白菜EST-SSR标记的通用性

EST-SSR是从表达序列标签(expressedsequencetag,EST)中开发的新型简单序列重复(simplesequencerepeat,SSR)标记。根据白菜EST设计了15对SSR引物,对白菜、油菜、玉米、高粱、水稻和茶树等进行了PCR,研究了白菜的EST-SSR标记在不同物种间的通用性。所设计的引物对不同白菜品种、近缘种油菜和远缘种玉米、高粱、水稻和茶树的扩增成功率分别为100%、93.3%、80%、93.3%、93.3%和86.7%。在15对引物中,有11对在远缘种中都有扩增产物,而且一些引物可显示多态性,多态性引物分别占了可扩增引物的33.3%、28.6%、28.6%和61.5%。这些结果表明,白菜EST-SSR引物具有较高的通用性,这对于比较基因组学研究有重要意义。     

RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。     

利用气孔保卫细胞周长及叶绿体数目鉴定油菜种间杂种研究

以白菜型冬油菜‘陇油7号’、‘陇油9号’(AA,2n=20),芥菜型油菜‘冬芥’(AABB,2n=36),甘蓝型油菜‘vision'(AACC,2n=38),种间杂种‘陇油7号’ב冬芥’、‘陇油9号’בvision'为试材,对其叶片气孔保卫细胞周长及叶绿体数目进行统计分析,探讨一种快速鉴定油菜种间杂种的简易方法,并用SSR标记法对气孔保卫细胞周长及叶绿体分界法可靠性进行进一步验证.结果显示:(1)油菜亲本及种间杂种气孔保卫细胞周长在不同叶片间变异程度接近,而同一叶片的气孔保卫细胞周长下部位变异程度较小.(2)不同类型油菜亲本及其种间杂种叶片气孔保卫细胞周长和叶绿体数存在明显的分界线,具体判定分界值为:周长＜58.90 μm为白菜型油菜、周长58.90～75.83 μm为种间杂种F1、周长＞75.83 μm为甘蓝型或芥菜型油菜;叶绿体数10～12个为白菜型油菜、14～16个左右为杂种F1、18～19左右为甘蓝型或芥菜型油菜.(3)随机选择经叶片气孔保卫细胞周长和叶绿体数分界特征值鉴定的油菜种间杂种单株40个,用SSR分子标记法对鉴定结果显示,有37个单株具有杂交种带型为真杂种,两种方法鉴定油菜真杂种的吻合率达97.5％.研究表明,白菜型、甘蓝型、芥菜型油菜及其种间杂种的叶片气孔保卫细胞周长及叶绿体存在特定的分界线值,据此可以方便有效地鉴定油菜种间杂种,即当植株气孔同时满足保卫细胞周长在58.90～75.83 μm、叶绿体数在14～16个左右时,则可判定其为真杂种.     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。     

基于苹果EST-SSR的梨种质资源遗传多样性分析

利用来自苹果的8对EST-SSR标记对48份梨(Pyrus)种质资源进行遗传多样性研究,以分析其在梨属植物上的通用性.结果表明,8对EST-SSR引物在供试材料上均能扩增出与苹果大小相似的产物,所有引物共检测到140个基因位点,其中多态性位点129个,多态性比例为92.14%,并且可成功区分不同品种.根据EST-SSR标记所揭示的多态性和UP-GMA法聚类分析,48份梨种质资源在相似系数0.62处可分为东方梨和西方梨两个种群,而中国的白梨(Pyrus bretschneideri Rehd.)、砂梨(P.pyrifolia Burm.f.Nakai)和秋子梨(P.ussuriensis Maxim.)相互交错在一起,没有独自成组.可见,苹果的EST-SSR标记在梨上具有高度的可转移性,可应用于梨属植物的资源评价及遗传关系研究.     

油菜3个栽培种发芽期耐盐性评价

对油菜种质资源进行耐盐性评价是培育耐盐油菜品种的基础性工作。本研究在室内分别采用3种盐(NaCl)浓度(86 mmol/L、170 mmol/L、256 mmol/L)对芥菜型油菜(Brassica juncea)、白菜型油菜(B.rapa)和甘蓝型油菜(B.napus)等3个栽培种的203份种质资源进行盐胁迫,结果表明盐胁迫对各栽培种的发芽率有不同程度抑制作用,白菜型油菜表现最好,甘蓝型油菜次之,芥菜型油菜最差。基于发芽率筛选出14份优异种质。进一步利用梯度浓度盐(NaCl)溶液对其进行胁迫,发现各栽培种发芽率均随着盐浓度的升高呈下降趋势,符合Boltzmann曲线,但白菜型油菜下降最为缓慢。通过比较盐胁迫下各栽培种6个性状值(胚轴长、根长、总鲜重、地上部分鲜重、总叶绿素含量和电导率)后发现,白菜型油菜发芽期耐盐性优于甘蓝型油菜和芥菜型油菜。对170 mmol/L NaCl胁迫下14份种质各性状值的主成分分析表明,白菜型油菜Br2、甘蓝型油菜Bn1和Bn3为最耐盐种质,可用于耐盐油菜品种的培育。     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

