Tom Blake

A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.

Quantitative trait locus effects and environmental interaction in a sample of North American barley germ plasm

Quantitative trait locus (QTL) and QTL x environment (E) interaction effects for agronomic and malting quality traits were measured using a 123-point linkage map and multi-environment phenotype data from an F1-derived doubled haploid population of barley (Hordeum vulgare). The QTL × E interactions were due to differences in magnitude of QTL effects. Highly significant QTL effects were found for all traits at multiple sites in the genome. Yield QTL peaks and support intervals often coincided with plant height and lodging QTL peaks and support intervals. QTL were detected in the vicinity of a previously mapped Mendelian maturity locus and known function probes forα- andβ-amylase genes. The average map density (9.6 cM) should be adequate for molecular marker-assisted selection, particularly since there were few cases of alternative favorable alleles for different traits mapping to the same or adjacent intervals.

Genetic analysis of the components of winterhardiness in barley (Hordeum vulgare L.)

Winterhardiness in cereals is the consequence of a number of complex and interacting component characters: cold tolerance, vernalization requirement, and photoperiod sensitivity. An understanding of the genetic basis of these component traits should allow for more-effective selection. Genome map-based analyses hold considerable promise for dissecting complex phenotypes. A 74-point linkage map was developed from 100 doubled haploid lines derived from a winter x spring barley cross and used as the basis for quantitative trait locus (QTL) analyses to determine the chromosome location of genes controlling components of winterhardiness. Despite the greater genome coverage provided by the current map, a previously-reported interval on chromosome 7 remains the only region where significant QTL effects for winter survival were detected in this population. QTLs for growth habit and heading date, under 16 h and 24 h light, map to the same region. A QTL for heading date under these photoperiod regimes also maps to chromosome 2. Contrasting alleles at these loci interact in an epistatic fashion. A distinct set of QTLs mapping to chromosomes 1, 2, 3, and 5 determined heading date under 8 h of light. Under field conditions, all QTLs identified under controlled environment conditions were determinants of heading date. Patterns of differential QTL expression, coupled with additive and additive x additive QTL effects, underscore the complexity of winterhardiness. The presence of unique phenotype combinations in the mapping population suggests that coincident QTLs for heading date and winter survival represent the effects of linkage rather than pleiotropy.

Linkage mapping of two mutations that reduce phytic acid content of barley grain

Abstract This study describes the inheritance and linkage map positions of two low phytic acid barley (Hordeum vulgare) mutations, lpa1-1 and lpa2-1, that dramatically reduce grain phytic acid content and increase inorganic seed phosphorus (P). Wide-cross, F2 mapping populations were constructed by mating six-rowed varieties, ‘Steptoe’ and/or ‘Morex’, with two-rowed ‘Harrington’lpa donor lines homozygous for either lpa1-1 or lpa2-1. The barley lpa1-1 mutation showed normal inheritance patterns, whereas a deficiency of homozygous lpa2-1/lpa2-1 F2 plants was observed. We identified a codominant, STS-PCR marker (aMSU21) that cosegregated with lpa1-1 in a population of 41 F2 plants. The aMSU21 marker was then mapped to a locus on barley chromosome 2H, using a North American Barley Genome Mapping Project (NABGMP) doubled haploid population (‘Harrington’בMorex’). We determined that lpa2-1 is located within a recombination interval of approximately 30 cM between two AFLP markers that were subsequently mapped to barley chromosome 7H by integration with the same NABGMP population. Recent comparative mapping studies indicate conserved genetic map orders of several homologous molecular marker loci in maize and the Triticeae species that also show corresponding linkage to the biochemically similar lpa2 mutations of maize and barley. This observation suggests that barley and maize lpa2 mutations may affect orthologous genes. No such evidence for correspondence of the phenotypically similar lpa1 mutations of barley and maize has been revealed.

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

Abstract Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications.

