玫瑰黄链霉菌Men-myco-93-63及其抗生素高产菌株差异蛋白分析

玫瑰黄链霉菌Men-myco-93-63是分离自马铃薯疮痂病自然衰退土壤中的一株拮抗菌。前期研究表明,该菌株及其发酵液对棉花黄萎病菌、瓜类白粉病菌等多种重要的植物病原真菌具有较强的抑制作用,具有良好的生防应用潜力。本文利用蛋白质双向凝胶电泳技术研究了Men-myco-93-63与其抗生素高产菌株D12-3之间的蛋白质表达差异。通过优化条件,建立了一套适合该生防菌的双向电泳体系。通过比较蛋白质图谱的差异,发现了四个差异蛋白,质普分析表明,四个蛋白点分别为聚羟基脂肪酸（PHAs）的包涵体蛋白（PhaP）,β-内酰胺酶(Beta-lactamase),甲基接受趋化蛋白(methyl-accepting chemotaxis protein)和ABC转运体(ABC transporter, ATP-binding protein),这些蛋白可能与该生防菌的抗性有关.     

黄瓜果皮品质性状AFLP分析条件的优化

以果皮性状差异明显的两个黄瓜自交系0349-5和0345-2为试材,提取高质量的DNA,利用AFLP技术和64对引物对两个亲本DNA进行了分析,共得到79条差异带,每对引物平均产生1.23条差异带,多态性带的比例为1.4%,其中有8对引物表现的差异带多于6条。     

利用RAPD标记鉴定山葡萄种质

采用修改后的CTAB法获得了高质量的基因组DNA.利用随机扩增多态性即RAPD标记对山葡萄7份种质进行鉴定,用4个引物(从30个引物中筛选)对试材进行PCR扩增,共扩增出30条谱带,平均每条引物产生7.5条谱带,其中21条谱带为多态性谱带,占总谱带数的70%.不同引物扩增的谱带数不同,范围在6～9条之间.利用4个引物扩增出的多态性谱带可以将7份山葡萄种质区分.     

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。     

离子束介导的水稻早熟变异株系AFLP分析

用AFLP分子标记技术对N+离子束介导玉米总DNA获得的2个稳定遗传至第6代的水稻早熟变异株系C(08-5-121-6-1)和D(08-5-121-6-2)进行分析。首先从64对引物中筛选出10对多态性较好的引物,然后用这10对引物对早熟变异株系C和D分别扩增。结果显示,变异株系C、D和阴性对照水稻B(豫粳6号)与阳性对照玉米A(郑单14)之间的AFLP扩增图谱相似率分别为15.7%、16.2%和11.6%,说明变异株系与对照豫粳6号的AFLP扩增图谱存在显著差异。变异株系C和D与阴性对照豫粳6号相比,分别扩增出50条和58条差异带,其中新带分别为25条和35条,缺失带分别为19条和15条,变异株系C和D分别扩增到与阳性对照玉米相一致的目的带6条和8条,说明变异株系C和D基因组DNA与玉米基因组DNA相似性高于对照水稻。     

利用AFLP技术鉴定凤凰单丛古茶树种质资源

本文采用AFLP技术对34个凤凰单丛古茶树资源进行了种质鉴定分析。结果显示,筛选出多态性较高的5对引物中,引物组合E41M42、E41M39和E41M33可鉴别出供试的全部资源,鉴别效率为100％;有23份资源具有特异带,占供试材料的67．6％。由此研究表明,AFLP技术可以通过特异带、特异的谱带类型、不同引物提供谱带的组合将凤凰单丛古茶树资源区分开来,这对保护凤凰单丛古茶树资源的育种产权、登录新品种、鉴定和检测其种子与苗木的真实性和纯度具有重要的现实意义。     

