Graham J. Scoles

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Abstract Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG)n, (AG/TC)n or (AAG/TTC)n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars.

Regulatory gene candidates and gene expression analysis of cold acclimation in winter and spring wheat

Freezing tolerance in plants develops through acclimation to cold by growth at low, above-freezing temperatures. Wheat is one of the most freezing-tolerant plants among major crop species and the wide range of freezing tolerance among wheat cultivars makes it an excellent model for investigation of the genetic basis of cold tolerance. Large numbers of genes are known to have altered levels of expression during the period of cold acclimation and there is keen interest in deciphering the signaling and regulatory pathways that control the changes in gene expression associated with acquired freezing tolerance. A 5740 feature cDNA amplicon microarray that was enriched for signal transduction and regulatory genes was constructed to compare changes in gene expression in a highly cold-tolerant winter wheat cultivar CDC Clair and a less tolerant spring cultivar, Quantum. Changes in gene expression over a time course of 14 days detected over 450 genes that were regulated by cold treatment and were differentially regulated between spring and winter cultivars, of these 130 are signaling or regulatory gene candidates, including: transcription factors, protein kinases, ubiquitin ligases and GTP, RNA and calcium binding proteins. Dynamic changes in transcript levels were seen at all periods of cold acclimation in both cultivars. There was an initial burst of gene activity detectable during the first day of CA, during which 90% of all genes with increases in transcript levels became clearly detectable and early expression differential between the two cultivars became more disparate with each successive period of cold acclimation.

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Abstract Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%).

AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.)

Abstract Rye (Secale cereale L.) is considered to be the most aluminum (Al)-tolerant species among the Triticeae. It has been suggested that aluminum tolerance in rye is controlled by three major genes (Alt genes) located on rye chromosome arms 3RL, 4RL, and 6RS, respectively. Screening of an F6 rye recombinant inbred line (RIL) population derived from the cross between an Al-tolerant rye (M39A-1–6) and an Al-sensitive rye (M77A-1) showed that a single gene controls aluminum tolerance in the population analyzed. In order to identify molecular markers tightly linked to the gene, we used a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis techniques to evaluate the F6 rye RIL population. We analyzed approximately 22,500 selectively amplified DNA fragments using 204 primer combinations and identified three AFLP markers tightly linked to the Alt gene. Two of these markers flanked the Alt locus at distance of 0.4 and 0.7 cM. Chromosomal localization using cloned AFLP and a restriction fragment length polymorphism (RFLP) marker indicated that the gene was on the long arm of rye chromosome 4R. The RFLP marker (BCD1230) co-segregated with the Alt gene. Since the gene is on chromosome 4R, the gene was designated as Alt3. These markers are being used as a starting point in the construction of a high resolution map of the Alt3 region in rye.

Immature embryo and anther culture of chromosome addition lines of rye in chinese spring wheat

Abstract Significant variation among Chinese Spring wheat ( Triticum aestivum L.) and a set of seven addition lines in which chromosomes from rye ( Secale cereale L.) were incorporated into the Chinese Spring background was observed for callus formation and plant regeneration from anther cultures and for plant regeneration from immature embryo cultures. Callus initiation from immature embryo cultures was uniformly high. Rye chromosome 4 contains factors which significantly increase both anther culture responses relative to Chinese Spring. Rye chromosomes 6 and 7 both contain positive factors for regeneration from immature embryo culture. While no correlation was found between anther culture and embryo culture responses, a positive correlation was observed between the two anther culture response variables.

Mapping quantitative trait loci associated with barley net blotch resistance

Net blotch of barley, caused by Pyrenophora teres Drechs., is an important foliar disease worldwide. Deployment of resistant cultivars is the most economic and eco-friendly control method. This report describes mapping of quantitative trait loci (QTL) associated with net blotch resistance in a doubled-haploid (DH) barley population using diversity arrays technology (DArT®) markers. One hundred and fifty DH lines from the cross CDC Dolly (susceptible)/TR251 (resistant) were screened as seedlings in controlled environments with net-form net blotch (NFNB) isolates WRS858 and WRS1607 and spot-form net blotch (SFNB) isolate WRS857. The population was also screened at the adult-plant stage for NFNB resistance in the field in 2005 and 2006. A high-density genetic linkage map of 90 DH lines was constructed using 457 DArT® and 11 SSR markers. A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in the 2005 and 2006 field trials. Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population. The major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada and could be routinely selected for using molecular markers in a practical breeding program.

Pigmentation in the developing seed coat and seedling leaves of Brassica carinata is controlled at the dihydroflavonol reductase locus

Flavonoid differences between near-isogenic lines of yellow- and brown-seeded Brassica carinata were used to identify a genetic block in seed coat and seedling leaf pigment biosynthesis. Seed coat pigment in the brown-seeded line consisted of proanthocyanidins (condensed tannins), while anthocyanin was absent. Dihydroquercetin, dihydrokaempferol, quercetin and kaempferol accumulated only in the mature seed coat of the yellow-seeded line, indicating dihydroflavonol reductase (DFR) as an element of genetic control in pigment biosynthesis. DFR transcripts from the developing seed coat in the yellow-seeded line were absent or less abundant at 5-30 days after pollination compared to transcript levels in the brown-seeded line. Seedling leaves of the yellow-seeded line exhibited reduced expression of DFR and contained less anthocyanin compared to the respective tissues from plants of the brown-seeded line when grown at 25/20 degrees C (day/night). Cooler (18/15 degrees C) growing temperatures affected seedling leaf pigmentation, mature seed coat colouration and DFR expression in the yellow-seeded line. Comparable brown-seeded line tissues were unaffected by these temperature changes. These results are suggestive of a temperature-sensitive regulator of DFR in the yellow-seeded line of Brassica carinata which ultimately affects the formation of pigments in the seedling leaves and in the mature seed coats.

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l−1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.

Isolation, characterization and expression analysis of a starch branching enzyme II cDNA from wheat

Abstract A full-length cDNA (2970 bp) encoding a starch branching enzyme II (SBEII; EC 2.4.1.18) in wheat (Triticum aestivum L. cv Fielder) kernel was isolated from a cDNA library. The translated region of the cDNA predicted a 823 amino acid primary product with a molecular mass of 91.4 kDa. A 54 amino acid transit peptide was postulated to be cleaved from the pre-protein to give a 769 amino acid (85.4 kDa) mature polypeptide, which showed extensive sequence similarity to SBEII sequences characterized from maize, rice and pea. Expression of the isolated cDNA in a BE-deficient E. coli strain demonstrated that it encoded a functional BE. RNA analysis of Sbe2 gene expression during seed development revealed that Sbe2 mRNA levels were highest in young kernels (5–10 days post-anthesis) and declined as the kernels matured.

The use of RAPD analysis to classify Triticum accessions

Abstract Crop germplasm collections contain a considerable percentage of misclassified accessions which may affect the use of germplasm for agricultural crop improvement. The objective of this study was to determine if random amplified polymorphic DNA (RAPD) analysis could be used to reclassify misclassified Triticum accessions. Twelve accessions suspected to be misclassified, based on morphological characters, as either macha or vavilovii wheat were studied using RAPD and cytological analyses. In the RAPD analysis, a dendrogram, based on Jaccard genetic similarity coefficients, grouped 5 dicoccum-like, 1 timopheevii-like, and 6 monococcum-like accessions with Triticum dicoccum, T. timopheevii, and T. monococcum accessions, respectively. These results were confirmed by the cytological analysis. A RAPD marker specific to the D genome was also detected. This study suggests that RAPD analysis can be used to classify germplasm and to distinguish some species in Triticum.