A wheat cDNA coding for a thaumatin-like protein reveals a high level of RFLP in wheat

 A cDNA clone that reveals a high level of polymorphism between wheat varieties was isolated from a wheat cDNA library. When hybridized to DraI-, EcoRV- and HindIII digested DNA this clone, gbx3832, enables us to distinguish 42 different patterns among 48 varieties: 37 varieties are clearly identified, the remaining 11 are divided into five groups. Base-sequence analysis of the clone reveals 72–74% sequence identity to mRNAs encoding thaumatin-like proteins from different cereals. Received: 27 January 1997 / Accepted: 18 April 1997     

Identification of a chromosome-specific probe that maps within the Ph1 deletions in common and durum wheat

 The Ph1 (pairing homoeologous) gene is the major factor that determines the diploid-like chromosome behavior of polyploid wheat. This gene, which is located on the long arm of chromosome 5B (5BL), suppresses homoeologous pairing at meiosis while allowing exclusive homologous pairing. In an effort to tag the specific chromosomal region where this gene is located, we have previously microdissected chromosome arm 5BL from bread wheat and produced a plasmid library by random PCR amplification and cloning. In this work we isolated from this library a 5BL-specific probe, WPG90, and mapped it within the interstitial deleted chromosome fragments carrying Ph1 in common and durum wheat. A PCR assay of Ph1 based on WPG90 was developed that allows an easy identification of homozygous genotypes deficient for this gene. Received: 19 June 1996 / Accepted: 18 October 1996     

用RFLP方法研究陕西省主要小麦品种遗传多样性及其演变

研究用ＲＦＬＰ方法,对我国主要小麦产区,陕西省４０年代以来广为种植的小麦品种的遗传多样性进行了研究,结果表明,占总带数２６．２％的ＨＩＮＤＩＩＩ酶的ＲＦＬＰ带具有遗传多态性。２４个小麦品种平均遗传相似系数为０．９４８。５３．８％的探针显示了多态性。平均多态性信息含量指数为０．２５２。陕西省小麦品种遗传多样性以７０年代为最高,之后开始降低。     

Detection of DNA sequence polymorphisms among wheat varieties

Summary A DNA marker detection strategy that allows the rapid, efficient resolution of high levels of polymorphism among closely related lines of common wheat (Triticum aestivum) has been developed to circumvent the apparent lack of restriction fragment length polymorphism in many important self-pollinated crop species. The technique of randomly amplified polymorphic DNA (RAPD) was combined with a denaturing gradient gel electrophoresis system (DGGE) to explore DNA sequence polymorphisms among different genotypes of wheat. Of the 65 primer combinations used for the polymerase chain reaction (PCR) amplifications, over 38% of them produced readily detectable and reproducible DNA polymorphisms between a spring wheat line, SO852, and a winter wheat variety, Clark. A high level of polymorphism was observed among a number of commercial varieties and breeding lines of wheat. This procedure was also used to detect polymorphisms in a recombinant inbred population to test the feasibility of its application in genome mapping. This DNA polymorphism detection system provides an opportunity for pedigree analysis and fingerprinting of developed wheat lines as well as construction of a high density genetic map of wheat. Without the need for ³²P and sophisticated DNA extraction procedures, this approach should make it feasible to utilize marker-based selection in a plant breeding program.     

The isolation,characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homoeologous chromosome arm

Summary To investigate the use of RFLP analysis in the Triticeae, a set of low copy number probes has been isolated from a wheat cDNA library. The probes identify each of the 14 homoeologous chromosome arms of wheat as determined by analysis of DNA fragments hybridizing to the probes in aneuploid lines of Chinese Spring. These probes can be used in RFLP analyses both for the assignment of homoeology of alien chromosomes or arms added to wheat, and for the determination of chromosome dosage in wheat aneuploids. Different chromosomes from various Triticeae species can therefore be followed in a wheat genetic background using a single technique. The potential uses of the set in facilitating the transfer of alien segments into wheat are outlined.     

Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene-enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene-rich regions in these species.     

