AFLP标记及在微生物和动物中的应用

AFLP是在PCR和RFLP基础上发展起来的新一代分子标记技术,具有稳定好、分辨率高和效率高等特点。AFLP技术现已广泛应用于微生物和动物方面的研究,在构造遗传图谱、遗传多态性研究、育种辅助选择等多领域中有着其它分子标记技术不可比拟的优势,广泛应用于微生物和动物的研究。     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997     

DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of mitochondrial DNA]

Restriction enzyme fragment patterns in the D loop and deletion-insertion polymorphism in the V noncoding region of human mitochondrial DNA (mt DNA) were analysed in Mongolian population using the polymerase chain reaction. Polymorphisms were detected and mt DNAs classified into 40 types using seven enzymes--AvaII, BamHI, CfrI131, KpnI, EcoRV, HaeIII RsaI and Asian specific deletion and insertion. The allele frequencies of the polymorphisms and gene diversity were determined. The data obtained for Mongolian population and the literature data were comparatively studied.     

Biochemical and biological characterization of four isolates of Spodoptera exigua nuclear polyhedrosis virus

Biological and biochemical properties of four nuclear polyhedrosis virus isolates from beet armyworm, Spodoptera exigua, were investigated. The isolates originated from the United States (SeNPV‐US), Thailand (SeNPV‐TH) and from two locations in Spain (SeNPV‐SP1 and SeNPV‐SP2). Restriction endonuclease analysis of the viral genomes revealed limited restriction fragment length polymorphism and indicated that these viruses contained distinct, but closely related, genotypes (variants). One BglII fragment from each isolate can serve as a restriction fragment length polymorphism marker for the identification of each isolate. The estimated genome size of the SeNPVs is approximately 134 kilobase pairs. The mobility profiles of the occluded virion polypeptides and polyhedrins of the four SeNPV isolates were very similar. Staphylococcus aureus V8 digestion of polyhedrin suggested that the polyhedrin from SeNPV‐US is distinct from the polyhedrins of the other isolates. Bioassays of the isolates in second‐instar S. exigua larvae showed that the SeNPV‐TH was the most potent SeNPV for beet armyworm with an LD₅₀ value of only 1.5 polyhedra per second‐instar larva.     

草莓DNA分子标记及其应用

综述了限制性长度多态性(RFLP)、随机扩增多态性DNA(RAPD)、扩增片段长度多态性(AFLP)、简单重复序列(SSR)等不同类型分子标记在草莓指纹图谱构建、品种鉴别、遗传多样性、进化、遗传作图以及相关性状的标记等方面的应用,分析了草莓分子标记研究中的关键问题,提出了今后研究方向。     

Identification of complex DNA polymorphisms based on variable number of tandem repeats (VNTR) and restriction site polymorphism

Summary Restriction fragment length polymorphisms (RFLPs) of genomic DNA are generally attributable to base changes that create or abolish restriction endonuclease sites or to nucleotide sequence insertions or deletions that alter the distance separating two restriction sites. Minisatellite or variable number of tandem repeats (VNTR) markers are prominent examples of the latter type of polymorphism. In this report, we describe complex DNA polymorphisms that are due both to the presence of VNTRs as well as to altered restriction endonuclease sites. A strategy for identifying such polymorphisms and resolving their component allelic fragments is demonstrated.     

Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.     

Identification and phylogenetic relationships of Vahlkampfia spp. (free-living amoebae) by riboprinting

Abstract The riboprinting technique (restriction fragment length polymorphism analysis of polymerase chain reaction amplified ribosomal DNA) was applied to 8 strains representing 7 species of amoebae from the Vahlkampfia genus (Family Vahlkampfiidae, Class Heterolobosea). The length of the 18S ribosomal gene was found to vary significantly between species. Restriction fragment length polymorphism analysis following digestion of the amplified ribosomal DNA with 18 restriction enzymes confirmed the separate identity of each species, originally based on morphological characteristics, and generated a phylogenetic tree. Two strains assigned to the same species yielded identical riboprints.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class IV (B-G) genotypes in meat-type chickens

Restriction fragment length polymorphism (RFLP) was used as a molecular genotyping approach to characterize differences in major histocompatibility complex class IV genes in meat-type chickens. A high level of polymorphism was observed following digestion with each of the two restriction endonucleases PvuII and BglII. Examination of DNA from 54 chickens revealed 23 polymorphic fragments. Application of RFLP techniques in the analysis of family groups should make possible the determination of B-G genotypes in the meat type chickens.     

Genetic variation in Lake Erie yellow perch (Percaflavescem) demonstrated by mitochondrial DNA analysis

Restriction fragment length polymorphism analysis of yellow perch ( Perca flavescens ) mitochondrial DNA from a Lake Erie population revealed seven variants in 22 fish (nucleon diversity 0.481). These fish were collected from an area where previous studies using allozymes had failed to reveal any genetic variation.     

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae , a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia . Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I–VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia . By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome- c -like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.     

