Mapping of RFLP probes characterized for their polymorphism on wheat

 Wheat anonymous probes were selected for their efficiency for providing a readable hybridization pattern and revealing RFLP among wheat varieties. We report the mapping of 132 such probes (20 wheat-leaf cDNA, 28 wheat-root cDNA and 84 genomic DNA) on the reference population of the International Triticeae Mapping Initiative (ITMI) derived from the cross W-7984 with Opata85. Each probe has been characterized for its polymorphism information content. The 132 probes allowed us to map 160 loci. Received: 7 July 1998 / Accepted: 19 October 1998     

Conversion of AFLP markers associated with FHB resistance in wheat into STS markers with an extension-AFLP method.

Amplified fragment length polymorphism (AFLP) has proven a powerful tool for tagging genes or quantitative trait loci (QTLs) of interest in plants. However, conversion of AFLP markers into sequence-tagged site (STS) markers is technically challenging in wheat owing to the complicated nature of its genome. In this study, we developed an "extension-AFLP" method to convert AFLP markers associated with Fusarium head blight (FHB) resistance into STS markers. When an AFLP marker of interest was detected with an EcoRI+3-MseI+4-selective primer combination, the PCR product was used as a template for an additional selective amplification with four primer pairs, in which one additional selective base (either A, C, G, or T) was added to the 3' end of one of the two primers. The extended primer pair that produced the targeted band was further extended by adding each of the four selective nucleotide bases for the next round of selective amplification. Extension selective amplification was performed until the target bands became clear enough for subsequent cloning and sequencing. By using the extension-AFLP method, we successfully converted two AFLP markers located on chromosome 3BS and associated with FHB resistance into STS markers. Our results indicated that the extension-AFLP method is an efficient approach for converting AFLP markers into STS markers in wheat. The developed STS markers might be used for marker-assisted selection (MAS) for FHB resistance in wheat breeding programs.     

Restriction fragment length polymorphism (RFLP) analysis in wheat. II. Linkage maps of the RFLP sites in common wheat.

Sixty-six F2 plants from the cross, Triticum aestivum cv. Chinese Spring (abbrev. CS) x T. spelta var. duhamelianum (Spelta), exhibiting the greatest number of RFLPs among eight common wheats, were analyzed for their RFLP genotypes using genomic DNA clones of CS as probes. In total, 204 RFLP loci were identified and their linkage relationships established. By nulli-tetrasomic analyses, all linkage groups were assigned to one another of the 21 wheat chromosomes. In addition, the carrier chromosomes of 228 non-RFLP loci were identified. The linkage maps of these RFLP loci have a total size of 1800 cM and exceed those of the classical genes in both size and locus number. Twenty loci show distorted segregation, four of which are clustered on chromosome 4A and three on the 2D chromosome. The CS alleles on 4A exhibit preferential transmission, while those on 2D exhibit depressed transmission, compared with Spelta alleles. This suggests the influence of gametic factors in those regions. RFLP loci are much fewer in the D genome than in the A and B genomes, but the numbers of non-RFLP loci are nearly the same in these three genomes. This suggests that Spelta wheat originated from a hybridization between T. dicoccum (spelt emmer) and T. aestivum.     

Restriction fragment length polymorphism (RFLP) analysis in wheat. I. Genomic DNA library construction and RFLP analysis in common wheat

To develop detailed linkage maps of restriction fragment length polymorphism (RFLP) sites in wheat chromosomes, it was necessary to construct a genomic DNA library and to characterize the clones obtained. Forty-nine per cent of the clones were of single or low copy number per genome. With 91 clones of this class, as probes, and with two to four restriction endonucleases, for DNA digestion, RFLPs were examined among eight common wheats and a single emmer wheat. About 20% of the probes, and 13% of the probe-enzyme combinations revealed genetic polymorphism among the common wheats. DNA deletions account for most of the genetic differences among these wheat genomes. Based on the RFLP data, phylogenetic distances among the nine polyploid wheats were estimated, and a dendrogram showing the genetic relationships among them was constructed.     

Detection of point mutations in the chloroplast genome by single-stranded conformation polymorphism analysis

Single-stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin-resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild-type DNA fragments, the bands of the single-stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild-type and mutants.     

Powdery mildew resistance genes in wheat: verification of STS markers

Five accessions of Aegilops speltoides and 67 European wheat cultivars (winter and spring) originating from the Czech Republic, Germany, Poland, Russia, Slovakia, United Kingdom, and 4 non-European wheat cultivars from Brazil and the USA were examined with molecular Sequence Tagged Site (STS) markers for resistance genes to powdery mildew: Pm 1, Pm 2, Pm 3 and Pm 13. All markers gave clear, repeatable results, although three of them (Pm 1, Pm 2 and Pm 3) appeared as not specific for resistance genes. Comparison of STS analysis results with Pm genes, postulated as the reaction type after inoculation with differential isolates of Erysiphe graminis f.sp. tritici (Blumeria graminis), revealed a high number of disparities. The marker for Pm 13 was not detected in any examined cultivar but was present in five accessions of Aegilops speltoides.     