利用RAPD和AFLP标记分析烟草种质资源的遗传多样性

从200条10 bp的RAPD引物中筛选获得28条多态性引物，对48份烟草材料的基因组DNA进行扩增。共获得184条DNA扩增片断带，其中多态性带86条，平均多态检出率为46.7%。用4对AFLP选择性扩增引物对48份烟草材料进行选择性扩增，共获得321条扩增带，其中174条具有多态性，平均多态检出率为54.2%。根据RAPD和AFLP标记的结果，采用UPGMA法进行聚类分析，两种方法获得了相似但不完全相同的结果，两者都可以将48份烟草资源分为两大类群，即黄花烟草(Nicotiana rustica)群和普通烟草(N.tobacum)群，普通烟草可进一步分为4组；48份材料的遗传距离变幅在1.4～11.0之间，其聚类结果与理论上所期望的结果基本一致，AFLP标记技术比RAPD标记技术能更好地从分子水平上揭示烟草种质资源的遗传背景、亲缘关系及演化规律。     

稻瘟病菌突变菌株的分子鉴定

摘要：利用Rep-PCR的两对引物Pot2-1/Pot2-2和PMC1-2/PMC1-3、 177条RAPD引物和MGR586为探针的3种RFLP 分子技术对温室的9个和自然病圃上的5个致病性突变菌株进行分析。3种技术鉴定了温室9个菌株与其起始菌株的DNA指纹基本一致，检测到7个菌株DNA条带缺失1~3条，2个菌株没有变化；分析了病圃上的侵染携带抗病基因Pi-1和Pi-2的5个突变株的起源，依据DNA指纹和致病类型证明后者起源于具有无毒基因Avr-1的菌株，排除了外来菌株的漂移作用，证实了突变是稻瘟病菌(Magnaporthe grisea )进化的主要动力。     

利用SSR和RAPD标记分析中国部分地区晚疫病菌群体遗传多样性

利用SSR和RAPD对来自福建、黑龙江、河北和内蒙古4个地理群体的80个马铃薯晚疫病菌（phytophthora infestans）株进行了遗传多样性分析。13对SSR引物共扩增出76条谱带,多态性条带比率78.9%,相似系数变化范围0.00～0.42之间;筛选出的14条RAPD引物共扩增出189条谱带,多态性条带比率95.2%,相似系数变化范围0.04～0.66之间。遗传多样性分析表明,在4个群体中,福建群体的多样性更为丰富。遗传相似性分析显示,黑龙江和内蒙古两个群体间的遗传相似性最高,而福建和河北两个群体间的遗传相似性最低。聚类分析显示,来自南方福建的菌株与来自北方黑龙江、河北和内蒙古的菌株亲缘关系较远,且福建群体分布于更多的聚类组,显示出更高的遗传变异度。     

玫瑰黄链霉菌Men-myco-93-63及其抗生素高产菌株差异蛋白分析

玫瑰黄链霉菌(Streptomyces roscoflavus)Men-myeo-93-63是分离自马铃薯疮痂病自然衰退土壤中的一株拮抗菌.该菌株及其发酵液对棉花黄萎病菌(Verticillium dahliae)和瓜类白粉病菌(Sphaerotheca fuliginea Poll)等多种重要的植物病原真菌表现出较强的抑制作用,具有良好的生防应用潜力.利用蛋白质双向凝胶电泳技术研究了Men-myco-93-63与其抗生素高产菌株D12-3之间的蛋白质表达差异.通过优化条件,建立了一套适合该生防菌的双向电泳体系.通过比较蛋白质图谱的差异,发现了4个差异蛋白.质谱分析表明,这4个蛋白点分别为聚羟基脂肪酸(PHAs)的包涵体蛋白(PhaP)、B-内酰胺酶(Beta-lac-tamase)、甲基接受趋化蛋白(methyl-accepting chemotaxisp rotein)和ABC转运体(ABC transporter,ATP-binding protein).这些蛋白可能与该生防菌的拮抗活性有关.     

