A duplex-PCR bioassay to detect a Trichoderma virens biocontrol isolate in non-sterile soil     

Sarah L. Dodd  Alison Stewart 《Soil biology & biochemistry》2004,36(12):1955-1965

This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346 bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400 bp that was easily distinguished from the 346 bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 spores g⁻¹ soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.  相似文献   

Hedysarum phylogeny mediated by RFLP analysis of nuclear ribosomal DNA – RFLP and Hedysarum phylogeny     

Trifi-Farah Neila  Marrakchi Mohamed 《Genetic Resources and Crop Evolution》2001,48(4):339-345

The RFLP technique has been employed to investigate phylogenetic relationships between species of the Hedysarum genus. Homologous DNA probes were used to generate patterns from a set of the Mediterranean group. The degree of band sharing was used to estimate genetic distances between species and to draw phylogenetic trees. These are in good agreement with phylogeny based on morphological and isozyme markers, but contain novel insights. The results are discussed in the context of current work in molecular biosystematics in Hedysarum complex. Attempts made in this approach to access the extent of variability at the DNA level occurring with the domestication process have been investigated. With the availability of the tested probes, it has been assumed that the genetic structure is not affected by the domestication process.  相似文献   

传染性支气管炎病毒中国地方分离株RFLP基因分型的研究   总被引：5，自引：1，他引：4  

步志高  祁贤 《中国兽医学报》1997,17(6):530-534

为初步确定我国不同地区流行的传染性支气管炎病毒（ＩＢＶ）的基因分型，对分离自国内８个不同地域疫区的ＩＢＶＱＤ、ＧＺ、ＺＺ、ＴＪ、ＤＬ、ＹＣ、ＪＳ１和ＪＳ２及参考株Ｍ４１、Ｈ５２和Ｔ的Ｓ１基因ＲＴ－ＰＣＲ扩增ｃＤＮＡ进行ＨａｅⅢ的ＲＦＬＰ分析。结果，ＱＤ与ＭＤ４１、Ｈ５２同属Ｍａｓｓａｃｈｕｓｓｅｔｅ基因型，ＧＺ、ＺＺ、ＹＣ与Ｔ的基因型相同，ＤＬ、ＪＳ１和ＪＳ２则表现为各自独立的基因型，而ＴＪ则为ＤＬ和Ｔ２种基因型毒株的混合感染，表明我国的广大地域内存在着Ｍａｓｓ基因型、Ｔ基因型和可能的变异株ＩＢＶ的流行。  相似文献   

Genotype identification and genetic diversity of Sugarcane yellow leaf virus in China     

M. Q. Wang  D. L. Xu  R. Li  G. H. Zhou 《Plant pathology》2012,61(5):986-993

  相似文献   

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes     

S. Kumari  S. A. Subbotin 《Plant pathology》2012,61(6):1021-1031

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular‐based phylogenetic inference and sequence analysis. Twenty‐two valid species and several species complexes were identified among nematodes included in the analysis. PCR‐RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR‐RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.  相似文献   

Molecular analysis of the genus Asparagus based on matK sequences and its application to identify A. racemosus, a medicinally phytoestrogenic species     

Boonsom T  Waranuch N  Ingkaninan K  Denduangboripant J  Sukrong S 《Fitoterapia》2012,83(5):947-953

The plant Asparagus racemosus is one of the most widely used sources of phytoestrogens because of its high content of the steroidal saponins, shatavarins I-IV, in roots. The dry root of A. racemosus, known as "Rak-Sam-Sip" in Thai, is one of the most popular herbal medicines, used as an anti-inflammatory, an aphrodisiac and a galactagogue. Recently, the interest in plant-derived estrogens has increased tremendously, making A. racemosus particularly important and a possible target for fraudulent labeling. However, the identification of A. racemosus is generally difficult due to its similar morphology to other Asparagus spp. Thus, accurate authentication of A. racemosus is essential. In this study, 1557-bp nucleotide sequences of the maturase K (matK) gene of eight Asparagus taxa were analyzed. A phylogenetic relationship based on the matK gene was also constructed. Ten polymorphic sites of nucleotide substitutions were found within the matK sequences. A. racemosus showed different nucleotide substitutions to the other species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the matK gene was developed to discriminate A. racemosus from others. Only the 650-bp PCR product from A. racemosus could be digested with BssKI into two fragments of 397 and 253-bp while the products of other species remained undigested. Ten commercially crude drugs were analyzed and revealed that eight samples were derived from A. racemosus while two samples of that were not. Thus, the PCR-RFLP analysis of matK gene was shown to be an effective method for authentication of the medicinally phytoestrogenic species, A. racemosus.  相似文献   

