SummaryDiscovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken.One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 × Q165). The sequencing yielded 96 755 (IJ76-514) and 86 241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases.Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program CAP 3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP -an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent -with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

Genetic variation present in wild and cultivated barley populations was investigated using two sources of microsatellite also known as simple sequence repeat (SSR) markers. EST-SSRs are derived from expressed sequences and genomic SSRs are isolated from genomic DNA. Genomic SSR markers detected a higher level of polymorphism than those derived from ESTs. Polymorphism information content was higher in genomic SSRs than EST-derived SSRs. This study showed that the EST-SSR markers developed in cultivated barley are polymorphic in wild and cultivated varieties and produced high quality markers. Ten of these functional markers were polymorphic across the accessions studied. EST markers indicated clearer separation between wild and cultivated barley than genomic SSRs. The EST-SSRs are a valuable source of new polymorphic markers and should be highly applicable to barley genetic resources, providing a direct estimate of functional biodiversity.

Abstract. Identification and deployment of disease resistance genes are key objectives of Australian barley breeding programs. Two doubled haploid (DH) populations derived from Tallon × Kaputar (TK) and VB9524 × ND11231 (VN) crosses were used to identify markers for net type net blotch (NTNB) (Pyrenophora teres f. teres). The maps included 263 and 250 markers for TK and VN populations, respectively. The TK population was screened with 5 pathotypes and the VN population with 1 pathotype of NTNB as seedlings in the glasshouse. In addition, the TK population was subjected to natural infection in the field at Hermitage Research Station, Qld. Analyses of the markers were performed using the software packages MapManager and Qgene. One region on chromosome 6H was strongly associated with resistance to NTNB in both populations (R 2 = 83% for TK and 66% for VN). In the TK population, 2 more quantitative trait loci (QTLs) were identified on chromosomes 2H and 3H, with R 2 values of 30% and 31%, respectively. These associations were consistent over all pathotypes studied during the seedling stage. The same QTL on chromosome 6H was also found to be highly significantly associated (R 2 = 65%) with the adult plant (field) response in the TK population. There are several very closely linked markers showing strong associations in these regions. Association of the 4 markers on chromosome 6H QTL with resistance to the NTNB has been validated in 2 other DH populations derived from barley crosses Pompadour × Stirling and WPG8412 × Stirling. These markers present an opportunity for marker assisted selection of lines resistant to NTNB in barley breeding programs.

SSR markers closely linked to 18 loci that control 16 important barley traits were assessed for their applicability in Australian barley breeding programs. A panel of 40 genotypes routinely used by the South Australian Barley Improvement Program (SABIP) was used to examine the usefulness of these SSR markers for marker assisted selection (MAS). The success of monitoring a trait locus from donor to recipient lines ranged from 10 to 98%, depending on the marker. SSRs with a high polymorphic information content (PIC) value were found to be the most useful for application in MAS. The assessment also indicated that SSRs derived from genomic sequences were more successful for MAS than those designed from expressed sequence tags. A total of 130 SSR markers were screened among 2 panels of Australian barley genotypes to determine which markers would be the most useful for discriminating Australian germplasm. PIC values generated by this screening were also compared with those generated using a panel of European barley genotypes. Using ordinary correlations (parametric), rank correlations (non-parametric), and partial correlations (multi-variate), a strong association was found between the 2 Australian panels, but no or weak correlation was observed between the 2 Australian panels and the European dataset. It can therefore be concluded that PIC values generated by SSR markers screened with European genotypes cannot be used to predict the usefulness of an SSR marker for discriminating Australian genotypes. From PIC values generated in this study, 36 SSR markers have been selected for the discrimination of Australian genotypes. These markers all show high and/or consistent PIC values among Australian and European barley genotypes.A R 0 2 1 7 8 S S R m a r k e r s f o r p l a n t b r e e d i n g i n b a r l e y A . K a r a k o u s i s e t a l .

Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae), was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD97) of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops.