大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。     

利用SSR技术鉴定西瓜甜瓜种子纯度

利用SSR分子标记技术对西瓜‘W1806’与甜瓜‘M1805’各3个批次及其亲本间的多态性进行引物筛选和种子的纯度鉴定。结果表明,在28对西瓜的SSR引物中,有5对引物在西瓜‘W1806’的F1代与亲本之间有很好的多态性。其中BVWS00839引物特异性好,条带清晰,父母本条带间隔明显,即作为3个批次的西瓜纯度鉴定的引物,其鉴定纯度分别为99.47%、98.96%和97.92%;在18对甜瓜的SSR引物中,只有1对CMBR052引物在F1代扩出的条带为典型的双亲互补型条带,故用该引物对甜瓜进行纯度鉴定,其纯度分别为97.90%、96.80%和97.40%。与田间鉴定结果的吻合率都在98%以上。这2个材料的吻合率说明SSR分子标记技术对西瓜和甜瓜纯度鉴定结果都是可靠的。     

正交设计优化青蒿SRAP-PCR反应体系及引物筛选

为了建立青蒿的SRAP最佳扩增体系,并筛选出SRAP多态性引物,本研究以青蒿叶片DNA为模板,采用正交试验设计,以Mg^2+、dNTP Mix、Taq DNA聚合酶、引物和DNA模板5种因素5个水平,对青蒿SRAP反应体系进行研究。结果表明,青蒿SRAP-PCR最佳反应体系为:引物0.6μmol/L、Mg^2+2.0 mmol/L、模板DNA 5.1 ng、Taq DNA聚合酶2.0 U、dNTPs 0.25 mmol/L,总体积为25μL。各因素对扩增反应均有不同影响,其中引物浓度的影响最大,dNTPs的影响最小。运用该体系对不同种质资源的青蒿进行验证,证明该体系稳定可靠,并在30个引物组合中筛选出了25对扩增条带清晰,多态性丰富的引物组合。这一结论为今后利用SRAP标记技术进行青蒿分子遗传学研究提供了科学依据。     

Development of a core set of KASP markers for assaying genetic diversity in Brassica rapa subsp. chinensis Makino

Single‐nucleotide polymorphisms (SNPs) are rapid, economical and reliable genotyping tools. Non‐heading Chinese cabbage (Brassica rapa L. subsp. chinensis Makino) is now an economically important vegetable crop worldwide. In this study, 1,167 SNPs were evaluated for 7polymorphism among 70 representative non‐heading Chinese cabbage inbred lines using a Kompetitive Allele Specific PCR (KASP) genotyping assay. On the basis of identified polymorphisms and the results of a principal component analysis, we selected 50 core SNPs that were balanced sufficiently to provide adequate information for genetic identification. The core SNPs were used for construction of a neighbour‐joining dendrogram that separated the 70 inbred lines into four main groups and several subgroups corresponding to Caixin, Heiyebaicai, Huangxinwu, Naibaicai, Taitsai, Pak‐choi, and Wutatsai. This categorization was superior to that achieved using a dataset of 479 polymorphic SNPs. To confirm the utility of the core SNP markers in genetic identification, we tested their stability and resolution using 162 commercial hybrid cultivars. The SNPs, which represent a cost‐effective, accurate marker set for germplasm analysis and cultivar identification, are suitable for molecular marker‐assisted breeding in non‐heading Chinese cabbage.     

Genetic diversity of herbicide-resistant and -susceptible Avena fatua populations in North Dakota and Minnesota

Genetic diversity within and among 20 herbicide-resistant (HR) and 16 herbicide-susceptible (HS) Avena fatua multi-field populations was determined using 82 polymorphic loci resulting from two intersimple sequence repeat (ISSR) primers and one long-primer random amplified polymorphic DNA (LP-RAPD) primer. Collections from the Red River Valley of North Dakota and Minnesota, sampled in 1964 and 2000, represented A. fatua populations before and after intensive exposure to herbicides. A 1995 collection from south-west North Dakota represented A. fatua exposed to low herbicide selection. Despite differences in years of herbicide exposure among collections, both HR and HS populations from every collection maintained nearly similar levels of ISSR and RAPD diversity. Genetic differentiation among populations (G_ST) varied from 11% to 13% among HR populations and from 9% to 16% among HS populations, indicating that 84–91% of total variation remained within HS or within HR populations. Minimal difference in gene diversity between HR and HS is consistent with multiple origins of resistance, where HR A. fatua most likely evolved from diverse founding individuals.     

分子标记用于赤眼蜂分子监测的研究

通过对松毛虫赤眼蜂（Trichogramma dedrolimi Matsumura）以及玉米螟赤眼蜂（Trichogramma ostriniae Pang et Chen）rDNA-ITS2基因的克隆测序，并联机检索GenBank核酸序列库中其它赤眼蜂的相关序列，利用核酸分析软件找到松毛虫赤眼蜂和玉米螟赤眼蜂ITS2序列中一段有鉴别意义的标志，并据此设计出蜂种的特异PCR引物，实现了松毛虫赤眼蜂和玉米螟赤眼蜂rDNA特异区带的PCR扩增。研究表明该特异区带具有种的特异性。我们认为该分子标记技术可用于目前我国生防中常用的松毛虫赤眼蜂和玉米螟蒌眼蜂的分子监测，如田间种群动态监控和寄生效果评估。     

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。     

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.     

SSR技术及其在果树上的应用

SSR(Simple sequence repeat)技术以其丰富的多态性、共显性遗传、重复性好和操作简便等优点日益受到重视,已成为植物遗传和育种研究中不可缺少的分子标记。对SSR技术的原理和特点作了简要的介绍,较详细地分析了如何获得SSR引物,特别是综述了果树上SSR引物的研究现状,同时将其与其它几种主要的分子标记进行了比较分析,认为SSR标记检测的位点多态性水平明显高于RFLP,而且重复性优于RAPD;着重介绍了SSR技术在果树种质资源和构建果树遗传图谱及基因定位等研究中的应用现状;指出SSR技术将在果树科研上起到重要的作用。     

Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markers

Screening of genotypes of melon ( Cucumis melo ) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth-chamber experiments. Disease incidence varied from 0 to 100% in a genotype-dependent manner. Using four combinations of light (60 and 90  µ E m⁻² s⁻¹) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker-assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence-characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom-2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker-assisted selection.