甘薯抗茎线虫病基因AFLP标记的开发

以甘薯(Ipomoea batatas)抗茎线虫病品种徐781和感茎线虫病品种徐薯18杂交F1分离群体的186株单株为材料,利用分离群体分组分析法(BSA法)和AFLP技术,在抗感池中共筛选了800对AFLP引物,结果表明,其中245对引物具有多态性.用这245对引物检测两亲本以及建池单株,发现引物组合E2M23和E33M20分别在抗病单株中扩增出l条在感病单株中未出现的特异条带,长度分别约为500和200 bp,认为这2个AFLP标记与甘薯抗茎线虫病基因连锁,分别命名为E2M23500和E33M20200.根据这2个AFLP标记对F1代186个单株的扩增结果,经Mapmaker 3.0软件分析,发现这2个分子标记与抗茎线虫病基因位于同一连锁群并紧密连锁,它们与抗茎线虫病基因间的遗传距离分别为6.9和11.1 cM.用这2个分子标记对10个中国甘薯主栽品种进行检测,所得结果与常规方法鉴定结果完全一致,表明2个分子标记可用于甘薯抗茎线虫病分子标记辅助育种.     

与黄瓜感花叶病毒病基因连锁的分子标记

本研究以黄瓜(Cucumis sativus L.)高感黄瓜花叶病毒(CMV)和高抗CMV亲本组合(HZL04-1×F-3)的F2分离群体为试材,采用极端个体分组法和AFLP技术筛选与黄瓜抗CMV基因连锁的分子标记.AFLP引物组合E22M88在抗病组和感病组间约200 bp处表现多态性.经F2单株分析,在感病亲本和感病单株中扩增出了约为200 bp的特异片段,而抗病亲本和抗病个体无此条带.该特异标记与CMV抗性基因相连锁,遗传距离为9.74 cM.测序结果显示,目标片段的大小为202 bp,并将该标记转换为了序列特征化扩增区域(SCAR)标记.提供了黄瓜材料CMV抗性鉴定的分子方法和手段,可用于分子标记辅助育种.     

甘薯抗茎线虫病基因的RAPD标记

以甘薯（Ipomoea batats）高抗品种徐781和高感品种徐薯18的200个后代为实验材料，对其进行抗病性鉴定和RAPD分析，获得与抗茎线虫病基因相连锁的RAPD标记OPD01-700。经13个高抗后代、5个高感后代、8个感病后代以及已鉴定的具有稳定抗性的10个品种验证表明，该标记可作为甘薯抗茎线虫病辅助育种的分子标记。     

甘薯肌醇-1-磷酸合成酶基因的克隆及序列分析

甘薯(Ipomoea batatas (L.) Lam.)新品系农大603是从感茎线虫(Ditylenchus destructor)病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。以农大603和徐薯18块根的mRNA为模板，根据植物抗线虫病基因NBS保守氨基酸序列设计引物，进行 RT-PCR分析，发现农大603的肌醇-1-磷酸合成酶(Myo inositol-1-phosphate synthase , MIPS)基因的表达量高于徐薯18。采用3'RACE技术扩增出MIPS基因的3'末端cDNA。根据植物MIPS基因 5'端一段保守的氨基酸序列设计兼并引物，并与3'端的特异引物组合，扩增出该基因的5'端cDNA序列。DNA序列比对表明，甘薯MIPS基因与大豆(Glycine max)、番茄(Lycopersicon esculentum )的MIPS基因同源性较高，分别达83.63 %和83.89 %。甘薯MIPS基因全长cDNA的克隆，有利于进一步研究该基因与抗甘薯茎线虫病的关系。     

