Theoretical and Applied Genetics

, Volume 126, Issue 1, pp 1–11

Accessing complex crop genomes with next-generation sequencing

Authors

  • David Edwards
    • Australian Centre for Plant Functional Genomics, School of Agriculture and Food SciencesUniversity of Queensland
    • Centre of Excellence for Integrative Legume Research, School of Agriculture and Food SciencesUniversity of Queensland
  • Rod J. Snowdon
    • Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and NutritionJustus Liebig University
Review

DOI: 10.1007/s00122-012-1964-x

Cite this article as:
Edwards, D., Batley, J. & Snowdon, R.J. Theor Appl Genet (2013) 126: 1. doi:10.1007/s00122-012-1964-x

Abstract

Many important crop species have genomes originating from ancestral or recent polyploidisation events. Multiple homoeologous gene copies, chromosomal rearrangements and amplification of repetitive DNA within large and complex crop genomes can considerably complicate genome analysis and gene discovery by conventional, forward genetics approaches. On the other hand, ongoing technological advances in molecular genetics and genomics today offer unprecedented opportunities to analyse and access even more recalcitrant genomes. In this review, we describe next-generation sequencing and data analysis techniques that vastly improve our ability to dissect and mine genomes for causal genes underlying key traits and allelic variation of interest to breeders. We focus primarily on wheat and oilseed rape, two leading examples of major polyploid crop genomes whose size or complexity present different, significant challenges. In both cases, the latest DNA sequencing technologies, applied using quite different approaches, have enabled considerable progress towards unravelling the respective genomes. Our ability to discover the extent and distribution of genetic diversity in crop gene pools, and its relationship to yield and quality-related traits, is swiftly gathering momentum as DNA sequencing and the bioinformatic tools to deal with growing quantities of genomic data continue to develop. In the coming decade, genomic and transcriptomic sequencing, discovery and high-throughput screening of single nucleotide polymorphisms, presence–absence variations and other structural chromosomal variants in diverse germplasm collections will give detailed insight into the origins, domestication and available trait-relevant variation of polyploid crops, in the process facilitating novel approaches and possibilities for genomics-assisted breeding.

Introduction

Polyploidy is a key evolutionary mechanism that has been a driving factor in the success of some of the most important domesticated plants (Leitch and Leitch 2008). Hexaploid wheat (Triticum aestivum) and oilseed rape/canola (Brassica napus) are prime examples of globally important cereal and oilseed plants with large, complex genomes. The bread wheat genome is allohexaploid, consisting of six sets of chromosomes originating from three distinct diploid genomes (termed A, B and D). The three diploid donor species each contributed seven pairs of chromosomes, resulting in 21 chromosome pairs. The donor species diverged between 2.5 and 6 MYA, and two distinct interspecies hybridisation events occurred which serially gave rise to the current polyploid bread wheat varieties. The first event, between 0.5 and 3 MYA, was a hybridisation between T. urartu (AuAu) and an unknown relative of Aegilops speltoides (BB), which produced tetraploid wild emmer wheat, T. turgidum (AuAuBB). The second hybridisation event produced allohexaploid T. aestivum (AuAuBBDD) through the hybridisation of T. turgidum and Ae. tauschii (DD) (Eckardt 2001; Huang et al. 2010; Chantret et al. 2005).

