Stephen J. Molnar

Identification of a RAPD marker linked to the oat stem rust gene Pg3.

SummaryThe feasibility of identifying molecular markers linked to disease resistance genes in oats was investigated utilizing random primers in conjunction with polymerase chain reaction technology. A pair of near-isogenic oat lines were screened for polymorphic DNA fragments linked to the stem rust resistance gene Pg3. Two primers were identified which amplified DNA fragments that were polymorphic between the lines analyzed. One primer (ACOpR-2) was shown to be completely linked to the Pg3 locus; the other primer was not linked to either the ACOpR-2 or the Pg3 loci. This type of analysis, combined with rapid leaf disc DNA extraction techniques, offers an effective means of identifying useful molecular markers and of applying them to plant breeding selection strategies.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species.

SummaryTandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

A molecular linkage map with associated QTLs from a hulless × covered spring oat population

In spring-type oat (Avena sativa L.), quantitative trait loci (QTLs) detected in adapted populations may have the greatest potential for improving germplasm via marker-assisted selection. An F6 recombinant inbred (RI) population was developed from a cross between two Canadian spring oat varieties: ‘Terra’, a hulless line, and ‘Marion’, an elite covered-seeded line. A molecular linkage map was generated using 430 AFLP, RFLP, RAPD, SCAR, and phenotypic markers scored on 101 RI lines. This map was refined by selecting a robust set of 124 framework markers that mapped to 35 linkage groups and contained 35 unlinked loci. One hundred one lines grown in up to 13 field environments in Canada and the United States between 1992 and 1997 were evaluated for 16 agronomic, kernel, and chemical composition traits. QTLs were localized using three detection methods with an experiment-wide error rate of approximately 0.05 for each trait. In total, 34 main-effect QTLs affecting the following traits were identified: heading date, plant height, lodging, visual score, grain yield, kernel weight, milling yield, test weight, thin and plump kernels, groat β-glucan concentration, oil concentration, and protein. Several of these correspond to QTLs in homologous or homoeologous regions reported in other oat QTL studies. Twenty-four QTL-by-environment interactions and three epistatic interactions were also detected. The locus controlling the covered/hulless character (N1) affected most of the traits measured in this study. Additive QTL models with N1 as a covariate were superior to models based on separate covered and hulless sub-populations. This approach is recommended for other populations segregating for major genes. Marker-trait associations identified in this study have considerable potential for use in marker-assisted selection strategies to improve traits within spring oat breeding programs.

Relationship between asparagine metabolism and protein concentration in soybean seed

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Identification of molecular markers for aluminium tolerance in diploid oat through comparative mapping and QTL analysis

The degree of aluminium tolerance varies widely across cereal species, with oats (Avena spp.) being among the most tolerant. The objective of this study was to identify molecular markers linked to aluminium tolerance in the diploid oat A. strigosa. Restriction fragment length polymorphism markers were tested in regions where comparative mapping indicated the potential for orthologous quantitative trait loci (QTL) for aluminium tolerance in other grass species. Amplified fragment length polymorphism (AFLP) and sequence-characterized amplified region (SCAR) markers were used to provide additional coverage of the genome. Four QTL were identified. The largest QTL explained 39% of the variation and is possibly orthologous to the major gene found in the Triticeae as well as Alm1 in maize and a minor gene in rice. A second QTL may be orthologous to the Alm2 gene in maize. Two other QTL were associated with anonymous markers. Together, these QTL accounted for 55% of the variation. A SCAR marker linked to the major QTL identified in this study could be used to introgress the aluminium tolerance trait from A. strigosa into cultivated oat germplasm.

Associations Among Oat Traits and Their Responses to the Environment

Abstract Desirable qualities of milling oat varieties include low hull content (high groat content), high beta-glucan content, high groat protein, low oil concentration, low kernel breakage, high grain yield, and superior yield stability. The objective of this study was to develop a graphical method for understanding the influence of environment on genetic relationships among traits. Associations among agronomic and quality traits in 67 oat (Avena sativa L.) performance trials conducted during 1996-2003 across Canada and some northern US states were studied using a trait-association by environment biplot, which allows visual study of pair-wise trait associations in multiple environments (year-location combinations). Based on the differential association of yield with days to heading and plant height, the North American spring oat growing regions can be divided into Northern mega-environment (Canadian Prairies plus North Dakota and Idaho) and Southern megaenvironment (Minnesota, South Dakota, and Ontario). We also found that the following trait associations were relatively stable across environments: (1) negative association of protein content vs. oat yield, (2) positive association of beta-glucan vs. groat oil, (3) positive association of beta-glucan vs. protein content, and (4) negative association of beta-glucan vs. breakage. All trait-associations were of moderate magnitude and were responsive to the environment. This suggests that breeding for superior oat varieties with desired trait combinations is possible, but it must be achieved through direct selection for multiple traits in representative environments.

