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.

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.