基于EST-SSR标记的莲属种质资源遗传多样性分析

利用EST-SSR分子标记对30个亚洲莲、6个美洲莲及14个亚美杂交莲品种(野生居群)进行遗传多样性分析,结果表明:从123对EST-SSR引物中筛选出52对(42.3%)扩增稳定、具有多态性的引物;利用这52对引物对50个不同类型代表的莲属品种进行扩增,共获得177条多态性条带,引物的等位基因数和多态性信息量(PIC)的范围分别为2~8个和0.63(NNFB-1059)-0.91(NNFB-750),平均为3.4个和0.79。利用NTSYS-pc2.11软件对扩增结果进行Jaccard相似性系数分析,50个荷花品种材料遗传相似系数为0.24~0.86;通过UPGMA法进行聚类分析,在遗传相似系数0.37处供试荷花材料可分为4大类群:亚洲莲品种均聚类在I、II类群中,III类群绝大部分为亚美杂交莲品种,IV类群为美洲莲,亚美杂交莲品种与亚洲莲品种的亲缘关系相对较近,与传统分类及前人研究结果相一致。     

西藏白菜型油菜遗传多样性的RAPD分析

通过利用22个10bp随机引物对来自西藏高原地区107份白菜型油菜种质资源材料的PAPD分析,探讨了西藏白菜型油菜品种之间的遗传分化关系,结果表明：（1）供试的107份材料共产生236条谱带,其中210条谱带有多态性,占88．98％,说明白菜型油菜在西藏高原地区具有较丰富的遗传多样性;（2）根据引物扩增出的DNA指纹图谱,运用UPGMA分析法,在遗传距离为0．078处,可将供试的107份白菜型油菜划分为11个类群,发现来自于同一地区或气候相似区的品种往往聚在一起,表明西藏高原白菜型油菜品种的相似性与其原产地的地理,气候背景密切相关,并在此基础上,结合西藏高原农业发展历史,气候背景以及地形地貌特点和植物地理学,植物区系学,植物进化论等方面的综合分析,提出西藏高原是世界白菜型油菜起源地的观点。     

用EST-SSR标记对茶树种质资源的研究

采用16对EST-SSR引物对42份茶树品种资源进行了分析。在这些引物中, 有13对可扩增出清晰条带, 其中10对引物具有多态性, 占76.9%。各多态性引物的PIC值变化范围为0.522~0.866, 平均PIC值为0.730。10对多态性引物共检测到84种等位基因型和74个等位变异, 每对引物可检测到基因型和等位基因数分别为4~12和3~10种。42份供试材料间遗传距离为0.074~0.667, 平均遗传距离为0.363, 说明本实验所测试的材料具有较广的遗传变异。在相似系数为0.55的水平可将这些材料聚为3类, 多数材料包含在第一类中。本研究表明, 利用EST-SSR标记进行茶树资源评价是有效的。     

Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris ssp. pekinensis) and oilseed rape (Brassica napus): Glucosinolate and IAA biosynthetic enzymes

The first steps in the biosynthesis of glucosinolates and indole-3-acetic acid (IAA) in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris ssp. pekinensis) involve the formation of aldoximes. In rape the formation of aldoximes from chain-extended amino acids, for aromatic and aliphatic glucosinolate biosynthesis, is catalysed by microsomal flavin-containing monooxygenases. The formation of indole-3-aldoxime from l-tryptophan, the potential precursor of both indole-3-acetic acid and indolyl-glucosinolates, is catalysed by several microsomal peroxidases. The biosynthesis of glucosinolates and indole-3-acetic acid was shown to be under developmental control in oilseed rape and Chinese cabbage. No monooxygenase activities were detected in cotyledons or old leaves of either species. The highest monooxygenase activities were found in young expanding leaves; as the leaves reached full expansion and matured the activities decreased rapidly. The indole-aldoxime-forming activity was found in all of the tissues analysed, but there was also a clear decrease in foliar activity with maturity in leaves of rape and Chinese cabbage. Partial characterisation of the Chinese cabbage monooxygenases showed that they have essentially identical properties to the previously characterised rape enzymes; they are not cytochrome P450-type enzymes, but resemble flavin-containing monooxygenases. No monooxygenase inhibitors were detected in microsomes prepared from either cotyledons or old leaves.Abbreviations DHMet dihomomethionine - FMO flavin-containing monooxygenase - HPhe homophenylalanine - IAA indole-3-acetic acid - l-Phe l-phenylalanine - l-Trp l-tryptophan - MO monooxygenase - IAALD indole-3-acetaldehyde - IAOX indole-3-aldoxime - THMet trihomomethionine     

Microsatellite markers for genome analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C genomes and genetic mapping in Brassica oleracea L.