Restriction fragment length polymorphism of human salivary alpha-amylase in a Japanese population

Restriction fragment length polymorphism of human salivary alpha-amylase after cleavage with restriction endonucleases PstI and BamHI was studied in a population from eastern Japan. Among 40 unrelated individuals, the frequencies of the 5.7- and 6.5-kilobasepair fragment alleles were 0.487 and 0.513, respectively.     

Mitochondrial DNA polymorphism in the natural populations of the Drosophila virilis species group

Restriction fragment length polymorphism (RFLP) analysis has been used to evaluate mitochondrial DNA (mtDNA) variation in 12 sibling species forming the Drosophila virilis species group. The variation thresholds corresponding to the interspecific and interstrain levels have been determined. The results indicate that interspecific hybridization has significantly contributed to the evolutionary history of the virilis species group.     

Lysogenic phage in Salmonella enterica serovar Heidelberg (Salmonella heidelberg): Implications for organism tracing

Abstract A phage typing system using a group of 11 closely related phage (as judged by Southern analysis and restriction fragment length polymorphism analysis) was able to distinguish at least six phage types in Salmonella heidelberg of human and animal origin. Restriction fragment length polymorphism analysis using cosmid probes from S. heidelberg confirmed that most S. heidelberg isolates belong to a single 'clonal' group. Southern analysis using DNA isolated from each of the testing phage group showed that phage types 4, 5 and 6 carry closely related endogenous or lysogenic phage. Induction of a lysogenic phage Hlp-4 (Heidelberg lysogenic phage) from type 4 could become lysogenic and convert phage types 1 and 3 to phage type 4 and phage type 5 to a non-typable phenotype.     

利用生化及分子标记确定长穗偃麦草(Elytrigia elongatum, EE. 2n=14)染色体与小麦染色体的部分同源性

利用限制性片段长度多态性（ＲＦＬＰ）及等电聚焦（ＩＥＦ）技术确定普通小麦中国春－二倍体长穗僵麦草７个异附加系所附加的外源染色体与小麦染色体的部分同源性,共有８个生化标记,１３个ＲＦＬＰ标记在亲本间揭示了多态性。结果表明：长穗堰麦草的ＩＥ、２Ｅ、３Ｅ、４Ｅ、 ５Ｅ、６Ｅ、７Ｅ ７条染色体分别与小麦染色体的 １、２、３、４、５、６、７ ７个部分同源群具有部分同源关系,堰麦草的ＩＥ与７Ｅ、５Ｅ与７Ｅ染色体间可能发生过重排。同时,研究还分别将Ｅｓｔ－Ｅ５、Ｅｓｔ－Ｅ８位点定位于３ＥＬ,Ｐｅｒ－Ｅ１定位于７Ｅ, Ｐｅｒ－Ｅ４定位于５Ｅ,β－Ａｍｙ－Ｅ１定位于４ＥＬ染色体,并进一步将α－Ａｍｙ－Ｅ１位点定位于６Ｅ染色体长臂上。     

Genetic variability among freshwater mussel Anodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) populations recently introduced in Poland

The alien Chinese mussel Anodonta woodiana was first reported in Poland in the system of heated lakes near Konin in 1993. Genetic studies with use of three molecular techniques (isoenzyme electrophoresis, PCR-RFLP and sequence analysis of a COI gene fragment) were carried out on the Polish first populations of A. woodiana. The studies have revealed low genetic variation between the populations (Nei's genetic distance for 12 loci ranged 0.000 to 0.007) as well as their considerable polymorphism. Each population averaged 2.28 alleles per locus, 2.72 alleles per polymorphic locus, and 75% polymorphic loci. Restriction analysis of the COI gene fragment have not revealed variability between the analysed specimens, including males and females. Restriction enzymes, ScrFI, Csp6I, and EcoRI used in the COI gene fragment PCR-RFLP generate distinct restriction patterns, which can be molecular markers for A. woodiana. The sequence obtained for COI fragment was the same in the examined female and male specimens and represents F mitotype (DNA was isolated from somatic tissues). The divergence between A. woodiana F and M mitotypes is high (34%), however it remains within the range of the general character of the DUI (doubly uniparental inheritance) phenomenon in freshwater bivalves (Unionidae).     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia

Restriction fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F. angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.     

Mitochondrial DNA polymorphism in male-sterile cytoplasm of rice

Summary Mitochondrial DNAs (mtDNAs) were isolated and purified from ten strains of rice plants with male-sterile cytoplasm. The mtDNAs were digested with the restriction endonuclease PstI and the fragment patterns produced were analysed by 0.7% agarose gel electrophoresis. Restriction fragment length polymorphism was observed among the mtDNAs analysed; there were seven different patterns for the ten examined. Our results indicate that there are a variety of mtDNAs in cytoplasmically male-sterile rice.     

Aldosterone deficiency II (CMO II deficiency) is not the result of a mutation of an MspI restriction site within the CYP11B gene

Summary We report our investigations of a German family with aldosterone deficiency (CMO II deficiency). Restriction fragment length polymorphism analysis using a P450c11 probe demonstrates that aMspI restriction site mutation within the CYP11B gene cannot be the underlying cause for this defect, as has been suggested previously.