C-banding polymorphism and linkage of nonhomoeologous RFLP loci in the D genome progenitor of wheat.

Chromosomes from four different accessions of Triticum tauschii, used as parents in generating F2 populations for RFLP genetic linkage map construction, were analyzed by C-banding. The accessions consist of the varietal taxa strangulata (AUS 21929) and meyeri (AUS 18911), and two genotypes of var. typica (AUS 18902 and CPI 110730 from Iran and Afghanistan, respectively). Chromosomes 1D and 7D of T. tauschii var. typica AUS 18902 are involved in a reciprocal interchange forming translocated chromosomes, T1DS.7DL and T7DS.1DL, with tbe breakpoints being located within the centrometric region. The formation of quadrivalent configuration in F1 hybrids provided further confirmation of the reciprocal translocation. Genetic linkage mapping of additional RFLP markers located on homoeologous group 1 and 7 chromosomes showed consistent linkage to a composite group of proximal markers on chromosomes 1D and 7D of a previously published map derived from the F2 progeny of AUS 18902 x AUS 18911. A high frequency of RFLP genotypes transmitted by the translocation parent was prevalent in the proximal regions of chromosomes 1D and 7D. Genotypic frequencies expected of the nontranslocated parental RFLP markers was evident only in the distal regions of these chromosomes.     

Development of a chromosomal arm map for wheat based on RFLP markers

Summary A chromosomal arm map has been developed for common wheat (Triticum aestivum L. em. Thell.) using aneuploid stocks to locate more than 800 restriction fragments corresponding to 210 low-copy DNA clones from barley cDNA, oat cDNA, and wheat genomic libraries. The number of restriction fragments per chromosome arm correlates moderately well with relative DNA content and length of somatic chromosomes. The chromosomal arm locations of loci detected with 6 different clones support an earlier hypothesis for the occurrence of a two-step translocation (4AL to 5AL, 5AL to 7BS, and 7BS to 4AL) in the ancestral wheat genomes. In addition, 1 clone revealed the presence of a 5AL segment translocated to 4AL. Anomalies in aneuploid stocks were also observed and can be explained by intrahomoeologous recombination and polymorphisms among the stocks. We view the development of this chromosomal arm map as a complement to, rather than as a substitute for, a conventional RFLP linkage map in wheat.Paper No. 802 of the Cornell Plant Breeding Series     

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.     

The conformation of single-stranded nucleic acids tDNA versus tRNA

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNA(Met). Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNA(Met), tDNA(Met) (a DNA oligomer having the sequence of tRNA(Met] and the anti-tDNA(Met) (the complement of tDNA(Met] suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double-stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2'-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.     

Physical mapping of restriction fragment length polymorphism (RFLP) markers in homoeologous groups 1 and 3 chromosomes of wheat by in situ hybridization.

Using wheat ditelosomic lines and in situ hybridization of biotin-labelled DNA probes, 18 restriction fragment length polymorphism (RFLP) markers were physically located on homoeologous groups 1 and 3 chromosomes of wheat. Most of the markers hybridized to chromosome arms in a physical order concordant with the genetic maps. A majority of the markers studied were clustered in non-C-banded, distal euchromatic areas, indicating the presence of recombination hot spots and cold spots in those regions. However, on IBS the markers were well dispersed, which could be due to the abundance of heterochromatin throughout the arm. An inversion between Xpsr653 and Xpsr953 was observed on 1AL. One new Xpsr688 locus, approximately 20-26% from the centromere, was found on 1AS and 1BS. The physical location of Xpsr170 on group 3 chromosomes probably represents an alternative to the loci on the genetic map. Finally, Xpsr313 was mapped to two physical loci on IDL. Five markers were located to bins consistent with the deletion-based physical maps.     

Development of STS markers linked to Hessian fly resistance gene H6 in wheat

Hessian fly is one of the world's most destructive insect pests of wheat Triticum aestivum L. We have used the combination of near-isogenic lines (NIL) and random amplified polymorphic DNA (RAPD) analysis to screen up to 2,000 primers to identify DNA markers that are linked to gene H6 that confers resistance to biotype B of the insect. This screen produced six primers that show polymorphic fragments associated with resistance by H6. We have screened 440 F₂ individuals from a cross of the susceptible cultivar Newton and a NIL that contains H6 to verify the linkage between these markers and the resistance gene. A high-resolution genetic map was constructed based on recombination frequency. Two of the markers were tightly linked to the gene with no recombination observed, three were within 2.0 cM, and one was 11 cM from the gene. Three of the six markers were successfully converted to sequence tagged site (STS) markers. Both RAPD and STS primers were used to screen for the presence or absence of the resistance gene in wheat varieties. The identification of markers and construction of the genetic high resolution map provide the first steps toward localization of this resistance gene.     