Groat yield of naked and covered oat

Near-isogenic covered (CN 18941) and naked (CN 18942) lines of the oat (Avena sativa L.) cultivar NO 141-1 were developed to determine if the contrasting spikelet morphologies of the two lines affected groat yields differentially. Molecular markers verified the high level of genetic similarity between the two lines. Their groat yields were not significantly different under field conditions. Key words: Hulless oats, Hulled oats, Avena sativa L., near- isogenic lines, groat yield, amplified fragment polymorphism

EC_oligos: automated and whole-genome primer design for exons within one or between two genomes

SUMMARY EC_oligos designs oligonucleotides (oligos) from exons of annotated genomic sequence information. It can automatically and rapidly select oligos that are conserved between two sets of sequence data, and can pair up oligos for use as PCR primers. It can do this on a whole-genome scale and according to user-defined criteria. AVAILABILITY The source code, executable program and user manual are available at ftp://ftp.ebi.ac.uk/pub/software/dos/EC_oligos/.

Novel Septoria Speckled Leaf Blotch Resistance Loci in a Barley Doubled-Haploid Population

The genetics of resistance to Septoria speckled leaf blotch (SSLB), caused by Septoria passerinii, was studied in the Leger × CIho9831 barley doubled-haploid population. The 140 lines in the population segregated as 102 resistant and 38 susceptible, approximating a 3:1 ratio. A recombination map was developed using diversity arrays technology and other molecular markers. Quantitative trait locus (QTL) analysis demonstrated that resistance is primarily conferred either by having the CIho9831 allele at a QTL on 6HS or by having the CIho9831 allele at both of two QTLs on 3H and 2HL. In addition, ≈1/16 of the lines were resistant for unidentified reasons. This model predicts a resistant/susceptible ratio of 11:5, which fits the phenotypic observations. Minor QTLs were detected on 2HS and 1H. DNA sequences of linked markers suggest that the 6HS, 3H, and 2HS QTLs are part of resistance gene clusters and that the 6HS and 3H QTLs share homology. The 6HS QTL is identical to or closely linked to the SSLB resistance locus Rsp4 and the 1H QTL to the Rsp2 or Rsp3 locus. The 3H and 2HS QTLs are unique and offer new opportunities for pyramiding resistance genes through marker-assisted breeding for resistance to S. passerinii.

Molecular marker linkage maps in diploid and hexaploid oat ( Avena sp.)

The genus Avena is organized into 14 taxa (8 diploid, 5 tetraploid, and 1 hexaploid) classified on the basis of chromosome number, genome, diaspore (unit of dispersal), flower morphology, and cross fertility (Ladizinsky 1989). Based on chromosome pairing and structure, diploid, tetraploid, and hexaploid species were given the genomic designations A or C, AABB or AACC, and AACCDD, respectively (Rajhathy and Thomas 1974). The primary cultivated oat species are hexaploid (2n = 6x = 42) A. sativa L. and A. byzantina C. Koch. Extensive cytological work has led to the development of karyotypes, as well as aneuploid stocks, (Rajhathy and Thomas 1974, Hacker and Riley 1965, Morikawa 1985, Linares et al. 1992, Jellen et al. 1993 and 1997). However, until recently identification of homoeologous groupings in hexaploid oat has been hindered by a lack of a complete aneuploid series, useful genetic markers, and easily identifiable chromosome morphology. The C-banding technique, and in certain cases the use of semi-automated digital image analysis/enhancement systems (Jellen et al. 1993), greatly facilitated the discrimination of individual chromosomes and/or genomes in diploid (Yen and Filion 1977, Fominaya et al. 1988a), tetraploid (Fominaya et al. 1988b), and hexaploid species (Linares et al. 1992, Jellen et al. 1993 and 1997). For instance, the C genome chromosomes are easily distinguished on the basis of their dark staining pattern from the A, B, or D genome chromosomes (Fominaya et al. 1988a, Jellen 1992). More recently, genomic in situ hybridization (GISH) and a demonstrated high hybridization specificity of DNA from C genome diploid species relative to A genome diploid species has allowed further delineation of C genome chromosomes from A and D genome chromosomes in AACC tetraploid and AACCDD hexaploids species. Several intergenomic translocations also have been identified within these allopolyploid species (Chen and Armstrong 1994, Jellen et al. 1994, Leggett and Markhand 1995).