利用芥蓝×青花菜DH群体构建AFLP连锁图谱

 利用青花菜(Brassica oleracea var. italica) 与芥蓝(Brassica oleracea var. alboglabra) 杂交后代双单倍体(DH) 群体为材料, 通过AFLP技术构建了一个甘蓝类作物较高密度的遗传连锁图谱。该图谱包括9个主要连锁群及2个小连锁群, 总图距801.5 cM, 含337个AFLP标记, 标记间平均距离为3.6 cM,为甘蓝类作物基因定位、比较基因组学及重要经济性状QTL分析提供了一个框架图谱。     

黄瓜全雌性基因连锁的AFLP和SCAR分子标记

 本研究以全雌品种‘戴多星’自交系和弱雌品种‘北京截头’自交系为双亲杂交获得F₁ ,然后得到F₂ 性型分离群体, 利用分离群体分组分析法(Bulked Segregant Analysis, BSA) 构建全雌和弱雌两个基因池, 筛选了64对AFLP选择性引物EcoR I-NN +Mse I-NNN组合, 发现EcoR I-TG +Mse I-CAC引物组合在全雌基因池中扩增出一条分子量为234 bp的特异带。经F₂ 代单株验证, 该特异条带能在全雌单株中稳定出现。以MAP MAKER (Version 310) 软件分析, 该标记与全雌性位点的连锁距离在617 cM。命名该连锁标记为TG/CAC₂₃₄。将该特异条带回收、克隆、测序, 设计特异SCAR引物, 再对F₂ 代单株基因组DNA进行扩增, 仅在全雌单株中扩增出1条分子量为166 bp 的特异带, 表明已成功地将与黄瓜全雌性连锁的AFLP标记转化为操作简便、表现稳定的SCAR标记, 该标记命名为SA₁₆₆。     

利用大白菜DH群体构建AFLP遗传连锁图谱

 以大白菜‘汴早- 26’和‘光90E16’的F₁ 代进行游离小孢子培养所获得的含有59个株系的DH群体为试材, 利用AFLP技术通过63对引物筛选共获得346个AFLP多态性标记, 运用JoinMap 3.0软件构建大白菜遗传连锁图谱。该图谱主要包括10个连锁群, 总图距为708 cM, 平均图距为2.0 cM。     

柑橘抗CTV转基因与分子标记研究进展

综述了柑橘抗衰退病基因工程中两方面的研究进展。介绍多种来源于柑橘衰退病毒(Citrustriztezavirus,CTV)核酸序列的转基因柑橘和抗性种质资源中抗性基因的分子标记,以及所涉及的方法和遇到的问题。目前研究表明,虽然已成功实现对病毒衣壳蛋白(CP)等基因的转化和Ctv等抗性基因的标记,但尚未获得对CTV有高度抗性的转基因柑橘,而抗性基因亦不能实现定点克隆和转化。因此上述两方面研究还有待深入。     

Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.     

ITS—RFLP分析进行昆虫病原线虫斯氏属和异小杆属的分类鉴定

本研究通过PCR扩增和六种限制性内切酶（AluⅠ，HinfⅠ，MboⅠ，RsaⅠ，HaeⅢ和PvuⅡ）酶切，对国内害虫防治上常用的几种昆虫病原线虫，包括斯氏属S.car-pocapsae,S.feltiae和S.glaseri以及异小杆属H.bacteriophora,H.zealandica,H.indicus和H.megidis等8个品系rDNA-ITS进行分析。建立起可以区分各线虫种的标准RFLP图谱。该方法快速简便，稳定可靠，需要的样品量少。可以用于新鲜的，或冻存的样品，甚至分析单条的线虫，不仅可进行昆虫病原线虫的快速分类鉴定。而且进一步可以应用于线虫田间释放的辅助监测。实际田间感染率的测定和线虫毒力的比较。     

Orobanche species and population discrimination using intersimple sequence repeat (ISSR)

Summary Orobanche species are commonly identified using morphological characteristics. In many cases, the distinction of closely related species is difficult, and a molecular tool is more suitable to differentiate them. In this study, genomic polymorphism between morphologically distinct species was investigated through amplification by polymerase chain reaction (PCR) of intersimple sequence repeat (ISSR) regions. Five primers were used to study genetic variation in the morphologically distinct species O. hederae and O. amethystea, as well as the closely related species O. cernua and O. cumana. For the first two species, all the primers detected genetic polymorphism. Anchored primers allowed the identification of more specific molecular markers than non‐anchored tri‐ and tetranucleotide primers. Genetic polymorphism was investigated among three O. hederae populations using the two types of primer. One non‐anchored and two anchored primers detected intraspecific variation, which was not correlated with the geographical location of those populations. The primer (GATA)₄ detected polymorphism between five specimens each of O. cernua and O. cumana species collected from different countries, permitting these two closely related species to be clearly differentiated. This study demonstrated that ISSR markers can be highly reliable for precise identification of Orobanche species.     

花椰菜类蔬菜自交系基因组间亲缘关系的AFLP分析

 利用AFLP 技术对花椰菜、青花菜、紫花菜、黄花菜自交系的遗传亲缘关系进行了研究。使用8 对选择性引物组合, 扩增出4901 条电泳带, 并对其进行聚类分析, 根据UPGMA 方法构建聚类树状图。结果表明: 花椰菜、青花菜、紫花菜、黄花菜明显分为四大类群; 青花菜与紫花菜亲缘关系较近, 花椰菜与其它花菜亲缘关系较远, 黄花菜居中。     

苹果柱型基因Co的一个AFLP 标记的SCAR转换

 将苹果柱型基因的一个AFLP 标记成功地转换成了简单实用的SCAR 标记。首先对AFLP 标记片段进行序列测定, 然后根据序列特点设计了两对特异引物CoA1/ CoA2 和CoA1/ CoA3 , 每条引物长20 bp。PCR 结果表明CoA1/ CoA2 可以扩增出216 bp 和148 bp 两条带, 其中216 bp 的为柱型性状的特征带; CoA1/CoA3 可以扩增出273 bp 和205 bp 的两条带, 其中273 bp 的为柱型性状的特征带。两对引物在杂交后代中扩增出的特征带与柱型性状的分离重组率都很低(CoA1/ CoA2 为6. 3 % ±2. 5 %; CoA1/ CoA3 为7. 3 % ±2. 6 %) , 所以它们都可以作为该SCAR 标记的特异引物所用。