3种小麦细胞质雄性不育系及其杂种线粒体DNA的RFLP分析

对细胞质分别来源于提莫菲维（Ｔ．ｔｉｍｏｔｈｅｅｖｉｉ）,粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）,偏凸山羊草（Ａｅ．ｖｅｎｙｒｉｃｏｓａ）的３种普通小麦的雄性不育系,相应保持系和恢复系及其上的ｍｔＤＮＡ用１２个线粒体基因探针进行了ＲＦＬＰ分析,结果为：⑴Ｔ、Ｋ、Ｖ型不育系的ｍｔＤＮＡ在组织结构上存在显著差异;⑵Ｔ、Ｋ、Ｖ不育系的ｍｔＤＮＡ与共同的保持系间显著不同,失测ｍｔＤＮＡ与小麦ｃｍｓ有关;⑶在     

Partial DNA sequencing of Douglas-fir cDNAs used for RFLP mapping

 DNA sequences from 87 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) cDNA RFLP probes were determined. Sequences were submitted to the GenBank dbEST database and searched for similarity against nucleotide and protein databases using the BLASTn and BLASTx programs. Twenty-one sequences (24%) were assigned putative functions; 18 of which were from plant species. Six sequences aligned with conifer genes, including genes from Douglas-fir. Similarities among the 87 sequences were revealed by analyses with FASTA, suggesting either redundancy or isoforms of the same gene. Assignment of putative functions to anonymous cDNA mapped markers will increase the understanding of structural gene organization of the Douglas-fir genome. Received: 10 April 1998 / Accepted: 29 April 1998     

PCR-RFLP of mitochondrial DNA reveals two origins of Apis mellifera in Taiwan

Beekeeping has been a highly valued industry in Taiwan. As a result, many subspecies of Apis mellifera have been introduced to Taiwan since 1911, leading to the hybridization of different subspecies. In order to know the matrilineal origins of Taiwan A. mellifera, a total of 280 samples collected from 33 apiaries throughout the island were examined. Using PCR-RFLP of four mitochondrial gene fragments, i.e., the non-coding region between tRNA^leu and cytochrome c oxidase subunit II (intergenic tRNA^leu-COII), cytochrome b (Cyt b), large subunit rRNA (Ls rRNA) and cytochrome c oxidase subunit I (COI), we only found two haplotypes exist in 280 samples. Haplotypes ababa and bbbaa account for 87% of these Western bees belonged to the Eastern European (C) lineage and 13% belonged to the Middle East (Z) lineage, respectively, with the latter being totally absent in northern Taiwan. African (A) and Mellifera (M) lineages, officially imported once in 1990s and 1930s respectively, were not detected. The identification of subspecies of A. mellifera and survey of their distribution on the island are expected to facilitate efficient breeding programs and establish a more booming beekeeping industry.     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Multiple sequence rearrangements accompanying the duplication of a tRNAPro gene in wheat mitochondrial DNA

In the course of isolating tRNA genes from wheat mtDNA, we have found the same tRNA^Pro gene in two different Hind III restriction fragments, H-P1 (0.7 kbp) and H-P2 (1.7 kbp). Sequences immediately flanking these duplicate genes are closely related, although not identical; sequence comparisons suggest that multiple rearrangements have occurred in the vicinity of the H-P2 tRNA^Pro gene, relative to the H-P1 version. The chimeric nature of H-P2 is emphasized by the presence of sequences that are also found upstream of the wheat mitochondrial 26S rRNA gene, as well as sequences derived from chloroplast DNA. Comparison of H-P2 with H-P1 plus upstream sequences provides some insight into possible molecular events that might have generated H-P2. In particular, such comparisons suggest a model in which the homologous sequences in H-P2 are seen to be derived from H-P1 plus upstream sequences as a result of an intragenomic, site-specific rearrangement event, followed by amplification of the product, its fixation in the mitochondrial genome, and subsequent sequence divergence (single base changes as well as insertions/deletions of up to 50 nucleotides). The results reported here implicate particular primary sequence motifs in certain of the rearrangements that characterize H-P2.     

Use of non-radioactive digoxigenin-labeled DNA probes for RFLP analysis in rice

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.     

Cloning and characterization of pejerrey mitochondrial DNA and its application for RFLP analysis

Probes were cloned, characterized, and developed for all regions of the mitochondrial DNA (mtDNA) of pejerrey Odontesthes bonariensis to provide the basis for the study of genetic diversity of South American atherinopsinii and to enable species identification from small amounts of tissue. The mtDNA was extracted from liver and cleaved with Eco RI, producing four fragments (7.4, 3.4, 3.1 and 2.9 kb) which were cloned using pUC118 plasmid vectors. Sequence analysis from both ends of the fragments showed that they encode tRNA (Asp, Phe, and Ser-TGA), 12 S rRNA, cytochrome oxidase (CO) II, NADH 4, 5, and 6, and the D-loop, and that the relative positions of these genes are identical to those in the mtDNA of other teleosts. A comparison of homology with carp mtDNA nucleotide sequences revealed that tRNA (Phe and Ser-TGA) and CO II were relatively conserved, whereas the D-loop region was highly divergent. The cloned mtDNA probes detected mtDNA fragments from about 800 ng of total DNA extracted from liver, muscle, and single embryos of O. bonariensis , and were effective for restriction length fragment polymorphism (RFLP) analysis of Patagonina hatcheri , the most distant atherinopsine relative of pejerrey. The cloned mtDNA probes may be useful for the analysis of genetic diversity and non-destructive species identification, including the examination of eggs, larvae and juveniles. The mtDNA sequences reported here provide the basis for the design of primers for PCR-based RFLP analysis.     

