Hordoindolines are associated with a major endosperm-texture QTL in barley (Hordeum vulgare).

Endosperm texture has a tremendous impact on the end-use quality of wheat (Triticum aestivum L.). Cultivars of barley (Hordeum vulgare L.), a close relative of wheat, also vary measurably in grain hardness. However, in contrast to wheat, little is known about the genetic control of barley grain hardness. Puroindolines are endosperm-specific proteins found in wheat and its relatives. In wheat, puroindoline sequence variation controls the majority of wheat grain texture variation. Hordoindolines, the puroindoline homologs of barley, have been identified and mapped. Recently, substantial allelic variation was found for hordoindolines among commercial barley cultivars. Our objective was to determine the influence of hordoindoline allelic variation upon grain hardness and dry matter digestibility in the 'Steptoe' x 'Morex' mapping population. This population is segregating for hordoindoline allele type, which was measured by a HinA/HinB/Gsp composite marker. One-hundred and fifty lines of the 'Steptoe' x 'Morex' population were grown in a replicated field trial. Grain hardness was estimated by near-infrared reflectance (NIR) and measured using the single kernel characterization system (SKCS). Variation attributable to the HinA/HinB/Gsp locus averaged 5.7 SKCS hardness units (SKCS U). QTL analysis revealed the presence of several areas of the genome associated with grain hardness. The largest QTL mapped to the HinA/HinB/Gsp region on the short arm of chomosome 7 (5H). This QTL explains 22% of the SKCS hardness difference observed in this study. The results indicate that the Hardness locus is present in barley and implicates the hordoindolines in endosperm texture control.

Identification of barley genome segments introgressed into wheat using PCR markers.

Barley has several important traits that might be used in the genetic improvement of wheat. For this report, we have produced wheat-barley recombinants involving barley chromosomes 4 (4H) and 7 (5H). Wheat-barley disomic addition lines were crossed with 'Chinese Spring' wheat carrying the phlb mutation to promote homoeologous pairing. Selection was performed using polymerase chain reaction (PCR) markers to identify lines with the barley chromosome in the ph1b background. These lines were self pollinated, and recombinants were identified using sequence-tagged-site (STS) primer sets that allowed differentiation between barley and wheat chromosomes. Several recombinant lines were isolated that involved different STS-PCR markers. Recombination was confirmed by allowing the lines to self pollinate and rescreening the progeny via STS-PCR. Progeny testing confirmed 9 recombinants involving barley chromosome 4 (4H) and 11 recombinants involving barley chromosome 7 (5H). Some recombinants were observed cytologically to eliminate the possibility of broken chromosomes. Since transmission of the recombinant chromosomes was lower than expected and since seed set was reduced in recombinant lines, the utility of producing recombinants with this method is uncertain.

Identification and mapping of a leaf rust resistance gene in barley line Q21861.

Barley line Q21861 possesses an incompletely dominant gene (RphQ) for resistance to leaf rust caused by Puccinia hordei. To investigate the allelic and linkage relations between RphQ and other known Rph genes, F2 populations from crosses between Q21861 and donors of Rph1 to Rph14 (except for Rph8) were evaluated for leaf rust reaction at the seedling stage. Results indicate that RphQ is either allelic with or closely linked to the Rph2 locus. A doubled haploid population derived from a cross between Q21861 and SM89010 (a leaf rust susceptible line) was used for molecular mapping of the resistance locus. Bulked segregant analysis was used to identify markers linked to RphQ, using random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and sequence tagged sites (STSs). Of 600 decamer primers screened, amplified fragments generated by 9 primers were found to be linked to the RphQ locus; however, only 4 of them were within 10 cM of the target. The RphQ locus was mapped to the centromeric region of chromosome 7, with a linkage distance of 3.5 cM from the RFLP marker CDO749. Rrn2, an RFLP clone from the ribosomal RNA intergenic spacer region, was found to be very closely linked with RphQ, based on bulked segregant analysis. An STS marker, ITS1, derived from Rrn2, was also closely linked (1.6 cM) to RphQ.

Transfer of sequence tagged site PCR markers between wheat and barley.

Transfer of mapping information between related species has facilitated the development of restriction fragment length polymorphism (RFLP) maps in the cereals. Sequence tagged site (STS) primer sets for use in the polymerase chain reaction may be developed from mapped RFLP clones. For this study, we mapped 97 STS primer sets to chromosomes in wheat and barley to determine the potential transferability of the primer sets and the degree of correspondence between RFLP and STS locations. STS products mapped to the same chromosome group in wheat and barley 75% of the time. RFLP location predicted STS location 69% of the time in wheat and 56% of the time in barley. Southern hybridizations showed that most primer sets amplified sequences homologous to the RFLP clone, although additional sequences were often amplified that did not hybridize to the RFLP clone. Nontarget sequences were often amplified when primer sets were transferred across species. In general, results suggest a good probability of success in transferring STSs between wheat and barley, and that RFLP location can be used to predict STS location. However, transferability of STSs cannot be assumed, suggesting a need for recombinational mapping of STS markers in each species as new primer sets are developed. Key words : sequence tagged sites, PCR, wheat, barley.