The conversion of RFLP markers to allele specific amplicons linked to QTLs governing malting quality in barley

Application of marker-assisted selection with RFLP based markers has been constrained by high cost and time requirements in situations involving a large number of plants. RFLP markers mapped on a Harrington/TR306 population have been identified elsewhere as linked to quantitative trait loci (QTL) governing malting quality. The probes ABG610, ABC622, as well as probes for the Nar1, Amy1 and Nar7 were sequenced and locus specific primers developed. These locus specific primers were applied to genomic DNA from both Harrington and TR306. Sequence analysis of the resultant monomorphic fragments revealed sequence divergence for the Xabg610, Xabc622, Amy1 and Nar1 loci, but not for the Nar7 locus. Application of a set of Hor2 primers to genomic DNA from the barley lines Harrington and TR306 led to the direct amplification of codominant alleles. Allele-specific primers were designed based on the sequence divergence identified among the Xabg610, Xabc622 and Nar1 alleles. Amplification conditions were optimized for each of these alleles such that only the favourable allele from Harrington was amplified. The usefulness of these primers for selecting Harrington alleles was demonstrated by their failure to amplify the corresponding alleles from the lines, Sterling, Stella and WM872. The Amy1 allele-specific amplicon was only capable of differentiating this locus between Harrington and TR306. The conversion of these markers into PCR amplifiable, allele-specific amplicons would greatly facilitate their application to barley breeding programs.     

Genetic variability of the wild diploid wheat Triticum urartu revealed by RFLP and RAPD markers

 Genetic variability among 49 accessions of Triticum urartu was estimated by RFLP and RAPD marker analyses, and the two data sets were compared. One T. timopheevii accession and two accessions of T. durum and T. aestivum, respectively, were included to identify T. urartu accessions closely related to these polyploid wheats. Twenty eight RFLP clones and 29 RAPD primers generated 451 and 155 polymorphic bands, respectively. The three accessions from Armenia clustered together and were well separated from all other accessions, which showed less pronounced geographical patterns. Genetic similarity and co-phenetic values calculated with RAPD markers were very similar to those calculated with RFLP markers for the intraspecific comparisons, but not for the interspecific comparisons. The identification of individual T. urartu accessions which are more related to polyploid wheats than others was not possible. Received: 14 May 1996 / Accepted: 13 September 1996     

Application of STS markers for leaf rust resistance genes in near-isogenic lines of spring wheat cv. Thatcher

Sequence tagged site (STS) markers for eight resistance genes against Puccinia recondita f. sp. tritici were used to screen a set of near-isogenic lines of wheat cv. Thatcher containing in total 40 different Lr genes and their alleles. Polymerase chain reaction (PCR) analysis was carried out by using STS, SCAR and CAPS primers specific for the leaf rust resistance genes Lr1, Lr9, Lr10, Lr19, Lr24, Lr28, Lr37 and Lr47. The STS, CAPS and SCAR markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr37 and Lr47 were found to be reliable in diverse genetic backgrounds. The amplification product of the Lr1 gene marker was detected in the susceptible cv. Thatcher and in all of the near-isogenic lines examined except Lr2a, Lr2b, Lr2c and Lr19. The sequence analysis of PCR products amplified in lines Lr1, Lr10, Lr28 and in cv. Thatcher indicated that the near-isogenic lines and cv. Thatcher contained in the targeted chromosome region an allele that differed from the original alleles corresponding to Lr1/6*Thatcher (TLR621) and susceptible Thatcher (TH621). The amplification product specific to the STS marker of the Lr1 gene was amplified in almost all Thatcher near-isogenic lines and in cv. Thatcher because their alleles possessed primer sequences identical to the original allele TLR621. The marker for the Lr28 resistance gene was identified in line Lr28, carrying gene Lr28, and in 21 other near-isogenic lines. The sequencing of PCR products specific to Lr28 and generated in lines Lr1, Lr10 and Lr28 indicated that the lines Lr1, Lr10 and Lr28 are heterozygous in this region.     

Identification of RFLP markers for resistance to wheat spindle streak mosaic bymovirus (WSSMV) disease.

Wheat spindle streak mosaic bymovirus (WSSMV) causes an economically important disease of winter wheat in Europe and North America. Artificial inoculation with this virus to identify resistant wheat genotypes is difficult. This study was conducted to identify restriction fragment length polymorphism (RFLP) markers associated with resistance to this disease. A population, consisting of 104 F5 recombinant inbred lines from a cross between hexaploid Triticum aestivum cultivars 'Geneva' (resistant) and 'Augusta' (susceptible), was evaluated for WSSMV symptoms under field conditions for four years. Two linked markers on the long arm of chromosome 2D, Xbcd1095 and Xcdo373, were determined to be associated with WSSMV resistance by bulked segregant analysis of the 10 most resistant and 10 most susceptible lines. Marker Xcdo373 accounted for 79% and Xbcd1095 for 73% of the phenotypic variation. Our results suggest that resistance to WSSMV in this population is qualitative in nature and is controlled by few genes. These markers should be useful in the development of wheat cultivars resistant to WSSMV and perhaps also to wheat yellow mosaic bymovirus (WYMV).