用于微生物多样性分析的棉田土壤总DNA的提取

目的：探讨适用于微生物多样性研究的棉田土壤微生物总DNA提取方法。方法：采用4种方法提取不同连作和轮作处理的棉田土壤微生物总DNA,比较其纯度、产率、片段大小,并应用ARDRA技术验证其质量。结果：其中3种方法均可获得23kb的DNA片段,但不同方法提取的DNA的产率和纯度上有明显差异。改良CTAB-SDS法提取的DNA完整性好,得率为24.20μg.g-1干土,纯化后A260/A280和A260/A230为分别为1.80和1.70,纯化回收率可达70.1%,完全适用于后续的PCR分析。结论：采用该法提取棉田土壤总DNA简便而高效。对该法提取获得的棉田土壤微生物总DNA进行ARDRA和DGGE分析,所得图谱能较全面地反映不同处理间微生物多样性及群落结构的差别,为不同栽培体系下棉田土壤微生物的分子生态学研究提供了基础。     

不同地理区域蛤蚧的RAPD分析

应用随机扩增多态性DNA（RAPD）技术,对6个不同地域（河池、南宁、桂林、百色、越南、泰国）的蛤蚧（Gekko gecko）进行了遗传多样性和系统发育分析。21个RAPD引物共扩增了218个位点,片段大小在200～2000bD,其中184个是多态位点,占84．4％。地域之间的遗传距离指数在0．0112～0．9631,遗传相似性系数在0．3817～0．9888之间,根据遗传距离指数和遗传相似性系数,用NTSYSpc 2．10软件包中的UPGMA法构建了系统聚类图,结果均显示南宁地区、桂林地区、百色地区和河池地区先聚在一起,再和越南聚在一起,最后和泰国群体聚类。这与形态地理分布特征相一致。     

Chromosome elimination and fragment introgression and recombination producing intertribal partial hybrids from <Emphasis Type="Italic">Brassica napus</Emphasis> × <Emphasis Type="Italic">Lesquerella fendleri</Emphasis> crosses

The intertribal sexual hybrids between three Brassica napus (2n = 38) cultivars and Lesquerella fendleri (2n = 12) with the latter as pollen parent were obtained and characterized for their phenotypes and chromosomal and genomic constitutions. F₁ plants and their progenies mainly resembled female B. napus parents, while certain characters of L. fendleri were expressed in some plants, such as longer flowering period, basal clustering stems and particularly the glutinous layer on seed coats related to drought tolerance. Twenty-seven F₁ plants were cytologically classified into five types: type I (16 plants) had 2n = 38, type II (2) had 2n = 38–42, type III (3) had 2n = 31–38, type IV (5) had 2n = 25–31, and type V (1) had 2n = 19–22. Some hybrids and their progenies were mixoploids in nature with only 1–2 chromosomes or some chromosomal fragments of L. fendleri included in their cells. AFLP (Amplified fragments length polymorphism) analysis revealed that bands absent in B. napus, novel for two parents and specific for L. fendleri appeared in all F₁ plants and their progenies. Some progenies had the modified fatty acid profiles with higher levels of linoleic, linolenic, eicosanoic and erucic acids than those of B. napus parents. The occurrence of these partial hybrids with phenotypes, genomic and fatty acid alterations resulted possibly from the chromosome elimination and doubling accompanied by the introgression of alien DNA segments and genomic reorganization. The progenies with some useful traits from L. fendleri should be new and valuable resource for rapeseed breeding.     

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region,<Emphasis Type="Italic"> RB</Emphasis>

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum "). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs.Communicated by R. Hagemann     

Genomic variations of Mycoplasma capricolum subsp. capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size range of 40-500 bp. The similarity between individual AFLP profiles, calculated by Jaccard's coefficient, ranged from 0.92 to 1.0. On the basis of the polymorphisms detected, the analysed strains can explicitly be grouped into two major clusters, equivalent to two evolutionary lines of the organism found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes.     