Microsatellites are highly polymorphic and efficient markers for the analysis of plant genomes. Primer specificity, however, may restrict the applicability of these markers even between closely related species for comparative mapping studies. We have demonstrated that the majority of microsatellites identified in oilseed rape (Brassica napus L; AC genome) correspond to loci which can be easily assigned to the A and C progenitor genomes. A study with 63 primer pairs has shown that 54% detect two loci, one from each genome, while 25% and 21%, respectively, are either A or C genome-specific. The distribution of rapeseed microsatellites in the C genome was investigated by genetic mapping in Brassica oleracea L. Ninety two dinucleotide microsatellites were screened for polymorphism in an F₂ population derived from a cross between collard and cauliflower, for which an RFLP map has been constructed previously. Thirty three primer pairs (35.7%) have yielded either unspecific or no PCR products whereas the remaining primer pairs amplified one or more distinct loci. The level of polymorphism found in the mapping population was 49.2%. A total of 29 primer pairs disclosed 34 loci of which 31 are evenly distributed on 8 of the 9 B. oleracea linkage groups. For the remaining three markers linkage could not be established. Our results showed that microsatellite markers from the composite genome of B. napus can serve as a useful marker system in genetic studies and for plant-breeding objectives in B. oleracea. Received: 14 April 2000 / Accepted: 3 July 2000     

Inheritance of microspore embryogenic ability in Brassica crops

Inheritance of microspore embryogenic ability in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris L. ssp. pekinensis) was examined by 4 × 4 diallel crosses using cultivars showing a different response. In both species, embryo yields of most F₁ hybrids were similar to, or over, the high responsive parent and some F₁s showed intermediate embryo yields between their parents. Diallel analysis showed that both additive and dominant effects were significant at the 1% level for the genetic control of microspore embryogenic ability in both species. Dominant genes had positive effects on microspore embryogenesis. In oilseed rape, the additive effects were important, while in Chinese cabbage the dominant effects were largely contributed. The broad- and narrow-sense heritabilities were 0.972 and 0.811 in oilseed rape, and 0.959 and 0.659 in Chinese cabbage, respectively. From the results of the segregation of embryo yields in the F₂ population of ’Lisandra’×’Kamikita’, it is considered that the microspore embryogenic ability is controlled by two loci with additive effects in oilseed rape. Received: 6 September 2000 / Accepted: 24 November 2000     

油菜EST-SSR标记的建立

在油菜17987条非冗余EST中,共发掘出了2083个EST-SSR,分布于2443条EST中,发生频率是13.58%,平均分布距离为4.34kb。在油菜EST-SSR中,二、三核苷酸重复是主要的重复类型,二者出现的频率相近,占总SSR的89.05%。AG/CT和AAG/CTT是二、三核苷酸中的优势重复类型,分别占二、三核苷酸重复类型的84.31%和37.71%。进一步设计了23对SSR引物,通过梯度PCR试验确定了各引物的适宜退火温度,并利用非变性聚丙烯酰胺凝胶银染对这些引物在10个油菜品种中的扩增情况和多态性进行了检测。有21对引物显示扩增,引物可用率为91.30%;有12对引物显现出多态性,占可扩增引物的57.14%。本研究结果证明根据油菜EST建立SSR标记是有效、可行的。     

