Peter C Bundock

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.

Adaptive climatic molecular evolution in wild barley at the Isa defense locus.

Wild barley (Hordeum spontaneum) represents a significant genetic resource for crop improvement in barley (Hordeum vulgare) and for the study of the evolution and domestication of plant populations. The Isa gene from barley has a putative role in plant defense. This gene encodes a bifunctional α-amylase/subtilisin inhibitor that inhibits the bacterial serine protease subtilisin, fungal xylanase, and the plants own α-amylase. The inhibition of plant α-amylases suggests this protein may also be important for grain quality from a human perspective. We identified 16 SNPs in the coding region of the Isa locus of 178 wild barley accessions from eight climatically divergent sites across Israel. The pattern of SNPs suggested a large number of recombination events within this gene, indicating that the low-outcrossing rate of wild barley is not a barrier to recombinant haplotypes becoming established in the population. Seven amino acid substitutions were present in the coding region. Genetic diversity for each population was calculated by using Neis diversity index, and a Spearman rank correlation was carried out to test the association between gene diversity and 16 ecogeographical factors. Highly significant correlations were found between diversity at the Isa locus and key water variables, evaporation, rainfall, humidity, and latitude. The pattern of association suggests selective sweeps in the wetter climates, with resulting low diversity and weaker selection or diversifying selection in the dryer climates resulting in much higher diversity.

SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids

Many plant species of great economic value (e.g., potato, wheat, cotton, and sugarcane) are polyploids. Despite the essential roles of autopolyploid plants in human activities, our genetic understanding of these species is still poor. Recent progress in instrumentation and biochemical manipulation has led to the accumulation of an incredible amount of genomic data. In this study, we demonstrate for the first time a successful genetic analysis in a highly polyploid genome (sugarcane) by the quantitative analysis of single-nucleotide polymorphism (SNP) allelic dosage and the application of a new data analysis framework. This study provides a better understanding of autopolyploid genomic structure and is a sound basis for genetic studies. The proposed methods can be employed to analyse the genome of any autopolyploid and will permit the future development of high-quality genetic maps to assist in the assembly of reference genome sequences for polyploid species.

Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences

Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.

Enrichment of genomic DNA for polymorphism detection in a non-model highly polyploid crop plant

Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8 megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270 000-280 000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13 000-16 000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.

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.

Application of large-scale sequencing to marker discovery in plants

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.

A novel highly differentially expressed gene in wheat endosperm associated with bread quality

Analysis of gene expression in developing wheat seeds was used to identify a gene, wheat bread making (wbm), with highly differential expression (~1000 fold) in the starchy endosperm of genotypes varying in bread making quality. Several alleles differing in the 5’-upstream region (promoter) of this gene were identified, with one present only in genotypes with high levels of wbm expression. RNA-Seq analysis revealed low or no wbm expression in most genotypes but high expression (0.2-0.4% of total gene expression) in genotypes that had good bread loaf volume. The wbm gene is predicted to encode a mature protein of 48 amino acids (including four cysteine residues) not previously identified in association with wheat quality, possibly because of its small size and low frequency in the wheat gene pool. Genotypes with high wbm expression all had good bread making quality but not always good physical dough qualities. The predicted protein was sulphur rich suggesting the possibility of a contribution to bread loaf volume by supporting the crossing linking of proteins in gluten. Improved understanding of the molecular basis of differences in bread making quality may allow more rapid development of high performing genotypes with acceptable end-use properties and facilitate increased wheat production.

Comparative mapping in the Poaceae family reveals translocations in the complex polyploid genome of sugarcane

BackgroundThe understanding of sugarcane genetics has lagged behind that of other members of the Poaceae family such as wheat, rice, barley and sorghum mainly due to the complexity, size and polyploidization of the genome. We have used the genetic map of a sugarcane cultivar to generate a consensus genetic map to increase genome coverage for comparison to the sorghum genome. We have utilized the recently developed sugarcane DArT array to increase the marker density within the genetic map. The sequence of these DArT markers plus SNP and EST-SSR markers was then used to form a bridge to the sorghum genomic sequence by BLAST alignment to start to unravel the complex genomic architecture of sugarcane.ResultsComparative mapping revealed that certain sugarcane chromosomes show greater levels of synteny to sorghum than others. On a macrosyntenic level a good collinearity was observed between sugarcane and sorghum for 4 of the 8 homology groups (HGs). These 4 HGs were syntenic to four sorghum chromosomes with from 98% to 100% of these chromosomes covered by these linked markers. Four major chromosome rearrangements were identified between the other four sugarcane HGs and sorghum, two of which were condensations of chromosomes reducing the basic chromosome number of sugarcane from x = 10 to x = 8. This macro level of synteny was transferred to other members within the Poaceae family such as maize to uncover the important evolutionary relationships that exist between sugarcane and these species.ConclusionsComparative mapping of sugarcane to the sorghum genome has revealed new information on the genome structure of sugarcane which will help guide identification of important genes for use in sugarcane breeding. Furthermore of the four major chromosome rearrangements identified in this study, three were common to maize providing some evidence that chromosome reduction from a common paleo-ancestor of both maize and sugarcane was driven by the same translocation events seen in both species.

DNA Banks and their role in facilitating the application of genomics to plant germplasm

Advances in genomics have provided technologies for high throughput analysis of plant genomes with potential for use in gene discovery in germplasm collections. The establishment of DNA banks facilitates this screening by making DNA from large numbers of plant accessions widely available. DNA banks require the development of appropriate policies for access and benefit sharing. Tools for automating sample and data handling are essential. Standard molecular methods for fingerprinting DNA accessions for international comparisons need to be determined. New screening technologies are required to take advantage of the emerging availability of large DNA collections. The Australian Plant DNA Bank aims to collect DNA from all Australian plant species and to sample the diversity within each species. DNA from all individuals of the species is being stored for rare species. Domesticated or economically important species from all countries are also being collected and stored. International networking of DNA banks will be a key step in linking genomics tools to global plant diversity.