西瓜抗炭疽病的遗传分析和抗性基因定位研究

西瓜炭疽病(Anthracnose)是由瓜类炭疽病菌(Colletotrichumorbiculare)引起的真菌性病害。本研究以抗病自交系PI189225和感病自交系Black Diamond杂交并自交获得F1、F2、F3为材料,采用炭疽病菌生理小种1接种,对西瓜抗炭疽病生理小种1进行遗传规律分析和基因定位研究。研究结果表明,西瓜炭疽病抗性基因由显性单基因控制,抗病对感病为显性,将此基因命名为Rco-1。用分离群体分组分析法(BSA)和AFLP分子标记技术对PI189225中的抗炭疽病基因进行分子标记鉴定,并利用MAPMAKER/Exp version 3.0软件进行了标记与目的基因间的遗传距离计算,发现E4/M19、E1/M8、E29/M5与抗炭疽病基因Rco-1连锁,遗传距离分别为34.8、23.4、6.9cM。为采用分子标记辅助选育抗炭疽病西瓜新品种奠定了基础。     

基于简化基因组测序技术的甘薯HRM分子标记开发及其应用

目前甘薯中针对SNP位点的分子标记开发及应用的报道较少。为开发甘薯特异SNP分子标记,本研究利用简化基因组测序技术对甘薯种质材料进行测序,筛选稳定的SNP位点,将其开发为基于HRM技术的分子标记,并在甘薯种质材料中进行验证。通过对23个甘薯种质材料简化基因组测序数据的分析,共发现835 756个SNP多态性位点,筛选其中的3 650个含有SNP多态性位点的高质量测序片段,成功设计了134对引物;初步验证发现,22对(16.42%)引物没有扩增产物,15对(11.19%)引物扩增产物2个以上,36对(26.87%)引物的扩增产物没有差异。61对(45.52%)引物在8个样本中的扩增特异性好,而且样本之间有差异。最后筛选34对扩增多态性丰富的引物对52份甘薯种质材料进行扫描,发现材料间多态性介于27.27%~90.91%之间,平均多态性达到59.35%。聚类分析表明,参试52份甘薯种质材料之间的差异较小,多数材料与骨干亲本南瑞苕、徐薯18之间关系较近,国外引进甘薯材料和地方品种差异较大。建议在今后甘薯育种中尽量选用地方品种和国外甘薯品种,从而更好地拓宽甘薯遗传背景。本研究初步建立了基于简化基因组技术和HRM技术开发甘薯SNP分子标记的新思路,为今后甘薯SNP分子标记开发提供了参考。同时本研究开发的甘薯分子标记,丰富了甘薯分子标记类型,为甘薯研究者开展快速的分子标记研究提供了支持。     

棉花极端矮化突变体AS98性状与分子标记分析

AS98是从一个棉花海陆种间杂交后代中发现的自然突变体,该突变体株高极端矮化,株高仅为25.6cm,叶片为正常阔叶。但是与正常棉花品种LHF10W99比,AS98的铃重、籽指显著减小,净光合速率降低,分别降低68.88%、49.45%和29.69%;遗传分析表明AS98的极端矮化性状受一对不完全显性基因控制;利用SSR和AFLP分子标记方法,以AS98与正常型品种LHF10W99杂交的F2分离群体为分子标记群体,发现一个与该极端矮化性状连锁的SSR标记(GH537)和一个AFLP标记E4M13(E-ACA,M-CGA),与AS98的矮化性状的遗传距离分别为4.9cM和9.7cM。通过对其农艺性状及其分子标记定位研究,可为该材料在棉花育种的应用提供理论指导。     

黄瓜抗西瓜花叶病毒性状的序列特征性扩增区域(SCAR)标记

以抗西瓜花叶病毒 (Watermelon mosaic virus, WMV)的黄瓜（Cucumis sativus）品种秋棚和感该病毒的欧洲八号为亲本,构建了120个F6重组自交系群体。根据苗期抗病接种鉴定结果进行遗传分析,发现黄瓜抗WMV的基因是由隐性单基因控制。利用集团分离分析法和相关分子标记技术,获得的特异片段E-ACT/M-CTT-427与WMV的抗性基因连锁,连锁距离为8 cM。双亲差异片段测序结果显示感病亲本目标片段存在6个T碱基的缺失。根据测序结果设计了1对与目的基因遗传距离为8 cM的序列特征性扩增区域(SCAR)引物,该引物可用于黄瓜抗WMV病毒的分子标记辅助育种。     

