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.     

三种主要黄瓜病毒抗性基因的定位

以黄瓜(Cucumis sativus L.)欧洲八号×秋棚为亲本构建的重组自交系(RIL)群体为材料,以病情指数为指标,对群体和亲本进行了小西葫芦(Cucurbitapepo L.)黄化花叶病毒(Zucchini yellow mosaic virus,ZYMV)、番木瓜环斑病毒西瓜株系(Papaya ringspot virus watermelon strain,PRSV-W)和西瓜花叶病毒(Watermelon mosaic virus,WMV)的抗病性鉴定.结果表明3种病毒的病情指数在黄瓜RIL群体呈双峰分布,表明对ZYMV、PRSV-W和WMV的抗性是受主基因控制的性状.利用Joinmap软件进行连锁分析,发现ZYMV和PRSV-W与WMV连锁,3个抗病基因的排列顺序和遗传距离是WMV-6cM-ZYMV-7cM-PRSV-W.根据AFLP、SSR和RAPD标记结果构建了一个黄瓜遗传图谱,3个抗病毒病基因被定位于第二连锁群.     

甘薯抗茎线虫病基因的AFLP标记的开发

以甘薯抗茎线虫病品种徐781和感茎线虫病品种徐薯18的杂交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1条在感病单株中未出现的特异条带,长度分别约为500 bp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6.94 cM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。     

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.     

黄瓜果实苦味(Bt)基因的插入缺失(Indel)标记

为了筛选与黄瓜果实苦味(Bt)基因紧密连锁的插入缺失(Indel)标记,本研究以黄瓜(Cucumis sativusL.)果实苦味和果实不苦的高代纯合自交系46GBt和931为亲本构建的F2分离群体为试材,明确了黄瓜果实苦味性状遗传是符合单显性基因控制的质量性状.同时在完成Bt基因初步定位和双亲重测序的基础上,利用生物信息学分析双亲在初步定位区域的序列差异,设计了7对InDel标记.通过对含有184个单株的F2群体分析,获得了与Bt基因连锁距离为0.8 cM的Indel标记Bt-InDel-1.利用不同于本研究且包含58个单株的黄瓜F2群体材料对Bt-InDel-1的验证分析表明,该标记检测的正确率达到94.8％.本研究结果可用于无苦味黄瓜的分子标记辅助选择(MAS)育种及Bt基因精细定位.     

小麦抗叶锈病基因Lr2c的SSR标记

选取抗叶锈病基因位于2D染色体上的TcLr2c等7个小麦（Triticum aestivum）近等基因系、感病亲本Thatcher及215株TcLr2c与Thatcher杂交F2代为材料,研究抗叶锈病基因Lr2c SSR分子标记。从筛选的29对位于小麦2D染色体的SSR引物中获得4对能够揭示Lr2c多态性的分子标记,通过215株TcLr2c × Thatcher F2群体验证,结果表明Xgwm261和Xgwm296与Lr2c紧密连锁,其距目的基因的遗传距离分别为1.9和3.6 cM,可用于小麦抗叶锈病分子辅助育种。     

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.     

用ISSR分析四川苎麻品种(系)间的遗传关系及雄性不育分子标记的建立

本文从100条ISSR引物中筛选出21条引物,对8个四川苎麻品种(系)间的遗传关系进行了分析。PCR扩增结果表明,21条引物在8份材料中共扩增出86条带,平均每条引物扩增出4.1条,其中多态性位点71个,各引物扩增出的位点数3~8个不等,平均每条引物可以检测到3.4个多态性位点。聚类分析和遗传距离分析结果表明,供试品种川7、川8与其亲本遗传距离较远。3个杂交品种(川7、川8、川9)均表现特有的偏父本遗传现象。此外,本研究用ISSR引物U835筛选到了1个雄性不育分子标记,并将其扩增产物克隆测序,结果表明该序列大小为658bp。根据该序列,此标记被转化成稳定的SCAR标记,可用于苎麻雄性不育分子标记辅助育种。     

小麦抗叶锈病基因Lr45的表达序列标签-酶切扩增多态性(EST-CAPS)分析

为了获得与小麦(Triticum aestivum L.)抗叶锈基因Lr45更多的分子标记,建立更丰富的遗传连锁图谱,本研究利用表达序列标签-酶切扩增多态性(EST-CAPS)标记技术,选择小麦2A染色体短臂的26对EST引物与4种限制性内切酶共104对组合,对小麦抗叶锈近等基因系(near-isogenic line,NILs)TcLr45和感病对照Thatcher进行了多态性分析.EST引物BE426158与BE442876的PCR产物在亲本间存在差异,多态性检出率为7.7％; 104个引物/酶切组合中,CD454036/Msp Ⅰ、CD454036/HaeⅢ、BE406923/Msp Ⅰ和BE425962/RsaⅠ共4组在Thatcher和TcLr45之间呈现多态性,多态性检出率为3.8％.将BE426158标记成功转化为一个更稳定的序列标签位点(sequence tagged site,STS)标记,命名为LR45-1,经在TcLr45×ThatcherF2群体、抗叶锈近等基因系及黑麦品种中验证,LR45-1与Lr45连锁,遗传距离为8.2 cM.研究结果提示,EST-CAPS技术可应用于小麦抗叶锈病基因的多态性分析及分子标记的开发.     