Modern bread wheat (AABBDD, 2n = 6x = 42) was domesticated some 10,000 years ago, its huge success being largely attributable to an increase in grain size early in the domestication process (Gegas et al. 2010). The amphidiploid crucifer B. napus (genome AACC, 2n = 4x = 38) on the other hand is one of the youngest major crop species, originating from a spontaneous hybridisation between the two closely related diploid progenitors B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) during mediaeval times (Iñiguez Luy and Federico 2011). The three wheat genomes display a high level of conserved synteny to the chromosomes of other grasses (International Brachypodium Initiative 2010), but display significant amplification of repetitive sequence elements and transposons that have expanded the genome to some 40 times that of rice. In contrast, the genome of B. napus is only around 10 times as large as that of the closely related crucifer Arabidopsis thaliana, but has a highly complex segmental structure in comparison to the model genome (Parkin et al. 2005; Wang et al. 2011a). Ancestral polyploidy (Lysak et al. 2005) and complex patterns of local gene losses and insertions within the diploid Brassica genomes (Town et al. 2006; Wang et al. 2011b), along with homoeologous non-reciprocal translocations between A and C genome chromosomes during polyploidisation (Udall et al. 2005; Gaeta and Pires 2010; Szadkowski et al. 2010), have given rise to a patchwork genome with considerable variation in gene copy number and syntenic conservation.

The structural complexity and size of the polyploid B. napus and T. aestivum genomes represent considerable obstacles to targeted implementation of germplasm in breeding. In particular the use of map-based cloning to identify genes underlying important quantitative trait loci (QTL) is extremely challenging in complex genomes, and genome-wide association approaches have had only limited success in these crops to date due to insufficient resolution of available genome-wide markers. Candidate gene approaches, implementing knowledge from extremely well-characterised, closely related model species, are also not trivial in wheat and oilseed rape, where multiple homologues and paralogues of any given gene of interest can impede a direct interpretation of their role in the crop genome. The combined pitfalls contributed by polyploidy, large gene families and widespread, abundant repetitive sequences complicate the detection and mapping of single-nucleotide polymorphisms (SNPs), an essential first step in genomics-based crop improvement (McKay and Leach 2011). Furthermore, although transcriptome profiling arrays are available for both Brassica and wheat, a general inability to distinguish between different homologous copies of specific expressed genes using oligonucleotide-based hybridisation methods can make interpretation of microarray expression data difficult. In this article, we review recent advances in next-generation DNA sequencing that may provide solutions to many of these problems, enabling considerably more targeted access to complex crop genomes and more efficient use of their underlying variation in breeding.

Next-generation sequencing of complex crop genomes

Efforts to unravel and understand crop genomes have been boosted in recent years by unprecedented developments in so-called “next-generation” DNA sequencing (NGS) technologies that present exciting new opportunities for crop genetics and breeding (Varshney et al. 2009; Duran et al. 2010; Edwards and Batley 2010). Although semi-conductor DNA sequencing technologies (e.g. Rothberg et al. 2011) are already heralding another new generation of DNA sequencing progress, most of the recent genome sequencing efforts in crop plants have implemented second-generation optical sequencing platforms (Edwards and Batley 2010; Imelfort et al. 2009a; Imelfort and Edwards 2009; Metzker 2010). Dramatic cost reductions accompanying the rapid increase in scale of these systems (Muers 2011) today make DNA sequencing an accessible and powerful option for high-throughput analysis even of more recalcitrant crop genomes. Since the introduction of advanced NGS technology, the bioinformatics analysis of this data has been a challenge (Batley and Edwards 2009), however an increasing number of tools are available to interrogate and analyse this data (Lai et al. 2012b; Lee et al. 2012; Marshall et al. 2010). Figure 1 gives a general overview of different areas in which NGS techniques can contribute to our understanding of complex crop genomes, their origins and structure, and can help identify and utilise useful genetic diversity for genetic analysis, trait dissection and breeding.
https://static-content.springer.com/image/art%3A10.1007%2Fs00122-012-1964-x/MediaObjects/122_2012_1964_Fig1_HTML.gif
Fig. 1

Next-generation sequencing (NGS) applications have vastly improved our ability to overcome the complexity of large or polyploid crop genomes. The approach of first unraveling the diploid progenitor genomes or individual chromosomes, and implementing synteny or annotation knowledge from less complex relatives, is key to the assembly of large crop genomes like those of oilseed rape or wheat. Whole-genome or reduced-representation resequencing are becoming routine tools for discovery of SNPs, PAVs and chromosome rearrangements that together contribute to variation. Our ability to relate sequence variation to complex traits using GWAS or genomic selection approaches depends increasingly on appropriate bioinformatic and statistical tools to deal which huge quantities of genotype data. On the other hand, the technologies to generate genome-wide DNA sequence data are quickly outpacing our ability to obtain high-quality phenotype data that accounts for strong environmental effects on complex traits