苦瓜AFLP分子标记反应体系的建立

利用AFLP分子标记技术,采用MseI/EcoRI酶切组合,从64对引物中筛选出6对带型分布均匀、多态性高且分辨能力强的引物,分别为:E1/M5、E4/M8、E6/M4、E7/M5、E7/M7、E8/M3,并确定了适用于苦瓜AFLP反应的最佳酶切-连接、预扩增和选择性扩增反应体系,从而为今后利用AFLP分子标记技术对苦瓜进行深入研究打下基础。     

我国“应县小黑豆”对SCN4号生理小种抗性的SSR及ISSR分子标记研究

本研究以"应县小黑豆"×"晋豆23"杂交组合F2群体为材料,用塑膜钵柱法进行抗性鉴定,利用分离体分组分析法(BSA)结合SSR和ISSR标记技术对抗性基因进行分子标记筛选和定位,旨在实现大豆孢囊线虫抗性育种中亲本和杂交后代的分子标记辅助选择.筛选到2个与抗SCN基因座位连锁的分子标记Satt038和ISSR811.Satt038片段含180bp和185 bp,为共显性标记,在F2群体中的分离比为121;ISSR标记ISSR811为显性标记,片段长350bp,F2群体分离比为13.2个标记在F2群体中呈孟德尔式遗传.通过JOINMAP分析,微卫星标记Satt038、ISSR标记ISSR811与抗SCN基因座位的遗传距离分别为26.9cM和10.2 cM.还探讨了分子标记Satt038和ISSR811用于大豆抗SCN育种进行分子标记辅助选择的可能性.     

与辣椒抗根结线虫基因Me1紧密连锁的EST-SSR标记开发

以含抗根结线虫病基因Me1的PM217与感线虫品种茄门,及其F1、F2作为试验材料,采用南方根结线虫（Meloidogyne spp.）人工接种鉴定,根据鉴定结果,利用分离群体分组分析法（bulked segregantanalysis,BSA）建立抗感池,共筛选到3对（118、141及211）多态性EST-SSR引物...     

辣椒抗疫病性状遗传及其相关AFLP标记分析

以93-100-17-1-0×茄门组合为基础，建立F2代作图群体。从感受性、诱导性和稳定性等3个方面进行遗传分析，发现93-100-17-1-0的抗疫病性状是由多基因或寡基因所控制的。根据AFLP分析，利用Mapmaker/exp (Version 3.0)作图软件构建辣椒的分子连锁图谱，结合winQTLCART(Version 1.15)，对辣椒抗疫病的基因定位，将与感受性有关的QTL位点定位在第5条连锁群上，与诱导性有关的QTL位点分别定位在第1、第2、第5、第8条连锁群，与稳定性有关的QTL位点定位在第1、第2、第5、第7条连锁群，各QTL位点可解释表型变异率在64%以上。     

小麦抗秆锈突变系龙辐03D51的筛选及其抗病性的遗传分析与RAPD标记

以龙6239幼胚为外植体,利用辐射诱变和组织培养相结合的方法,选育出突变系龙辐03D51,经秆锈接种鉴定,发现其对优势小种21C3CPH免疫,而亲本龙6239对21C3CPH高度感染。遗传分析表明,抗病性由显性单基因控制。该突变系还具有其亲本的优质、高产特点,已成为优异的后备品系。在RAPD检测中,所用的60个随机引物中有3个引物在龙辐03D51和其亲本龙6239中具有多态性,引物E07、E11、E17在龙辐03D51中分别扩增出380bp、700bp和600bp的特异带,初步认为这些谱带可能与秆锈抗性有关。     

Identification of sex-specific DNA markers in betel vine (Piper betle L.)

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in male and female plants of Piper betle L. Two bulks of DNA were made drawing one each from male and female, by pooling an equal volume of DNA samples from each group of individual contributing to the bulk analysis. Fifty different random decamer primers were screened with the two bulks to identify markers associated with sex expression of which only four primers were found to be associated with sex expression. These four primers were then tested with individual plant DNA samples where sex-associated RAPD markers were identified. A ~1,400 and ~850?bp fragment from the primer OPA04 and OPN 02 respectively was found to be present in all the male individuals and absent in all the female plants. In another primer, a ~980?bp amplification product from the primer OPC 06 was present only in the female individuals. A common primer OPA 08 showed both male and female specific markers of 650 and 1,200?bp respectively. Thus, the three male- specific RAPD markers OPA04₁₄₀₀, OPA08₆₅₀ and OPN02₈₅₀ and two female-specific markers OPA08₁₂₀₀ and OPC06₉₈₀ can reliably differentiate the male and female plants of P. betle L. Ploidy comparison also showed the differences in male and female plants.     