Genetic diversity analysis of hulless barley from Shangri-la region revealed by SSR and AFLP markers

For adding the hulless barley resources of Shangri-la region to the global barley resource library, a basic work was done by us to assess their genetic diversity of this region. The genetic diversity of 60 hulless barley samples collected from three counties in Shangri-la region of Yunnan Province, were studied using SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) markers. A total of 70 alleles were detected for 19 pairs of SSR primers, and 525 band containing 464 polymorphic bands were revealed for 5 pairs of AFLP primers. The value of polymorphism information content (PIC) ranged from 0.03 to 0.86 for SSR primers. The total numbers of alleles were 51, 55, 43 in three populations and the polymorphic bands were 188, 205 and 141. The genetic distances and genetic identity among the three populations showed their close relationship. The gene diversity among populations relative to the total population diversity (Gst) was 0.13 for SSR markers and 0.02 for AFLP markers and indicated that just 13 and 2% variations were among populations, respectively. The UPGMA cluster analysis revealed that all of the samples grouped randomly rather than clustered into distinct groups corresponding to their populations, row types and spring/fall types. We concluded that there was high genetic diversity in the population of Shangri-la region and the formation of diversity was related to complex environment and inhabitants’ traditional practices.     

Genetic diversity and differentiation in Ethiopian populations of Phytolacca dodecandra as revealed by AFLP and RAPD analyses

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analyses were performed on six populations (a total of 89 individuals) of Phytolacca dodecandra (endod) collected in Ethiopia. The populations were selected based on our previous investigation to represent two altitude groups: lowland/central-highland (1600–2500 m) and highland (2501–3000 m). A total of 197 AFLP and 68 RAPD markers were scored from 5 primer pairs and 12 random primers, respectively. The overall patterns obtained for AFLPs and RAPDs from diversity, cluster and principal component analyses were very comparable. However, the moderate correlation (r = 0.56) between AFLP and RAPD similarity matrices as well as the discrepancies in diversity estimates between the two techniques in some populations and in the lowland/central-highland plants could be due to differences in sensitivity of reaction conditions, bias in scoring of bands, number of markers used for analyses, and/or parts of the genome surveyed. For both AFLP and RAPD, the lowland/central-highland populations showed higher polymorphism and Shannon information measure (H) than the highlands. Cluster and principal component analyses performed for both marker types have also clearly demonstrated the differentiation of all the lowland/central highland plants from those of the highlands, in agreement with our previous conclusion. Markers scored from any of the five AFLP primer pairs were sufficient to clearly distinguish the two altitude groups; with RAPD, selection of about 8 informative markers produced by seven random primers was needed for the same purpose.     

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.     

马铃薯遗传资源多样性的SRAP分析

采用SRAP分子标记，对44份马铃薯品种的遗传多样性进行了分析。结果显示, 随机抽取27对SRAP引物，对基因组DNA进行特异扩增，其中23对引物扩增出多态性条带，共获得104个多态性条带, 多态性引物比率达85.2%, 平均每对引物产生4.5个多态性条带，表明SRAP标记具有较高的多态性比率。44份种质资源的SRAP标记遗传距离为0.147-0.741。当遗传距离D=0.67时，将44份种质资源分为四大类群，包括一个复合类群和三个独立类群。其中复合类群可进一步分为7个亚群。从而在DNA水平上证明了所研究马铃薯种质资源具有丰富的遗传多样性。     

Constructing DNA fingerprinting of Hemarthria cultivars using EST-SSR and SCoT markers

DNA fingerprinting of known cultivars may provide much-needed data to assist with the identification of such cultivars. From 86 pairs of expressed sequence tag SSRs (EST-SSR) and 45 start-codon targeted polymorphism (SCoT) primers, eight pairs of EST-SSR primers and seven SCoT primers were chosen to construct the DNA fingerprinting of six Hemarthria cultivars in this study. Using genomic DNA from six cultivars, a total of eight pairs of EST-SSR primers were able to amplify 193 bands, producing an average of 24.1 bands per primer pair. The percentage of polymorphic bands (PPB) was 83.4 %, and the polymorphism information content (PIC) ranged from 0.480 to 0.695, with an average of 0.602. A total of seven SCoT primers amplified 105 bands with an average of 15 bands per sample. The PPB was 84.8 %, and the PIC ranged from 0.471 to 0.758, with an average of 0.612. The unweighted pair-group method with arithmetic mean dendrogram from EST-SSR and SCoT markers grouped the six Hemarthria cultivars into two major groups of the same. These clusters are in accordance with their known species and origin. Our results indicate a rich genetic diversity in these six Hemarthria cultivars. The six cultivars we assayed could be easily identified using these eight pairs of EST-SSR and seven pairs of SCoT primers.     

