Integration of sequence tagged microsatellite sites to the chickpea genetic map

Fifty sequence-tagged microsatellite site (STMS) markers and a resistant gene-analog (RGA) locus were integrated into a chickpea ( Cicer arietinum L., 2n = 2 x = 16 chromosomes) genetic map that was previously constructed using 142 F(6)-derived recombinant inbred lines (RILs) from a cross of C. arietinum x Cicer reticulatum Lad. The map covers 1,174.5 cM with an average distance of 7.0 cM between markers in nine linkage groups (LGs). Nine markers including the RGA showed distorted segregation ( P < 0.05). The majority of the newly integrated markers were mapped to marker-dense regions of the LGs. Six co-dominant STMS markers were integrated into two previously reported major quantitative trait loci (QTLs) conferring resistance to Ascochyta blight caused by Ascochyta rabiei (Pass.) Labr. Using common STMS markers as anchors, three maps developed from different mapping populations were joined, and genes for resistance to Ascochyta blight, Fusarium wilt (caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris), and for agronomically important traits were located on the combined linkage map. The integration of co-dominant STMS markers improves the map of chickpea and makes it possible to consider additional fine mapping of the genome and also map-based cloning of important disease resistance genes.     

Sequence tagged microsatellite profiling (STMP): improved isolation of DNA sequence flanking target SSRs

Sequence tagged microsatellite profiling (STMP) enables the rapid development of large numbers of co-dominant DNA markers, known as sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5′-ends of the microsatellites. It is also possible to convert STMs into conventional microsatellite, or simple sequence repeat (SSR), markers that are amplified using a pair of primers flanking the repeat sequence. Here, we describe a modification of the STMP procedure to significantly improve the capacity to convert STMs into conventional SSRs and, therefore, facilitate the development of highly specific DNA markers for purposes such as marker-assisted breeding. The usefulness of this technique was demonstrated in bread wheat.     

Direct cloning by covalent attachment of probe DNA to target DNA.

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.     

Transferability of microsatellite and sequence tagged site markers in Oryza species

The genus Oryza comprises 22 species which are potentially useful as a source of genetic variability that can be introgressed into the worldwide cultivated rice, Oryza sativa. Molecular markers are useful tools for monitoring gene introgressions and for detecting polymorphism among species. In this study, cross-amplification was estimated among 28 accessions of 16 Oryza species, representing the genomes AA, BB, CC, BBCC and CCDD, using 59 microsatellite (OG, OS and RM series) and 15 STS (Sequence Tagged Sites) markers. All markers amplified at least one Oryza species, indicating different levels of transferability across species. Markers based on microsatellite sequences amplified 37 % of the accessions, with an average of 6.58 alleles per locus and an average polymorphism information content (PIC) of 70 %. For STS markers, the amplification level was 53.3 %, and the average number of alleles and PIC values were 1.6 and 10 %, respectively. These Results showed that although the STS markers detected a reduced level of genetic diversity, the transferability was higher, indicating that they can be used for genetic analysis when evaluating less genetically related species of Oryza. Among the microsatellite markers, an analysis of species with an AA genome showed that the OG markers produced the highest level of polymorphic loci (54.6 %), followed by RM markers (48 %). Highly polymorphic and transferable molecular markers in Oryza can be useful for exploiting the genetic resources of this genus, for detecting allelic variants in loci associated with important agronomic traits, and for monitoring alleles introgressed from wild relatives to cultivated rice.     

Polymorphism identification within 50 equine gene-specific sequence tagged sites

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.     

A panel of human chromosome 22-specific sequence tagged sites

A panel of 29 sequence tagged sites (STSs) covering the long arm of chromosome 22 has been assembled. STS primer pairs were synthesized using available chromosome 22 sequence derived from the GenBank and EMBL DNA sequence databases, as well as published cDNA and genomic sequence, or from previously published and communicated primer pairs. Each STS was optimized for the polymerase chain reaction using a chromosome 22-only hybrid and human genomic DNA. Further STS content analysis on a panel of somatic cell hybrids that incorporated two chromosome 22 translocations resulted in the mapping of the X-box binding protein (XBP), D22S156, and transcobalamin II (TCN2) genes to 22q11-q13.1. The panel of STSs was used for the rapid determination of the STS content and thus the chromosomal DNA content of a new irradiation hybrid.     

Direct DNA sequence determination from total genomic DNA.

It is possible to perform a combined amplification and sequencing reaction ('DEXAS') directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily determined directly from total genomic DNA.     

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transferability of sequence tagged microsatellite site (STMS) primers across four major pulses

The transferability of genome-specific sequence tagged microsatellite site (STMS) primers from field pea (P. sativum) and chickpea (C. arietinum) to other major pulses was examined. Overall, field pea STMS primers amplified products in most of the accessions in comparison to that of the chickpea STMS primers, which amplified products in relatively few accessions. The highest level of successful amplifications with a single primer was 89% for field pea and 33% for chickpea primers respectively. The potential transferability of the STMS primers among species, expressed as the total mean percentage of positive amplifications, was 53% for the field pea STMS primers and 9% for the chickpea STMS primers. The individual mean percentage of successful transferability of field pea STMS primers across lentil, vetch and chickpea/Cicer sp. accessions was 60%, 39% and 62%, respectively. Whereas, for the chickpea STMS primers successful transferability was 5%, 3% and 18% for lentil, vetch and field pea, respectively. The trnasferability of these STMS primers indicates a high level of sequence conservation in these regions across species. Together with their locus-specificity, co-dominant nature and potential to amplify multiple alleles, their transferability makes STMS markers a powerful tool for genetic mapping, diversity analysis and genotyping.     

Immobilization of DNA for affinity chromatography and drug-binding studies.

A method is described in which double-stranded DNA is alkylated with 4-bis-(2-chloroethyl)amino-L-phenylalanine and the product immobilized on an insoluble support via the primary amino group of the phenylalanine moiety. The DNA is hence irreversibly bound to the matrix by both strands at a limited number of points.