Highly polymorphic AFLP markers as a complementary tool to ITS sequences in assessing genetic diversity and phylogenetic relationships of sweetpotato (Ipomoea batatas (L.) Lam.) and its wild relatives

Comparative analyses of genetic diversity and phylogenetic relationshipsof sweetpotato (Ipomoea batatas (L.) Lam.) and its wildrelatives in Ipomoea series Batataswere conducted using amplified fragment length polymorphism (AFLP) and sequencedata from the internal transcribed spacer (ITS) region of the ribosomal DNA. LowITS divergence among thirteen species of ser. Batatasresulted in poorly resolved relationships. More variable AFLP characters werefound to be more efficient in characterizing genetic diversity and phylogeneticrelationships at both intra- and interspecific levels within ser.Batatas. Highly informative AFLP fingerprints of 36accessions representing 10 species of ser. Batatas weregenerated using only six primer combinations. Of the species examined,I. trifida was found to be the mostclosely related to I. batatas, whileI. ramosissima andI. umbraticola were the most distantlyrelated to I. batatas. The highlypolymorphic AFLP markers are a valuable tool in assessing genetic diversity andphylogenetic relationships of sweetpotato and its wild relatives.     

人工合成小麦衍生品种川麦47的抗条锈病SSR分子标记定位

条锈病是我国小麦最重要的病害之一，严重威胁小麦生产。川麦47是利用高抗条锈硬粒小麦-节节麦人工合成种基因资源与四川高产小麦绵阳26杂交、有限回交育成的高抗条锈病小麦新品种。为明确川麦47抗条锈性遗传基础，将川麦47分别与高感条锈小麦品种台长29杂交，获得杂交F1、F2群体；对川麦47与台长29构建的F2群体(355株)进行了条锈病抗性鉴定和遗传分析，结果表明，川麦47携带一个显性抗条锈病基因；利用SSR分子标记技术和F2分离群体分组分析法研究表明，该抗条锈病基因位于小麦1B染色体上，与微卫星分子标记Xgwm11、Xgwm18、Xgwm273和Xgwm498紧密连锁，遗传距离分别为2.2CM，2.2CM，4.5CM，3.9CM。川麦47可用于分子标记辅助育种。     

Exploring genetic diversity and disease response of cultivated rice accessions (<Emphasis Type="Italic">Oryza</Emphasis> spp.) against <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> under rainfed upland conditions in Benin

The main goal of this study is to gain insight into the relationship between the genetic profile of cultivated rice (Oryza spp.) accessions and their resistance to rice blast. Therefore, the genetic and phenotypic variability of a set of 350 cultivated rice accessions originating from Africa (Benin, Mali and Nigeria, Ivory Coast etc.) was examined. Seventy-seven fluorescent amplified fragment polymorphism (AFLP) markers were used to gain insight into the genetic variation and to classify the germplasm collection. In addition, the rice germplasm was assessed for its resistance to blast disease caused by Pyricularia oryzae in upland field conditions. Huge differences in responses of rice accessions to P. oryzae were observed, ranging from highly susceptible to highly resistant. Twelve percent of all accessions were highly resistant to P. oryzae. Based on their AFLP marker profile these highly resistant accessions could be separated from the other accessions. Stepwise regression revealed that the best prediction of the blast resistance level was achieved with a maximum number of 13 AFLP markers. Marker CTA22 was the most important for accurate prediction of blast resistance, this marker was present in all highly resistant accessions. It can be concluded that AFLP markers are a valuable tool to screen rice accessions for their susceptibility towards blast disease and that, based on a subset of markers, it is possible to predict the resistance to rice blast.