Extreme DNA sequence variation in isolates of Aspergillus fumigatus

DNA sequence analysis of the alkaline protease gene was used to investigate two Aspergillus fumigatus strains isolated from ostriches (QLD1 and NSW3) and one environmental isolate (FRR 1266) that have shown genetic variation in previous analyses. The results showed that the QLD1 sequence was virtually identical to the published sequences for three human isolates but NSW3 differed in >6% and FRR 1266 in >10% of the nucleotides that were analysed. An RFLP assay was designed to determine the distribution of these (and other) genetic variants among environmental and clinical isolates of A. fumigatus.     

Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich)

Background

Tetraploid cotton contains two sets of homologous chromosomes, the At- and Dt-subgenomes. Consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. Chromosome-specific markers could solve this problem. Recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the At- and Dt-subgenomes to tetraploid cotton. These sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. In this study, simple sequence repeats (SSRs) on each chromosome in the two diploid genomes were characterized. Chromosome-specific SSRs were developed by comparative analysis and proved to distinguish chromosomes.

Results

A total of 200,744 and 142,409 SSRs were detected on the 13 chromosomes of Gossypium arboreum L. and Gossypium raimondii Ulbrich, respectively. Chromosome-specific SSRs were obtained by comparing SSR flanking sequences from each chromosome with those from the other 25 chromosomes. The average was 7,996 per chromosome. To confirm their chromosome specificity, these SSRs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. The chromosome-specific SSRs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific SSRs were unique and could distinguish homologous chromosomes in tetraploid cotton. Because longer dinucleotide AT-rich repeats were the most polymorphic in previous reports, the SSRs on each chromosome were sorted by motif type and repeat length for convenient selection. The primer sequences of all chromosome-specific SSRs were also made publicly available.

Conclusion

Chromosome-specific SSRs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. The SSRs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among Gossypium species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1265-2) contains supplementary material, which is available to authorized users.     

Nucleotide sequence of a major satellite DNA element isolated from a saltwater fishSillago japonica

A member of satellite repetitive DNA was isolated and sequenced from a saltwater fishSillago japonica (Percoidei). This sequence consists of several oligo-dA/dT tracts and two inverted repeats which resemble each other. Dot blot hybridization analysis using a satellite DNA clone pSJ2 among the species in the suborder Percoidei revealed that the pSJ2 sequence was amplified at least after the family Sillaginidae had been derived.     

大豆疫霉菌一个DNA指纹分析重复序列探针的鉴定

【目的】大豆疫霉菌指纹分析的建立和黑龙江与新疆大豆疫霉菌群体的群体遗传分析。【方法】利用生物信息学方法寻找大豆疫霉菌(Phytophthora sojae)的中度重复序列,并对黑龙江和新疆大豆疫霉菌进行DNA指纹分析。【结果】分析得到一个中度重复序列,定名为PS1227。Southern blot分析表明PS1227在大豆疫霉菌基因组中约有34条可辨的介于1.5-23kb之间的杂交条带,其中21个杂交条带在49个供试菌系中表现多态性。单游动孢子分析表明PS1227指纹特征在病菌无性生殖阶段表现稳定。利用PS1227标记,本实验发现采自黑龙江HP4002、SY6和GJ0105菌系分别与新疆的DW303、71228和71222菌系具有完全相同的指纹特征。【结论】获得一个可用于大豆疫霉菌流行学和群体生物学研究的指纹分析序列PS1227,在分子水平证实了新疆大豆疫霉菌可能由黑龙江传入。     

Evaluation of a DNA pooled-sampling strategy for estimating the RFLP diversity of maize populations

Molecular characterization by means of RFLPs of large sets of populations is presently limited by experimental costs. In order to reduce costs, we have evaluated a method based on the RFLP analysis of balanced bulks of DNA from several individuals. The precision of this approach for estimating allele frequencies within each population was investigated using (i) DNA extracted from controlled bulks of leaf tissues of maize inbred lines, (ii) data obtained from individual analyses of 10 maize populations. Both approaches showed that allele frequencies can generally be estimated with a high precision (coefficients of determination up to 0.99 for some probe-enzyme combinations assayed), relative to the variation in allele frequencies observed among maize populations. Although further efforts are needed to define a set of probe-enzyme combinations that can be routinely image processed, these results, and preliminary results from a 65 maize populations project, suggest that this approach could provide highly informative data for large sets of populations, and at relatively low costs.