Identification of a microsatellite on chromosomes 6B and a STS on 7D of bread wheat showing an association with preharvest sprouting tolerance

Abstract In bread wheat, the transfer of tolerance to preharvest sprouting (PHS) that is associated with genotypes having red kernel colour to genotypes with amber kernels is difficult using conventional methods of plant breeding. The study here was undertaken to identify DNA markers linked with tolerance to PHS as these would allow indirect marker-assisted selection of PHS-tolerant genotypes with amber kernels. For this purpose, a set of 100 recombinant inbred lines (RILs) was developed using a cross between a PHS-tolerant genotype, SPR8198, with red kernels and a PHS-susceptible cultivar, ‘HD2329’, with white kernels. The two parents were analysed with 232 STMS (sequence-tagged microsatellite site) and 138 STS (sequence-tagged site) primer pairs. A total of 300 (167 STMSs and 133 STSs) primer pairs proved functional by giving scorable PCR products. Of these, 57 (34%) STMS and 30 (23%) STS primer pairs detected reproducible polymorphism between the parent genotypes. Using these primer pairs, we carried out bulked segregant analysis on two bulked DNAs, one obtained by pooling DNA from 5 PHS-tolerant RILs and the other similarly derived by pooling DNA from 5 PHS-susceptible RILs. Two molecular markers, 1 STMS primer pair for the locus wmc104 anda STS primer pair for the locus MST101, showed apparent linkage with tolerance to PHS. This was confirmed following selective genotyping of individual RILs included in the bulks. Chi-square contingency tests for independence were conducted on the cosegregation data collected on 100 RILs involving each of the two molecular markers (wmc104 and MST101) and PHS. The tests revealed a strong association between each of the markers and tolerance to PHS. Using nullisomic-tetrasomic lines, we were able to assign wmc104 and MST101 to chromosomes 6B and 7D, respectively. The results also indicated that the tolerance to PHS in SPR8198 is perhaps governed by two genes (linked with two molecular markers) exhibiting complementary interaction.

Evaluation of "sequence-tagged-site" PCR products as molecular markers in wheat.

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.

Sequence-tagged-site-facilitated PCR for barley genome mapping

SummarySpeed, efficiency, and safety considerations have led many genome mapping projects to evaluate polymerase chain reaction (PCR) sequence amplification as an alternative to Southern blot analysis. However, the availability of informative primer sequences can be a limiting factor in PCR-based mapping. An alternative to random amplified polymorphism detection (RAPD) is the sequence-tagged-site (STS) approach. If informative primer sequences could be derived from known sequences, then current maps, which are based on both known function and anonymous clones, might be easily converted to maps utilizing PCR technology. In this paper, four pairs of primer sequences were obtained from published sequences, and four pairs were obtained by sequencing portions of DNA clones from genomic clones derived from a random genomic library used in the North American Barley Genome Mapping Project (NABGMP). These primers were used to screen for polymorphisms in the progeny of a winter x spring and a spring x spring barley cross. Two types of polymorphisms were distinguished using these primer sets: (1) insertion/deletion events that could be read directly from agarose gels, and (2) point mutation events. The latter were identified using polyacrylamide-gel electrophoresis of PCR products following digestion with restriction endonucleases (four-base cutters). To determine whether the PCR-based polymorphisms were allelic to polymorphisms identified by the clones from which the primer sequences derived, chromosomal assignments and (when possible) co-segregation analysis was performed.

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of β-glucan content and β-glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt β-glucan content and for green and finished malt β-glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley β-glucan, 6 QTL for malt β-glucan, 3 QTL for β-glucanase in green malt and 5 QTL for β-glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley β-glucan, malt βglucan, green malt β-glucanase and finished malt βglucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt βglucan content, green and finished malt β-glucanase activity, and other malting quality parameters.

STS-PCR markers appropriate for wheat-barley introgression

Introgression of chromosomal segments across large taxonomic distances has long been an objective of scientists interested in understanding the relationships between genes and their effect on phenotype. Barley and wheat represent cultivated members of the Triticeae with different zones of adaptation, different responses to pathogens, and different end-use characteristics. Introduction of small, well-characterized chromosomal segments among grass relatives presents an opportunity to both better understand how genes perform in novel genomic environments and to learn more about the evolutionary novelties which differentiate related species. Since the distribution of the wheat-barley addition lines, the potential power and value of a comprehensive series of wheat/barley translocation lines has been widely appreciated. A scarcity of easy-touse markers which unambiguously distinguish barley loci from their wheat homologues has limited the ability of scientists to identify the relatively rare inter-chromosomal recombination events which are the necessary antecedents of these lines. Since the single most critical pathogen affecting U.S. wheat producers is Karnal bunt (Tilletia indica) and since barley carries a gene conferring immunity, molecular markers may prove practically and immediately important. In this report we describe a series of 135 barley-specific markers amplified by 115 primer sets developed from sequences from previously mapped restriction fragment length polymorphism (RFLP) markers. These easily distinguish the cognate barley products from their wheat counterparts and should find ready use in the identification of lines which contain wheat/barley translocation events.