Application of Molecular Marking Technologies in Heredity Breeding of Eucalyptus     

Liu Guo  Xie Yaojian 《中国林业科技(英文版)》2012,(3):49-49

Two clonal trial stands of Chinese Fir （Cunninghamia lanceolata） were used in this study, one was 19-year-old stand which included 38 clones, and the other was 17-year-old stand including 102 clones.The statistical analyses showed that there were very significant genetic variations in height, DBH,volume and ratio of heartwood(R_hw),wood basic density（ρ_b ） of the clones in the two stands. The repeatability of clones was in median to high level,and the genetic CV was different over the all five traits.There were very significant phenotypic and genetic correlations among height,DBH and volume,and negative correlations among growth, R_hw andρ_b.The selection method experiment indicated that index selection could improve volume, R_hw andρ_b,showing synthetically superior selection effects compared to any individual trait selection methods.  相似文献   

菊花绿变病植原体在中国的发生及分子鉴定     

李正男  鞠明岫  马强  张磊  孙平平  相宁 《植物检疫》2020,(3):37-42

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。  相似文献   

Prototheca zopfii genotype 2: the causative agent of bovine protothecal mastitis?     

Möller A  Truyen U  Roesler U 《Veterinary microbiology》2007,120(3-4):370-374

In order to clarify the epidemiology of bovine protothecal mastitis, 30 Prototheca zopfii mastitis isolates were genetically investigated. Based on the 18S rDNA, which allows a differentiation of the former species P. zopfii in two distinct P. zopfii genotypes and Prototheca blaschkeae sp. nov., newly developed genotype-specific PCR-assays as well as RFLP-assays were applied.

All mastitis isolates investigated could be assigned to P. zopfii genotype 2 suggesting that this genotype is the aetiological agent of bovine Prototheca mastitis.  相似文献   

Molecular cytogenetic identification of wheat-Elymus tsukushiense introgression lines   总被引：3，自引：0，他引：3  

S.L. Wang  L.L. Qi  P.D. Chen  D.J. Liu  B. Friebe  B.S. Gill 《Euphytica》1999,107(3):217-224

Elymus tsukushiense Honda (syn. Roegneria kamoji C. Koch) (2n = 6x = 42, S^tsS^tsH^tsH^tsY^tsY^ts) is a hexaploid species, distantly related to bread wheat Triticum aestivum L. em Thell (2n = 6x = 42, AABBDD). Apart from the delineation of evolutionary relationships, this species is a potential source of resistance to scab, a devastating disease of wheat caused by Fusarium graminearum Schw. A standard C-banded karyotype was established identifying all 21 chromosome pairs of E. tsukushiense. By using C-banding and genomic in situ hybridization analyses, three wheat-E. tsukushiense chromosome addition lines, one ditelosomic addition line, and one disomic substitution line were identified in BC₂ progenies from wheat × E. tsukushiense hybrids. Twenty DNA markers specific for the seven homoeologous groups of the Triticeae were used to determine the homoeology of the added E. tsukushiense chromosomes. The E. tsukushiense chromosomes in the addition lines NAU702, NAU703, and NAU701 were identified as belonging to homoeologous groups 1, 3, and 5, and thus, were designated as 1Ets#1, 3Ets#1, and 5Ets#1, respectively. NAU751 was identified as a disomic substitution line with chromosome 3A of wheat replaced by chromosome 3Ets#1. Line NAU702 has a high level of resistance to scab and will be used in chromosomal engineering and development of improved wheat germplasm for scab resistance breeding. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献