生物遗传多样性和系统学研究中的RAPD分析

论述了物种遗传多样性的概念,并对随机扩增多态性ＤＮＡ分析方法的原理、特点以及应用范围进行了介绍,并对其优点和缺点进行了评论,总结了国内外这方面的研究进展,认为随机扩增多态性ＤＮＡ技术是研究生物遗传多样性的重要方法。     

濒危树种金钱松RAPD体系的建立和遗传多样性分析

通过优化随机扩增多态性DNA（RAPD） 反应体系,对浙江省不同来源的金钱松Pseudolarix amabilis进行遗传多样性分析。结果表明：18条随机引物在62个样品中可检测到172个可重复位点,其中多态性位点有170个,多态性比率为99.2%。聚类分析的结果表明：同一来源金钱松材料聚为一类,说明金钱松天然群体的RAPD多态性分类和其地理分布有一定的关系,但也有特殊个体不一致,表明金钱松天然种群体存在较高的遗传多样性,并建议对金钱松进行就地保存和迁地保存。图2表3参31     

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.     

Random amplified polymorphic DNA markers reveal low genetic variation and a single dominant genotype in Eichhornia crassipes populations throughout China

Genetic variation within and between 34 populations of Eichhornia crassipes (water hyacinth) in China was surveyed using random amplified polymorphic DNA (RAPD) markers. A total of 1009 individuals were analysed, for which 12 RAPD primers amplified 69 reproducible bands, with 22 (32%) being polymorphic. The percentage of polymorphic loci (p) within a population ranged from 4.4% to 17.4%, and the mean Nei's gene diversity (H_e) was 0.046 ± 0.0145, indicating a low genetic diversity of E. crassipes in China. Each population contained at least four RAPD phenotypes (genotypes), and the same particular genotype was invariably dominant in all the populations sampled. The mean proportion of distinguishable genotypes was 0.29. Analysis of molecular variance revealed a large proportion of genetic variation (83.9%) residing within populations and a slightly larger proportion (88.1%) within localities, indicating a low genetic differentiation of E. crassipes populations, both locally and regionally. Human-mediated dispersal, vigorous clonal growth, and a generally low level of sexual reproduction were thought to be responsible for such a pattern of genetic structure.     

A molecular marker linked to the Prv1 gene that confers resistance to Papaya ringspot virus-type W in melon

Papaya ringspot virus‐type W (PRSV‐W) is the most prevalent and important viral pathogen of cucurbits in Brazil. It can be effectively controlled by the incorporation of genetic resistance into susceptible melon cultivars. The present study identified amplified fragment length polymorphic (AFLP) markers linked to the PRSV‐W resistance Prv¹ allele. The susceptible yellow‐fleshed melon‐breeding line AF426^prv1 and its nearly isogenic‐resistant line AF426^Prv1, which carries the Prv¹ allele resident in the Indian cantaloupe U.S. Plant Introduction (PI) 180280, were screened for AFLP marker polymorphisms. Of 30 251 AFLP loci, only three were polymorphic between the nearly isogenic lines. Segregation analyses for these three polymorphic markers and the Prv¹ allele using a BC₁ population of 197 plants indicated close linkage (0.5% recombination frequency) between marker EK190 (HindIII‐CGA and MseI‐GTG; 190 bp) and Prv¹. Thus, EK190 might be a useful marker in breeding programmes aiming to develop melon cultivars resistant to PRSV‐W. The other two markers are closely linked to each other, but distantly linked to Prv¹.     

瓜实蝇DNA甲基化的MSAP体系建立与优化

瓜实蝇[Bactrocera cucurbitae (Coquillett)]是中国重要的蔬菜害虫,但其DNA甲基化研究尚未见报道。甲基化敏感扩增多态性是研究DNA甲基化的重要技术之一。通过对酶切反应、连接、PCR扩增和引物筛选等条件优化,建立瓜实蝇MSAP反应体系,即：①20 μL酶切体系中加入10 U的限制性内切酶与600 ng基因组DNA,于37℃酶切反应过夜;②20 μL连接体系中加入T4 连接酶1 U,HpaⅡ-MspⅠ-adapter接头50 pmol,EcoR I-adapter接头5 pmol,并于16℃反应12 h;③连接产物稀释后进行PCR预扩增和选择性扩增,再经6%变性聚丙烯酰胺凝胶电泳和银染检测结果。通过该体系筛选出适用于瓜实蝇基因组DNA甲基化多态性研究的6对引物;瓜实蝇MSAP体系为瓜实蝇的表观遗传学研究提供了技术支持。     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.     

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.