鲮微卫星DNA分子标记的筛选与遗传多样性分析

采用磁珠富集法对已建立的生长较快的西江段野生浅色鲮群体的极大、极小群体各31尾基因组DNA进行了微卫星序列的筛选,128个阳性克隆成功测序93个,其中80个为的微卫星序列,微卫星序列完美型占85％,非完美型占6．25％,混合型占8．75％。利用微卫星序列合成了23对微卫星引物,扩增结果表明,等位基因数为1～10个,扩增片段大小为92～283bp,其中14对引物扩增条带清晰,为中度或高度多态位点,极大、极小群体的平均有效等位基因数和平均多态信息含量分别为3．0784、3．2079和0．5675、0．5974,反映出2个群体遗传多样性均较丰富;高度多态性引物4的位点b,片段大小为119bp,在极大群体中出现频率为61．3％,在极小群体占22．6％,出现频率极大群体高于极小群体近3倍,初步可作为候选差异性分子标记。     

甘蔗抗褐锈病基因定位亲本间多态性SSR标记筛选

为获得更多用于构建甘蔗遗传连锁图谱可用的多态性SSR标记,本研究以6个高抗甘蔗褐锈病品种和6个高感甘蔗褐锈病品种为亲本,根据感病母本×抗病父本选配12个杂交组合,筛选在亲本间条带清晰、多态性明显、重复性较好的引物。结果表明,组合柳城03-1137×德蔗93-88、Mex105×粤糖00-236亲本间的多态性引物数最多,分别占总数的52.38%和47.62%;其中,10对引物(18-mSSCIR34、22-mSSCIR38、25-mSSCIR48、32-mSSCIR67、51-mSSCIR50、57-MSSCIR21、71-SMC1490CL*、73-SMC278CS*、75-SMC31CUQ*、77-SMC336BS*)在组合柳城03-1137×德蔗93-88中多态性最好,7对引物(22-mSSCIR38、25-mSSCIR48、45-mSSESTC04、51-mSSCIR50、67-mSSCIR9*、76-SMC334BS*、80-SMC486CG*)在Mex105×粤糖00-236中多态性最好,利用筛选得到的引物,更有利于构建分子遗传图谱。本研究结果为抗褐锈病新基因定位和开发与其紧密连锁的分子标记研究提供了一定的理论依据。     

两个罗非鱼杂交组合亲本群体与子一代的AFLP分析

运用AFLP指纹技术分别对尼罗罗非鱼和奥利亚罗非鱼及它们的杂交子一代、橙色莫桑比克罗非鱼和荷那龙罗非鱼及它们的杂交子一代的遗传结构进行分析。从90对选扩引物组合中筛选出8对进行PCR扩增，8对引物组合共检出250个扩增位点（片段大小100～500bp）。每一个体的每对引物扩增条带数在20～47之间。群体内遗传相似度从高到低依次为：奥利亚罗非鱼（0.913），尼罗罗非鱼（0.871），荷那龙罗非鱼（0.829）和橙色莫桑比克罗非鱼（0.784）。奥利亚罗非鱼和尼罗罗非鱼间的遗传相似度是0.811，遗传距离为0.189；奥利亚罗非鱼和尼罗罗非鱼与杂交子一代的遗传距离分别为0.165、0.142。橙色莫桑比克罗非鱼和荷那龙罗非鱼间的遗传相似度0.756，遗传距离为0.244；橙色莫桑比克罗非鱼和荷那龙罗非鱼与杂交子一代的遗传距离分别为 0.148、0.162。引物组合E-AGG/M-CTT在尼罗罗非鱼、E-ACA/M-CAC在尼奥鱼中扩增出差异的DNA片段，可作为区分奥利亚罗非鱼和尼罗罗非鱼与杂交子一代的分子标记。