Establishing workable reference genome assemblies is the key prerequisite for discovery of DNA sequence variation by resequencing using powerful NGS platforms. The 2.3 Gbp genome of maize, a paleopolyploid with extensive proliferation of long terminal repeat retrotransposons, was the first large crop genome to be published (Schnable et al. 2011). By next-generation resequencing of the gene fraction of genetically diverse maize lines, Gore et al. (2009) were able to generate a haplotype map as a foundation for linking DNA sequence variants to phenotypic variation in agronomically important complex traits. Resequencing in maize also demonstrated for the first time the huge diversity in the DNA sequence and genome structure among varieties of the same crop species (e.g. Springer et al. 2009), with inter-genomic differences between different inbred lines being found on a scale seen only between different species in mammals. These examples from maize demonstrate the enormous power of next-generation sequencing to elucidate the structure of complex, highly repetitive genomes and associate DNA sequence and structural genome variation to important agronomic traits.

Other somewhat less complex crop genomes that have recently been sequenced include the 1.1 Gbp soybean genome (Schmutz et al. 2010) and the 844 Mbp autotetraploid genome of potato (The Potato Genome Sequencing Consortium 2011). The soybean genome, which derived from two ancient genome duplication events followed by extensive gene diversification, gene loss and chromosome rearrangement, was sequenced using a whole-genome shotgun approach, while the relatively small potato genome was deciphered by sequencing a homozygous doubled-monoploid potato clone. The availability of genome sequences has enabled tremendous progress in trait dissection and sequence mining in these and other crops (Hayward et al. 2012b; Hayward et al. 2012c; Raman et al. 2012; Tollenaere et al. 2012). On the other hand, the huge size of the T. aestivum genome and the elevated complexity of the B. napus genome, coupled with a particularly high prevalence of gene duplication due to their more recent polyploidy events, represent difficult obstacles that have hindered the development of reliable reference sequences and genome-wide SNP resources for wheat and oilseed rape. Whole-genome DNA sequences for both wheat and oilseed rape were reported in the media during the past two years, however in neither case was a public, whole-genome reference assembly available at the time these articles were written. Numerous international initiatives are currently involved in individual and/or joint efforts to generate reference genome sequences for B. napus (for details see http://www.brassica.info/resource/sequencing.php). For wheat, the sheer size of the genome necessitates alternative approaches to whole-genome de novo sequencing (http://www.wheatgenome.info/wiki/).