Evaluation of barley chromosome-3 yield QTLs in a backcross F2 population using STS-PCR.

Chromosome 3 displayed the two largest yield QTLs in a previous study of 150 doubled haploid lines derived from a cross of Steptoe and Morex barley varieties. Low-copy number RFLP markers, detected using Southern analysis, are excellent tools for generating robust linkage maps as demonstrated by the Steptoe and Morex map produced by the North American Barley Genome Mapping Project (SM NABGMP). However, this technique can be cumbersome when applied to practically oriented plant breeding programs. In the present report, we demonstrate the conversion of RFLPs to more practically useful PCR-based markers that are co-dominant and allelic to the barley chromosome-3 RFLP markers from which they derive. We have used these sequence-tagged-site (STS) PCR markers to evaluate the putative yield QTL components of the Steptoe chromosome 3 in a Morex backcross population. Headshattering, plant lodging, and yield measurements are reported from five replicated field experiments conducted under diverse growing conditions in Montana. Our study detected significant effects for all three traits in a chromosomal region that evidently corresponds to the larger of the two previously reported chromosome-3 QTLs. However, we failed to detect any yield or other effects which might be coincidental to the second largest yield QTL. The genetic effects of the yield QTL identified in our first backcross breeding population show similar magnitude, environmental interactions, and association with lodging and headshattering QTLs observed in the SM NABGMP experiments. Our study elucidates complex environmental conditioning for headshattering and plant lodging which probably underlie the variable yield effects observed under different growing conditions.

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.

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

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

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.

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.

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

Speed, 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.

Biparental inheritance of chloroplast DNA and the existence of heteroplasmic cells in alfalfa.

Mapping of chloroplast DNA (ctDNA) restriction fragment patterns from a chlorophyll deficient mutant and two phenotypically normal alfalfa genotypes (Medicago sativa L.) has demonstrated the existence of a distinct ctDNA genotype from each source. These unique restriction fragment patterns were utilized to identify maternal or paternal origin of ctDNA in hybrid plants from crosses involving the normal alfalfa genotypes as females and the yellow-green chlorophyll deficient sectors as males. Progeny from these crosses expressing the yellow-green sectored phenotypes contained paternal ctDNA in the chlorophyll deficient sectors and maternal ctDNA in the normal sectors, confirming biparental plastid inheritance. The existence of mixed cells containing both mutant and normal plastids at various stages of sorting-out was observed by transmission electron microscopy of mesophyll cells in mosaic tissue from hybrid plants. This observation verified the biparental transmission of plastids in alfalfa.

Comparison of growth, exogenous auxin sensitivity, and endogenous indole-3-acetic acid content in roots of Hordeum vulgare L. and an agravitropic mutant.

The chemically induced barley (Hordeum vulgare L.) mutation, agr, was found to be a simple recessive trait resulting in agravitropic roots and normal gravitropic shoots. The total seedling root growth was similar for mutant and wild-type roots, although the mutant had fewer roots per seed and greater elongation per root. Although the concentration of exogenous indole-3-acetic acid (IAA) required to reduce root growth by 50% (GR50) was 12 times greater for the agravitropic mutant, agravitropic and gravitropic roots were equally sensitive to exogenous applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). Root IAA contents, determined by high-pressure liquid chromatography (HPLC), were not different for gravitropes and agravitropes. The greater root elongation rates, lack of sensitivity to exogenous IAA, and normal endogenous IAA levels indicate that auxin-controlled growth regulation may be altered in the mutant.

Mapping of the Hor-3 locus encoding D hordein in Barley.

The hordein storage proteins of barley (Hordeum vulgare L.) are of intense interest due to their genetic diversity and prominence and impact on the industrial and agricultural uses of the seed. Two major hordein loci have been previously mapped on chromosome 5 (Hor-1 and Hor-2 encoding the C and B hordeins, respectively). A third major locus, Hor-3, which codes for D hordein, has been located in the centromeric region of chromosome 5, probably on the long arm. Two allelic variants with apparent molecular weights of 83,000 and 91,000 and similar isoelectric points of 8.0 comprise the products of this locus in the barley varieties 'Advance' and 'Triple Awned Lemma'. The D hordein examined is similar in molecular weight and isoelectric point to the high molecular weight (HMW) glutenin proteins encoded by the 1B chromosome of wheat (Triticum aestivum L.).