与大白菜TuMV抗病基因TuRBCS01紧密连锁的分子标记开发

分子标记辅助选择可大大加快育种进程,然而由于大白菜抗病毒病遗传规律的复杂性,目前所定位的基因和筛选的连锁标记远不能满足育种需要,为了更好地对抗病毒病白菜(Brassica campestris ssp.pekinensis)进行分子标记辅助选择,本研究筛选了与大白菜抗芜菁花叶病毒(Turnip mosaic virus,TuMV)抗性基因TuRBCS01紧密连锁的分子标记.本研究在对该基因进行定位的基础上,利用回交1代(backcross1,BC1)分离群体,采用分离群体分组分析法(bulked segregation analysis,BSA)、简单重复序列(simple sequence repeats,SSR)和序列特异引物(sequence specific primer,SSP)标记技术,筛选与该基因紧密连锁的分子标记.通过对13对SSP引物和16对SSR引物的分析表明,有8对引物在两亲本间表现多态性,有5对引物扩增出的标记与基因TuRBCS01紧密连锁或共分离,分别为mBr4072、Bra025493-1、Bra025493-2、Bra025467-4和Bra025467-5.筛选出与大白菜TuMV抗性基因TuRBCS01紧密连锁的分子标记2个,分别为SAAS_mBr4072_240 (1.5 cM)和Bra025493-1(1.0 cM),另有3个标记与基因TuRBCS01共分离,分别为SAAS—Bra025493-2_749、SAAS_ Bra025467-4—780和SAAS—Bra025467-5_ 956.上述标记丰富了大白菜抗TuMV分子标记的数量和种类,有望用于大白菜抗病毒病分子标记辅助选择.     

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.     

与南瓜矮生基因连锁的分子标记

以中国南瓜(Cucurbita moschata Duch)矮生突变体(矮10)为供体亲本,以印度蔓生南瓜(C.maxima Duch)(蒙日南瓜)为轮回亲本,经6代回交和2代自交选育出S1~S9共9个南瓜矮生近等基因纯合株系,其中S2株系再与蒙日南瓜杂交获得F2分离群体共306株。利用RAPD技术,采用近等基因系(near isogenic lines,NILs)分析法,结果表明,在640条随机引物中,发现RAPD标记S1225-548与矮生单基因D呈现连锁关系,遗传距离为2.29cM。RAPD标记成功转化成更为稳定的SCAR标记SCAR3-398。     

与南瓜矮生基因连锁的分子标记研究

本研究以世界上首次发现的中国南瓜矮生突变体（矮10）为供体亲本，以印度蔓生南瓜（蒙日南瓜）为轮回亲本，经6代回交2代自交选育出S1—S9共9个南瓜矮生近等基因纯合株系，其中S2株系再与蒙日南瓜杂交获得F2代分离群体共306株。利用RAPD技术, 采用近等基因系（Near isogenic lines, NILs）分析法，对上述材料进行分析。在640条随机引物中，发现RAPD标记S1225-548与矮生单基因D呈现连锁关系，遗传距离为2.29cM。并且本研究进一步将此RAPD标记成功转化成更为稳定的SCAR标记SCAR3-398。     

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。     

Potide-G derived from potato (Solanum tuberosum L.) is active against potato virus YO (PVYO) infection

A PVYO virus-resistant potato (Solanum tuberosum L. cv. Golden Valley) was identified, and further, from its tubers, a small (5.57 kDa) antiviral peptide potide-G was isolated. Application of potide-G on virus susceptible potato (cv. Winter valley) expressed robust resistance to PVYO infection and showed no virus infected morphology. We found that PVYO infection spreads up completely within 3 days post inoculation (dpi) in susceptible cultivar. PVYO was more accumulated toward the basal leaves, when infection occurred longer. Combined results of morphology of PVYO infection, ELISA, RT-PCR, and real-time PCR showed the resistance to the PVYO infection depends on the expression of Ry gene. Indeed, the real-time PCR result showed that the Ry gene up-regulated to 3 times higher in PVYO infected cv. Golden valley. Golden crude protein was found to be active against PVYO infection in the in vivo test. In addition, application of potide-G in a virus susceptible potato potently reduced the viral infection actively with 50 times lower concentration than that of the Golden protein. Further identification of a host-specific resistant gene in a plant and the peptide derived from it offers new opportunities for the development of novel bio-pesticides against plant virus.     

利用分子标记辅助选择Pigm-1基因改良恢复系R20的稻瘟病抗性

水稻稻瘟病是水稻的重要病害之一,给我国甚至全世界的水稻安全生产带来了巨大隐患.选育优质抗稻瘟病恢复系,进而培育稻瘟病水稻杂交组合,是选育抗稻温病品种的有效途径之一.本研究以双抗77009为抗性基因Pigm-1的供体亲本,以感病恢复系R20为受体亲本,通过杂交和连续回交方法,并利用分子标记辅助选择技术,定向改良优质水稻恢...     

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。     

Resistance to wheat leaf rust in Aegilops tauschii Coss. and inheritance of resistance in hexaploid wheat

A collection of 164 Aegilops tauschii accessions, obtained from Gatersleben, Germany, was screened for reaction to leaf rust under controlled greenhouse conditions. We have also evaluated a selection of synthetic hexaploid wheats, produced by hybridizing Ae. tauschii with tetraploid durum wheats, as well as the first and second generation of hybrids between some of these resistant synthetic hexaploid wheats and susceptible Triticum aestivum cultivars. Eighteen (11%) accessions of Ae. tauschii were resistant to leaf rust among which 1 was immune, 13 were highly resistant and 4 were moderately resistant. Six of the synthetic hexaploid wheats expressed a high level of leaf rust resistance while four exhibited either a reduced or complete susceptibility compared to their corresponding diploid parent. This suppression of resistance at the hexaploid level suggests the presence of suppressor genes in the A and/or B genomes of the T. turgidum parent. Inheritance of leaf rust resistance from the intercrosses with susceptible bread wheats revealed that resistance was dominant over susceptibility. Leaf rust resistance from the three synthetics (syn 101, syn 701 and syn 901) was effectively transmitted as a single dominant gene and one synthetic (syn 301) possessed two different dominant genes for resistance.