Genetic diversity of three endangered species of <Emphasis Type="Italic">Echium</Emphasis> L. (Boraginaceae) endemic to Cape Verde Islands

Echium hypertropicum, E. stenosiphon and E. vulcanorum are the three endemics representative of the genus Echium (Boraginaceae) in Cape Verde archipelago. The aim of this study is to provide a first attempt at estimating genetic diversity among natural populations of these endangered Echium species based on RAPD, so as to provide data available for future appropriate strategies for their conservation. PCO and UPGMA of RAPD analysis suggests a close genetic proximity between the Southern endemic species (E. hypertropicum and E. vulcanorum) and shows that the levels of polymorphism strongly differ between these two Echium species (27 and 29% respectively) and E. stenosiphon (74%), the Northern endemic species. Mantel test also corroborates a close genetic proximity between genetic and geographic data. Population genetic analysis of E. stenosiphon revealed low levels of gene flow between islands (Nm = 0.32) being S. Nicolau the most isolated as evident in PCO. Furthermore the differentiation between groups of individuals belonging to putative subspecies was tested by AMOVA. According to our results there is no genetic basis to consider the two subspecies of E. stenosiphon namely ssp. stenosiphon and ssp. lindbergii. Our results enable us to suggest that E. stenosiphon must be ranked as a Threatened species. Measures aiming at conservation of E. hypertropicum and E. vulcanorum must be implemented at short-term taking into account the small number of existing plants and its low genetic variability.     

Genetic structure of <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> ssp. <Emphasis Type="Italic">condensatus</Emphasis> (Poaceae) on Miyake Island: implications for revegetation of volcanically devastated sites

We investigated the genetic structure of Miscanthus sinensis ssp. condensatus on Miyake Island, which was devastated by a volcanic eruption in 2000, by amplified fragment length polymorphisms (AFLPs) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for chloroplast DNA (cpDNA) variation, to develop recommendations for the revegetation of devastated sites. Genetic differentiation among populations was significant, and five populations were classified into three regional groups. The aspect ratios of leaf-blades varied significantly among populations, but both geographical proximity and morphological similarities did not precisely reflect genetic similarities. In the airport population, we found a rare haplotype that may have been transmitted from outside the island. These findings will assist the revegetation of the island.     

Genetic differentiation and hybridization between greater and lesser spotted eagles (Accipitriformes:Aquila clanga, A. pomarina)

Greater and lesser spotted eagles (Aquila clanga, A. pomarina) are two closely related forest eagles overlapping in breeding range in east-central Europe. In recent years a number of mixed pairs have been observed, some of which fledged hybrid young. Here we use mitochondrial (control region) DNA sequences and AFLP markers to estimate genetic differentiation and possible gene flow between these species. In a sample of 83 individuals (61 pomarina, 20 clanga, 2 F1-hybrids) we found 30 mitochondrial haplotypes which, in a phylogenetic network, formed two distinct clusters differing on average by 3.0% sequence divergence. The two species were significantly differentiated both at the mitochondrial and nuclear (AFLP) genetic level. However, five individuals with pomarina phenotype possessed clanga-type mtDNA, suggesting occasional gene flow. Surprisingly, AFLP markers indicated that these mismatched birds (originating from Germany, E Poland and Latvia) were genetically intermediate between the samples of individuals in which mtDNA haplotype and phenotype agreed. This indicates that mismatched birds were either F1 or recent back-cross hybrids. Mitochondrial introgression was asymmetrical (no pomarina haplotype found in clanga so far), which may be due to assortative mating by size. Gene flow of nuclear markers was estimated to be about ten times stronger than for mtDNA, indicating a sex-bias in hybrid fertility in accordance with Haldanes rule. Hybridization between the two species may be more frequent and may occur much further west than hitherto assumed. This is supported by the recent discovery of a mixed pair producing at least one fledgling in NE Germany.     

Genetic analysis of inbreeding of two strains of the parasitic nematode Haemonchus contortus

Haemonchus contortus is a sheep parasitic nematode that causes severe economic losses. Previous studies have indicated a high degree of genetic heterogeneity, which is hardly affected by selection for drug resistance. As a tool for the analysis of the population dynamics of H. contortus and its response to drug resistance, we designed a strategy to study the inbreeding of a benzimidazole-sensitive and a benzimidazole-resistant strain. After 15 generations, a theoretical inbreeding coefficient of 0.87 was achieved. The different stages of inbreeding were analysed using restriction fragment polymorphism, microsatellite variability and amplified fragment length polymorphism. Model-based clustering of the amplified fragment length polymorphism genotypes showed that the allele frequencies of the benzimidazole-resistant strain were stable during the last eight generations. In the sensitive strain a gradual shift of allele frequencies was observed, which led to a temporary increase of the genetic diversity around the eight generations.