基于AFLP标记的野生梅种质的鉴定

梅Prunus mume是我国原产的名花佳果。为有效保护和利用野生种质资源,文中采用了AFLP标记技术,结合形态表型特征分析,对65份野梅种质进行试验分析。首先从64对引物中筛选出MseⅠ-EcoRⅠ8对引物组合,扩增出1 002条多态性条带。按照Nei’72距离系数进行聚类,在Nei’72=0.26处,可区分西山野梅原变种、西山野梅原种、西山毛梅、厚叶梅、南大坪桃梅、嵩明小梅、曲梗常绿梅、蜡叶梅以及匍匐梅,这与形态学上变种或变型的划分一致。群体内变种单株样本遗传多样性丰富。基于梅花种质资源的遗传变异,建议今后需要对所有变种与变型进行有效的保护。     

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg²⁺ in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.     

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.     

斜带髭鲷高质量DNA提取及AFLP分析体系建立

目的:提取斜带髭鲷高质量基因组DNA并建立适用于AFLP分析的体系。方法:以斜带髭鲷(Hapalogenys nitens)为材料提取高质量基因组DNA,并进行AFLP扩增。对AFLP分析体系中几个关键环节(基因组双酶切、连接、预扩增及选择性扩增、变性聚丙烯酰胺凝胶电泳和银染)做了优化。结果:提取的斜带髭鲷全基因组DNA纯度高,无拖尾。采用9组选择性引物共检测出350个不同的扩增位点,其中多态位点为175个,多态性比例为49.58%。聚丙烯酰胺凝胶电泳图像染色均匀,条带清晰且无背景干扰。结论:该分析体系的建立为斜带髭鲷的群体遗传多态性、种质资源、遗传图谱、遗传育种等研究奠定基础。     

Comparative analysis of genetic relationship and diagnostic markers of several taxa of Guizotia Cass. (Asteraceae) as revealed by AFLPs and RAPDs

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) were employed to examine the genetic relationship between Guizotia taxa, to suggest the taxonomic status of some of these taxa, and to identify their diagnostic markers. Results from AFLPs and RAPDs share some features in common, both revealing G. scabra ssp. schimperi as the most closely related taxon to G. abyssinica, and indicating that G. arborescens and G. zavattarii are the most divergent taxa. Most of the diagnostic markers revealed in this study were specific to G. arborescens and G. zavattarii. Our analysis suggests that G. scabra ssp. scabra, G. scabra ssp. schimperi, Chelelu and Ketcha are separate species. In this study, AFLP was found to be superior to RAPD in detecting genetic variation, in internal consistency of the data and in the fitness of its clusters to genetic similarity data. AFLPs revealed genetic relationship between Guizotia taxa that is more inline with the cytogenetic and hybridization studies than that revealed by RAPDs.     

Genetic diversity and differential in vitro responses to Ni in <Emphasis Type="Italic">Cenococcum geophilum</Emphasis> isolates from serpentine soils in Portugal

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the non-serpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 microg g(-1) Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 microg g(-1) Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The non-serpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.     

Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae)

The origin of polyploid Veronica cymbalaria (Plantaginaceae) was investigated using DNA sequence data and amplified fragment length polymorphism (AFLP) fingerprints to reveal the parentage of this taxon. The use of AFLP fingerprints in phylogenetic analysis is problematic and various methods have therefore been compared. DNA sequence data (for the internal transcribed spacer (ITS) region and the plastid trnL-F region (trnL intron, 3'exon, and trnL-F spacer)) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ITS region suggested a reliable hypothesis for the evolution of the V. cymbalaria complex. This hypothesis allowed evaluation of the effect of different distance measures (Jaccard and Nei-Li) in phenetic, character-state weighted parsimony, and Bayesian analyses of AFLP markers. The study establishes that tetraploid V. cymbalaria originated at least twice in the eastern Mediterranean, with one parent differing in the two separate origins. Hexaploid V. cymbalaria originated even more often. The results illustrate that even subtle differences in the analyses of AFLP markers can lead to drastically different conclusions. The study reveals multiple origins of a Mediterranean polyploid species. Furthermore, it demonstrates that the analysis of a complex marker system such as AFLP fingerprints using only one type of analysis can easily be misleading.     

Origin of new <Emphasis Type="Italic">Brassica</Emphasis> types from a single intergeneric hybrid between <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> by rapid chromosome evolution and introgression

Many novel lines were established from an intergeneric mixoploid between Brassica rapa (2n = 20) and Orychophragmus violaceus (2n = 24) through successive selections for fertility and viability. Pedigrees of individual F₂ plants were advanced to the 10th generation by selfing. Their breeding habit was self-compatible and different from the self-incompatibility of their female parent B. rapa, and these lines were reproductively isolated to different degrees from B. rapa and B. napus. The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate contents in seed meal. These lines had 2n = 36, 37, 38, 39 and 40, with 2n = 38 being most frequent (64.56%), and no intact O. violaceus chromosomes were detected by genomic in situ hybridization (GISH) analysis. Amplified fragment length polymorphism (AFLP) analyses revealed a high extent of variation in genomic compositions across all the lines. O. violaceus-specific bands, deleted bands in B. rapa and novel bands for two parents were detected in these lines, with novel bands being the most frequent. The morphological and genetic divergence of these novel types derived from a single hybrid is probably due to rapid chromosomal evolution and introgression, and provides new genetic resources for rapeseed breeding.     

5种松树的遗传多样性和遗传分化研究

利用cpSSR和AFLP标记,比较了巴山松、黄山松、油松、马尾松以及云南松的遗传多样性与遗传分化结果.发现5种松树都表现出较高水平的遗传多样性与种间分化,但cpSSR比AFLP标记提供了更多的种内种间分化,而展示的遗传多样性相对要少.在对不同方法指标的对比研究后,认为将cpSSR的单倍体关系考虑在内,能够更有效地分析植物居群遗传分化造成的地理结构.对于显性标记AFLP,传统的平方根法会低估遗传参数,而Lynch和Milligen的方法因样本量的限制也可能高估遗传多样性,Bayesian法被认为是最精确的方法,提供了与分子方差分析(AMOVA)近似的遗传分化值.     

Identification of QTL for sugar-related traits in a sweet × grain sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench) recombinant inbred population

QTL for stem sugar-related and other agronomic traits were identified in a converted sweet (R9188) × grain (R9403463-2-1) sorghum population. QTL analyses were conducted using phenotypic data for 11 traits measured in two field experiments and a genetic map comprising 228 SSR and AFLP markers grouped into 16 linkage groups, of which 11 could be assigned to the 10 sorghum chromosomes (SBI-01 to SBI-10). QTL were identified for all traits and were generally co-located to five locations (SBI-01, SBI-03, SBI-05, SBI-06 and SBI-10). QTL alleles from R9188 were detected for increased sucrose content and sugar content on SBI-01, SBI-05 and SBI-06. R9188 also contributed QTL alleles for increased Brix on SBI-05 and SBI-06, and increased sugar content on SBI-03. QTL alleles from R9403463-2-1 were found for increased sucrose content and sucrose yield on SBI-10, and increased glucose content on SBI-07. QTL alleles for increased height, later flowering and greater total dry matter yield were located on SBI-01 of R9403463-2-1, and SBI-06 of R9188. QTL alleles for increased grain yield from both R9403463-2-1 and R9188 were found on SBI-03. As an increase in stem sugars is an important objective in sweet sorghum breeding, the QTL identified in this study could be further investigated for use in marker-assisted selection of sweet sorghum.