphisms, were further analysed in all individuals of the F₂ mapping population. Seventy‐one out of 80 polymorphic loci could be integrated into the linkage map, thereby increasing the total number of markers to 182. However, 46% of the mapped AFLP markers constituted four major clusters located on chromosomes 2R, 5R and 7R, predominantly in proximity to the centromere. The integration of AFLP markers caused an increase of 215 cM, which resulted in a total map length of almost 1100 cM.     

Mapping of EST-derived CAPS markers in Italian ryegrass (Lolium multiflorum Lam.)

New molecular markers derived from expressed sequence tag (EST) sequences were mapped on linkage maps of Italian ryegrass by a two-way pseudo-testcross strategy. cDNA sequences were obtained from various tissues of Italian ryegrass ( Lolium multiflorum ) and converted into cleaved amplified polymorphic sequence (CAPS) markers. Of 260 EST primer pairs that amplified a single band, 74 generated bands that showed clear polymorphisms among individuals of an F₁ mapping family. Of the 74 polymorphic marker loci, 69 were mapped on an Italian ryegrass linkage map previously constructed using amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) markers. The newly-developed EST-CAPS markers would be useful as an efficient tool to identify genetic markers and to identify candidate genes for quantitative trait loci (QTLs) associated with important traits in Italian ryegrass.     

Mapping a new source of resistance to powdery mildew in mungbean

Both restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) analyses were employed to map a new source of resistance to powdery mildew in mungbean. Disease scores of an F₂ population derived from the cross between a moderately resistant breeding line VC1210A and a susceptible wild relative (Vigna radiata var. sublobata, accession TC1966) showed a continuous distribution and was treated as a quantitative trait. Although no significant quantitative trait loci (QTL) that can explain the variation was detected by QTL analysis based on the reconstructed RFLP linkage map, new marker loci associated with resistance were discovered by AFLP analysis. The RFLP loci detected by two of the cloned AFLP bands are associated with resistance and constitute a new linkage group. A major resistance quantitative trait locus was found on this linkage group that accounted for 64.9% of the variation in resistance to powdery mildew. One of the probes developed in this study has the potential to assist in breeding for powdery mildew resistance in mungbean.     

AFLP标记在植物遗传多样性研究中的应用

遗传多样性是生物多样性的基础。AFLP技术由于具有众多分子生物学分析方法无法比拟的特点,近几年来在遗传多样性研究中广泛应用,就其在植物种质鉴定、遗传图谱构建、目的基因定位、遗传多态性检测、群体遗传结构、分子标记辅助育种等方面的应用进行了综合评述,并对AFLP的原理、特点及发展前景进行了阐述     

高温胁迫下不同玉米花粉组蛋白Zm-H3基因的克隆及表达分析

为了解组蛋白H 3基因(Zm-H3)在不同耐高温玉米(Zeamays L.)材料中的差异表达情况,以中地88(耐高温)和先玉335(不耐高温)2个玉米品种为材料,在高温条件下取其花粉提取总RNA,再通过mRNA纯化、反转录、文库构建及高通量测序等步骤,克隆了一个玉米组蛋白Zm-H3.生物信息学分析表明,该基因位于玉米第...     

基于10个棉花腺体相关材料转录组的EST-SSR标记开发

棉花是重要的经济作物,但有毒棉酚的存在,使棉子得不到充分的利用,所以低酚棉育种是棉花育种的重要内容之一。色素腺体是棉酚的储存器官,开发功能型分子标记,加密棉花遗传图谱对色素腺体和其他重要性状相关基因的研究具有重要作用。本试验以10个有腺体和无腺体材料转录组信息为基础开发EST-SSR标记,在12895条1 kb以上的Unigene中共搜索到1546个SSR位点,发生频率和平均距离分别是11.99%和15.99 kb。得到的EST-SSR中,二碱基和三碱基重复是主要的重复类型,分别占30.85%和48.97%,AT/TA和GAA/CTT分别是二碱基和三碱基的优势重复类型。对长度≥20 bp的SSR共设计合成了56对引物,在这10个材料中进行检测,能扩增出清晰条带的有38对,占67.86%,其中呈多态性的有9对,占23.68%,并对其所在的Unigene功能和表达量进行了初步分析。本研究进一步证明了棉花EST-SSR标记开发的可行性,并且为棉花高密度遗传图谱和腺体相关基因的研究奠定了基础。     

A genetic linkage map of rhodesgrass based on an F1 pseudo-testcross population

The linkage relationships between 164 polymorphic amplified fragment length polymorphism (AFLP) and 25 restriction fragment length polymorphism (RFLP) fragments assayed in a pseudo‐testcross population generated from the mating of single genotypes from two divergent cultivars were used to construct female, ‘Katambora’ (‘Kat’) and male, ‘Tochirakukei’ (‘Toch’) parental genetic maps for rhodesgrass. The ‘Kat’ genetic map consists of 84 marker loci (72 AFLP and 12 RFLP markers) distributed on 14 linkage groups and spans a total length of 488.3 cM, with an average distance of 7.8 cM between adjacent markers. The ‘Toch’ genetic map consists of 61 marker loci (52 AFLP and nine RFLP) mapped on 12 linkage groups spanning a total length of 443.3 cM, with an average spacing of 9.0 cM between adjacent markers. About 23% of the markers remained unassigned. The level of segregation distortion observed in this cross was 11.1%. In both maps, linked duplicated RFLP loci were found. These linkage maps will serve as a starting point for linkage studies in rhodesgrass with potential application for marker‐assisted selection in breeding programmes.     

Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.