Genome sequencing in Brassica crops

When the diploid progenitors of a polyploid species are known, a logical approach to simplify de novo genome sequencing is to begin by assembling the respective diploid genome sequences. In the case of B. napus and its progenitors, the A genome of B. rapa was chosen as the first Brassica genome to be sequenced, based on its smaller size and lower quantity of repetitive DNA elements compared to the C genome of B. oleracea. The B. rapa v1.1 genome sequence (http://www.brassicadb.org/brad/) was published in 2011 by the Multinational Brassica rapa Genome Sequencing Project Consortium (Wang et al. 2011b). Completion of the published assembly used a combination of sequential sequencing of physically annotated BAC clones spanning the 10 B. rapa chromosomes, supplemented after the origin of next-generation sequencing by high-depth, short-read shotgun sequences. The B. rapa genome sequence is a valuable resource to investigate polyploid genome evolution, and also represents a major step towards truly genomics-based breeding of Brassica oil and vegetable crops. Whereas the triplication of diploid Brassica genomes relative to Arabidopsis (Lysak et al. 2005) was confirmed by the completed genome sequence, B. rapa was found to contain only 41,174 protein-coding genes instead of the approximately 90,000 that would have been expected in an ancestral crucifer hexaploid. Interestingly, different ancestral subgenomes appear to have been preferentially retained during the evolutionary gene loss process that led to fractionation of the ancestral polyploid genome to what is today B. rapa (Wang et al. 2011b). Furthermore, despite their close relationship, a total of 7 % of B. rapa genes were found to be not shared by A. thaliana. This underlines the enormous value of genome analysis in crop species themselves, rather than our previous reliance on genome information from more or less closely related model species. Completion of the B. oleracea C genome is expected to unveil a similar pattern of differential genome fractionation (Town et al. 2006) on a genome-wide scale. The consequence in B. napus is that genomic resequencing is highly likely to uncover widespread presence–absence polymorphism caused by removal or retention of different copies of homoeologous genes. In contrast to maize, where fixation of presence–absence variation in different heterotic breeding pools (Springer et al. 2009) results in strong additive effects on heterosis, the different B. napus subgenomes appear to have a higher level of plasticity with less structured patterns of gene loss and retention. This is presumably an important driving force determining the degree of “intersubgenomic” or “fixed” heterosis (Basunanda et al. 2010; Zou et al. 2010) in homozygous oilseed rape/canola genotypes. In addition to the biased gene fractionation among subgenomes, subgenome dominance has been observed in both the duplicated maize genome (Schnable et al. 2011) and the triplicated Brassica A genome (Cheng et al. 2012). An ability to comprehensively survey such variation by DNA sequencing will allow us to much more effectively harness additive–additive gene interactions and thereby predict and manage heterosis.

Sequencing in the genome of wheat

The challenges of sequencing the T. aestivum genome arise from its large size, polyploidy and abundance of repetitive elements. The genome consists of around 17 billion nucleotides (Paux et al. 2008) and current estimates of gene abundance vary from 77,000 to 295,900 genes (Rabinowicz et al. 2005; Paux et al. 2006; Berkman et al. 2011a, b). The presence of multiple genomes with sequence identity between homoeologous chromosomes often confounds genetic and genomic analysis and complicates the assembly of a reference genome. In addition to the presence of multiple genomes, it is estimated that between 75 and 90 % of the bread wheat genome comprises repetitive sequences (Flavell et al. 1977; Wanjugi et al. 2009), predominantly consisting of nested transposable elements (Li et al. 2004). The abundance of repetitive sequences and the associated increase in genome size further complicates genome sequencing, as the genic and low copy regions are interspersed with long stretches of repeats which are difficult to deconvolute during genome assembly. Together, these factors make wheat one of the more challenging genomes to sequence and analyse.

The establishment of an international wheat genome sequencing project was suggested at a wheat genomics workshop held in November 2003. Initially a BAC-by-BAC approach was adopted, starting with a 5-year pilot project to produce a high-resolution physical map with anchored BACs, followed by the sequencing of a BAC minimum tiling path (Gill et al. 2004). The International Wheat Genome Sequencing Consortium (IWGSC) was established the following year with the goal of producing a complete and annotated genome sequence for T. aestivum cv. Chinese Spring (http://www.wheatgenome.org). In the 7 years since its establishment, the consortium has produced BAC libraries for all individual wheat chromosome arms (Šafář et al. 2010), physical maps of chromosomes 3B (Paux et al. 2008) and 3DS (Fleury et al. 2010), data analysis pipelines (Sabot et al. 2005) and subgenome-specific molecular markers (You et al. 2011; Nie et al. 2012).