普通小麦SSR和EST-SSR引物对冰草通用性的比较分析

选用定位于普通小麦7个部分同源群的534对SSR引物和351对EST-SSR引物分别对普通小麦品种‘Fukuho’和四倍体冰草‘Z559’的基因组DNA进行扩增,结果显示:有475对(89.0%)SSR引物和314对(89.5%)EST-SSR引物对‘Fukuho’能有效扩增,226对(42.3%)SSR和258对(73.5%)EST-SSR引物对‘Z559’能有效扩增,表明小麦EST-SSR对冰草的通用性明显高于SSR;扩增强带比率SSR和EST-SSR引物分别为76.1%、84.1%,说明小麦EST-SSR在冰草上扩增带的质量亦优于SSR。选择上述在‘Fukuho’和‘Z559’基因组DNA之间有多态性扩增且带谱清晰的SSR和EST-SSR引物各60对,对‘Fukuho’、‘中国春’、‘北京8号’和二、四、六倍体冰草‘Z804’、‘Z559’、‘Z1075’的基因组DNA再行PCR扩增,结果显示,40对(66.7%)SSR和22对(36.7%)EST-SSR引物在‘Fukuho’、‘中国春’和‘北京8号’间扩增产物表现多态性,且前者高于后者;50对(83.3%)SSR和52对(86.7%)EST-SSR引物在冰草‘Z804’、‘Z559’和‘Z1075’间扩增产物表现多态性,两者相当。通用性、多态性和扩增强带比率综合比较表明,普通小麦EST-SSR和SSR经筛选虽都能转用于冰草,但两者相比EST-SSR更优。     

SRAP、ISSR技术的优化及在甘蓝类植物种子鉴别中的应用

将SRAP与ISSR 2种分子标记技术应用于8种甘蓝类植物（Brassica oleracea L.）的种子鉴别中。先以甘蓝（Brassica oleracea var. capitata）基因组DNA为模板,通过对SRAP、ISSR反应体系中各影响因素的逐一筛选,优化了甘蓝类植物SRAP、ISSR反应体系。进而采用30个SRAP引物组合和15个ISSR引物对白甘蓝、皱叶甘蓝、红甘蓝、羽衣甘蓝、花椰菜、青花菜、抱子甘蓝、球茎甘蓝的基因组DNA进行了PCR扩增,结果表明：M3E5与M4E5两个SRAP引物组合可以在8种甘蓝类植物之间显示较高的多态性;844和888两个ISSR引物也可在8种甘蓝类植物之间产生很好的多态,特别是844引物单独应用即可区分所有材料。     

小菜蛾幼虫对不同寄主的取食嗜好性及其适宜性

对不同寄主种类、不同寄主形态和不同寄主饲喂的小菜蛾(Plutella xylostella)幼虫之间的取食嗜好性比较试验表明,小菜蛾幼虫优先取食大白菜、萝卜或菜心幼苗,其次为油菜和甘蓝幼苗,在大白菜与油菜幼苗之间的取食选择比例是93.33%和6.67％;在甘蓝与菜心幼苗之间的取食选择比例是16.67%和83.33%.小菜蛾幼虫的取食嗜好性受饲喂寄主种类的影响,偏食大白菜或菜心幼苗.小菜蛾幼虫选择寄主取食的次序与寄主体内可溶性糖或淀粉含量没有明显关系,但与两者的相对量呈一定的负相关.取食大白菜或菜心幼苗的小菜蛾生长良好,单头取食达0.583～0.637 cm², 单头体重达2.07～2.18 mg, 与取食甘蓝或油菜幼苗的幼虫在取食量、个体发育方面有明显差异.小菜蛾幼虫也喜好取食已经被虫危害过的幼苗.     

Effect of a turnip rape <Emphasis Type="Italic">(Brassica rapa)</Emphasis> trap crop on stem-mining pests and their parasitoids in winter oilseed rape <Emphasis Type="Italic">(Brassica napus)</Emphasis>

Concerns about the negative effects of chemical control of oilseed rape (Brassica napus L.) pests on non-target species, human safety, and development of insecticide resistance, require alternative control strategies such as the use of trap crops and biocontrol to be developed. Psylliodes chrysocephala(L.) (Coleoptera: Chrysomelidae) (cabbage stem flea beetle) and Ceutorhynchus pallidactylus (Marsh.) (Coleoptera: Curculionidae) (cabbage stem weevil) are two major stem-mining pests of oilseed rape. This study investigated the phenology of these pests and their main parasitoids in the UK, the potential use of turnip rape (Brassica rapa L.) as a trap crop to reduce oilseed rape infestation, and the effects of insecticide treatment on pest incidence and larval parasitism. Water trap samples, plant dissections and pest larval dissections were done to determine: the incidence of adult pests and their parasitoids, the level of plant infestation by the pests and percentage larval parasitism, respectively. The turnip rape trap crop borders reduced P. chrysocephalabut not C. pallidactylus infestation of oilseed rape plots. Treatment of the trap crop with insecticide had little effect on either pest or parasitoid incidence in the oilseed rape. TersilochusmicrogasterSzép. andT. obscurator Aub. (Hymenoptera: Ichneumonidae) were the main larval parasitoids of P. chrysocephalaand C. pallidactylus, respectively. Tersilochus microgasteris reported for the first time in the UK. The implications for integrated pest management are discussed.