Gary A Ablett

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discovering 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 x 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 cap3. 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.

EST versus Genomic Derived Microsatellite Markers for Genotyping Wild and Cultivated Barley

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.

Mapping and validation of the genes for resistance to Pyrenophora teres f. teres in barley (Hordeum vulgare L.)

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 (R2 = 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 R2 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 (R2 = 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.

Potential of SSR markers for plant breeding and variety identification in Australian barley germplasm

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.

Single nucleotide polymorphisms in cytochrome P450 genes from barley

Abstract.Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.

Genetic structuring in the spotted gum complex (genus Corymbia, section Politaria)

Spotted gums (genus Corymbia, section Politaria) occur as a species replacement series along the eastern seaboard of Australia, their distributions marked by regions of disjunction and sympatry. Their taxonomy remains controversial, with species assignment often challenging and reliant on knowledge of geographic origin as well as subtle morphological or leaf-oil variation. In the present paper, we explore a classification for spotted gums, without assuming predefined geographic or taxonomic groups but instead using genetic structure at microsatellite marker loci (n = 9) and a Bayesian model-based clustering approach implemented in Structure software. The C. torelliana outgroup (n = 21; section Cadagaria) formed a well resolved cluster (minimum pairwise Fst = 0.19). Four populations were evident within the spotted gums (n = 93) but structure was weak (pairwise Fst range 0.13–0.05). Geographic distance, topography and distribution disjunction were major determinants of structure, with migration among populations approximating a linear stepping-stone model. Corymbia maculata was resolved as a taxon and had the greatest genetic distance from any other population (minimum pairwise Fst 0.08). Three clusters were evident within the northern taxa but alignment with taxonomic groupings was poor. C. citriodora material from north of a major disjunction in central Queensland formed a Northern population. C. citriodora, C. variegata and C. henryi material south of this disjunction but north of the Border Range, formed a Central population, whereas a Southern population comprised C. variegata and C. henryi from predominately south of the Border Range.

Sequence Polymorphism Discovery in Wheat Microsatellite Flanking Regions using Pyrophosphate Sequencing

Sequence polymorphisms such as insertion/deletions (indels) and single nucleotide polymorphisms (SNP) are suitable for automated analysis of molecular markers and useful for cultivar identification, genetic mapping and trait association. While they are abundant, their initial discovery, comprising detection, validation and characterisation of sequence polymorphisms, is time consuming and expensive. This is especially true for multi-allellic hexaploid wheat. We investigated simple sequence repeat (SSR) flanking regions as a source for sequence polymorphisms in wheat. SSRs have a potentially high polymorphic frequency, there are a large number of highly characterised markers available, tested primers are published and most are single locus. Of 126 markers investigated, polymorphisms were found in 33 (26%) when tested in 10 wheat varieties. No new primers needed to be designed, the published primer sequences were used as PCR primers and then as sequencing primers. Polymorphism was detected by resequencing using a modification of pyrophosphate sequencing (Pyrosequencing®) which yielded quality sequencing from the first base after the primer with up to 80 bases of information. Our method of pyrophosphate sequencing of SSRs, although not suitable for full-length sequencing, is an attractive method to directly find sequence polymorphism in varieties of interest using the abundant, well characterised and published SSR markers.

Mapping and QTL analysis of the barley population Tallon x Kaputar

A genetic map of barley with 224 AFLP and 39 simple sequence repeat (SSR) markers was constructed using a doubled haploid (DH) mapping population from a cross between the varieties Tallon and Kaputar. Linkage groups were assigned to individual barley chromosomes using the published map locations of the SSR markers as reference points. This genetic map was used to identify markers with linkage to agronomic, disease, and quality traits in barley. The population, which comprised 65 lines, was tested in a range of environments across Australia. Quantitative trait loci (QTLs) analyses were performed using software packages MapMaker, MapManager, and Qgene. Significant associations with markers were found for several traits. Grain yield showed significant association with regions on chromosomes 2H, 3H, and 5H over a range of sites throughout Australia. Regions on chromosomes 2H and 3H explained 30% and 26% of variation in lodging, respectively. Among quality traits, diastatic power was associated with regions on chromosomes 1H, 2H, and 5H (R2 = 37%). Hot water extract was associated with a region on chromosome 6H and a marker not assigned to a chromosome (R2 = 45%). There were also environment-specific QTLs for the traits analysed. The markers identified here present an opportunity for marker assisted selection of lines for these traits in barley breeding programs.

High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.

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.

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae)

Premise of the study: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow. Methods and Results: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species—M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii—and in commercial macadamia cultivars including hybrids. Conclusions: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.