Maximising recombination across macadamia populations to generate linkage maps for genome anchoring.

The Proteaceae genus Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.

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

BACKGROUND: The 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. RESULTS: Comparative 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. CONCLUSIONS: Comparative 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.

A comprehensive genetic map of sugarcane that provides enhanced map coverage and integrates high-throughput Diversity Array Technology (DArT) markers.

BACKGROUND: Sugarcane genetic mapping has lagged behind other crops due to its complex autopolyploid genome structure. Modern sugarcane cultivars have from 110-120 chromosomes and are in general interspecific hybrids between two species with different basic chromosome numbers: Saccharum officinarum (2n = 80) with a basic chromosome number of 10 and S. spontaneum (2n = 40-128) with a basic chromosome number of 8. The first maps that were constructed utilised the single dose (SD) markers generated using RFLP, more recent maps generated using AFLP and SSRs provided at most 60% genome coverage. Diversity Array Technology (DArT) markers are high throughput allowing greater numbers of markers to be generated. RESULTS: Progeny from a cross between a sugarcane variety Q165 and a S. officinarum accession IJ76-514 were used to generate 2467 SD markers. A genetic map of Q165 was generated containing 2267 markers, These markers formed 160 linkage groups (LGs) of which 147 could be placed using allelic information into the eight basic homology groups (HGs) of sugarcane. The HGs contained from 13 to 23 LGs and from 204 to 475 markers with a total map length of 9774.4 cM and an average density of one marker every 4.3 cM. Each homology group contained on average 280 markers of which 43% were DArT markers 31% AFLP, 16% SSRs and 6% SNP markers. The multi-allelic SSR and SNP markers were used to place the LGs into HGs. CONCLUSIONS: The DArT array has allowed us to generate and map a larger number of markers than ever before and consequently to map a larger portion of the sugarcane genome. This larger number of markers has enabled 92% of the LGs to be placed into the 8 HGs that represent the basic chromosome number of the ancestral species, S. spontaneum. There were two HGs (HG2 and 8) that contained larger numbers of LGs verifying the alignment of two sets of S. officinarum chromosomes with one set of S. spontaneum chromosomes and explaining the difference in basic chromosome number between the two ancestral species. There was also evidence of more complex structural differences between the two ancestral species.

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.

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.

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.

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 alpha-amylase/subtilisin inhibitor that inhibits the bacterial serine protease subtilisin, fungal xylanase, and the plant's own alpha-amylase. The inhibition of plant alpha-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 Nei's 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.

SAGE of the developing wheat caryopsis.

Understanding the development of the cereal caryopsis holds the future for metabolic engineering in the interests of enhancing global food production. We have developed a Serial Analysis of Gene Expression (SAGE) data platform to investigate the developing wheat (Triticum aestivum) caryopsis. LongSAGE libraries have been constructed at five time-points post-anthesis to coincide with key processes in caryopsis development. More than 90,000 LongSAGE tags have been sequenced generating 29,261 unique tag sequences across all five libraries. Tag abundance, generated from cumulative tag counts, provides insight into the redundancy and diversity of each library. Annotation of the 500 most abundant tags spanning development highlights the array of functional groups being expressed. The relative frequency of these more abundant transcripts allows quantitative analysis of patterns of expression during grain development. We have identified activities of cellular proliferation/differentiation, the accumulation of storage proteins and starch biosynthesis. The abundance of calcium-dependent protein kinases indicate their importance in signalling across development. Acquisition of a broad array of defence coincides with storage accumulation and is dominated by inhibitors of amylase activity. Differential expression profiles of abundant tags from each library reveal the coordinated expression of genes responsible for the cellular events constituting caryopsis development. This SAGE platform has also provided a resource of novel sequence and expression information including the identification of potentially useful promoter activities. Further investigations into both the abundant and low expressing transcripts will provide greater insight into wheat caryopsis development and assist in wheat improvement programmes.

Abundant transcripts of malting barley identified by serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) was applied to the major cereal crop barley (Hordeum vulgare) to characterize the transcriptional profile of grain during the malting process. Seven SAGE libraries were generated from seed at different time points during malting, in addition to one library from dry mature seed. A total of 155,206 LongSAGE tags, representing 41,909 unique sequences, was generated. This study reports an in-depth analysis of the most abundant transcripts from each of eight specific time points in a malting barley time course. The 100 most abundant tags from each library were analysed to identify the putative functional role of highly abundant transcripts. The largest functional groups included transcripts coding for stress response and cell defence, ribosomal proteins and storage proteins. The most abundant tag represented B22EL8, a barley metallothionein, which showed significant up-regulation across the malting time course. Considerable changes in the abundance profiles of some of the highly abundant tags occurred at 24 h post-steeping, indicating that it may be an important time point for gene expression changes associated with barley seed germination.