The key to facilitate the assembly of the extremely large hexaploid wheat genome is the ability to physically isolate individual chromosomes (Vrána et al. 2000; Kubaláková et al. 2002), which can be size-separated using flow cytometry (Laat and Blaas 1984; Kubaláková et al. 2000). This method of chromosome isolation has been successfully applied to rice (Lee and Arumuganathan 1999), chickpea (Vlacilova et al. 2002), rye (Kubaláková et al. 2003), barley (Lysak et al. 2005) and wheat (Vrána et al. 2000). Initially only wheat chromosome 3B could be isolated due to its relatively large size, however using cytogenetic stocks exhibiting structural chromosome changes Kubaláková et al. (2002) demonstrated that it is possible to isolate individual chromosome arms from potentially all chromosomes. This approach enables a physical partitioning of the genome into more manageable segments, consequently reducing the complexity of the sequence assembly process.

Whole chromosome shotgun (WCS) sequencing was initially suggested as an alternative to the BAC by BAC approach for wheat genome sequencing (Gill et al. 2004), however the BAC by BAC approach was adopted as the most advanced technology at the time to achieve a thorough and robust reference genome sequence. With the advent of next-generation sequencing technologies, however, several groups have started to apply WCS to large and complex crop genomes. Mayer et al. (2009) assembled Roche 454 sequencing data from DNA from the isolated barley chromosome 1H. To produce a gene-rich scaffold of ordered genes, a “genome zipper” approach was developed based on the syntenic relationship between barley and the sequenced genomes of rice and sorghum (Mayer et al. 2009). This approach has since been extended to the entire barley genome, utilising syntenic comparisons with rice, sorghum and Brachypodium to determine the order of ~ 86 % of the predicted barley genes (Mayer et al. 2011). When applied to wheat, the isolation and sequencing of DNA from individual chromosome arms reduces both effective genome size and complexity. This in turn assists downstream bioinformatic assembly and analysis, which is broadly understood to be the current bottleneck in genome sequence assembly. The effectiveness of the WCS approach has been demonstrated for wheat chromosomes 7A, 7B and 7D (Berkman et al. 2011a, b; Lai et al. 2011) as well as chromosome arm 4AL (Hernandez et al. 2012).

Sequence-based transcriptome profiling

Whole-transcriptome profiling via total or reduced-representation mRNA sequencing represents a powerful technique to interrogate gene expression on a global scale (Wang et al. 2010). In comparison to array-based expression analyses, digital gene expression (DGE) methods offer the advantage that expression levels can be quantified even for low-abundance transcripts or previously unknown genes by sequencing at sufficient depth. Sequencing-based whole-transcriptome profiling can also enable large-scale characterisation of transcription start sites and alternative splicing.

Deep mRNA sequencing was demonstrated by Trick et al. (2009) to be an extremely powerful reduced-representation method to distinguish SNPs within expressed genes from A and C genome B. napus transcripts, based on their sequence divergence. In the same way, Bancroft et al. (2009) profiled leaf transcriptomes of doubled-haploid (DH) lines from a genetic mapping population to simultaneously discover and map over 20,000 sequence-annotated SNPs in B. napus, generating valuable knowledge on genetic linkage that helped improve previous draft genome assemblies of B. rapa and B. oleracea. In the process, they also estimated transcript abundance by counting aligned reads per kbp along the lengths of chromosomes, demonstrating that even relatively low-coverage mRNA sequencing was able to provide semi-quantitative data on relative gene expression levels. Upscaling of this method to more recent sequencing platforms today enables a considerably higher sequencing depth and accordingly greater accuracy of transcript quantification. Current NGS-based transcriptome analysis techniques can demonstrably overcome the limitations of microarray techniques with regard to previously unknown genes, and in distinguishing between expression of homoeologous and paralogous gene copies (Parkin et al. 2005). With increasing sensitivity of NGS platforms it is becoming increasingly feasible to quantify even very low-abundance transcripts, or to profile global gene expression even with very small quantities of RNA down to the single-cell level (Ozsolak et al. 2010).

Despite the demonstrated value of NGS technology in transcriptome analysis in other plant species, these have not yet been widely applied to transcriptome analysis in wheat. However, a number of research groups are currently working to analyse wheat transcriptome NGS data. The combination of wheat transcriptome data and the availability of isolated wheat chromosome arm data assemblies will likely lead to a greater understanding of the structure, expression and evolution of the wheat genome, at the same time providing valuable data for annotation of emerging genome sequences.

Applying NGS data for genome-wide association mapping

The application of genome-wide association studies (GWAS) to plants (e.g. Breseghello and Sorrells 2006; Nordborg and Weigel 2008; Gore et al. 2009; Atwell et al. 2010; Huang et al. 2010) has greatly increased the resolution of QTL detection, particularly for species like Arabidopsis, rice and maize where a very high density of SNP markers covering the entire genome was available. In the meantime, NGS platforms have made SNP discovery a more or less routine task for most major crops and genotyping-by-sequencing techniques are now commonly applied in crop species (e.g. Miller et al. 2007; Huang et al. 2010; Chutimanitsakun et al. 2011; Elshire et al. 2011; Pfender et al. 2011). As sequencing costs continue to drop, it may be only a matter of time until whole-genome or reduced-representation resequencing techniques replace array-based SNP screening as the method of choice for GWAS in crop plants (Chia and Ware 2011). This might be particularly true for polyploid crops in which assignment of genic SNPs to specific homologous loci can often be more reliable using haplotype-oriented sequencing data than on array platforms. Furthermore, because they can theoretically access novel allelic variants in genetically diverse association panels, NGS-based genotyping methods (see Davey et al. 2011 for a comprehensive review) have a potential advantage over array-based SNP screening methods that only survey known alleles in a given panel of SNPs (Chia and Ware 2011). Use of SNPs discovered by genomic resequencing for GWAS was shown by Huang et al. (2010) to be highly effective for uncovering genes involved in important agronomic traits in rice.

Standard GWAS approaches still have the drawback of a lower statistical power of QTL detection, however, meaning that they are generally less suitable for complex traits like seed yield to which numerous minor-effect QTL might contribute. To overcome this drawback, Yu et al. (2008) introduced the concept of nested-association mapping (NAM), an approach which improves the power of genome-wide association studies for the detection of genes underlying complex traits. The success of the NAM technique in maize (McMullen et al. 2009) has prompted efforts in many other major crops to begin developing large, immortal NAM populations for genomic dissection of complex traits. In crops with complex genomes, however, the ability to accurately identify causal genes by GWAS and NAM will ultimately also depend on high-density SNP markers that are able to efficiently distinguish homologous loci.

A major challenge facing plant breeders in coming years is the so-called “phenotyping bottleneck”. Whereas we are now able to generate huge quantities of genotype and sequence data in a short space of time for relatively small amounts of money, phenotyping of complex traits for which such genotyping capacities might give tangible solutions remains a costly and difficult challenge. Increasing abiotic stress tolerance, nutrient efficiency, heterosis, oil content and seed yield in the face of changing environments and emerging production constraints remain challenging goals in this respect. Innovative new approaches are required to analyse and dissect complex traits and associate their components to the underlying genetic mechanisms.

SNP discovery/allele mining in wheat and canola

SNP discovery from NGS data is challenging due to high error rates and short reads (Duran et al. 2009c; Imelfort et al. 2009b; Azam et al. 2012). In one of the first examples of cereal SNP discovery from next-generation genome sequence data, Barbazuk et al. (2007) identified more than 7,000 candidate SNPs between maize lines B73 and Mo17, with a validation rate of over 85 % using 454 data. Large numbers of wheat expressed-gene SNPs have also been identified from Roche 454 data using autoSNPdb (http://www.autosnpdb.appliedbioinformatics.com.au/), software which was originally developed for rice, barley and Brassica Sanger sequence data (Duran et al. 2009a, b).

The large data volumes from the Illumina sequencing platform provide the potential to discover very large numbers of genome-wide SNPs (Imelfort et al. 2009a). More than 1 million SNPs have been identified between six inbred maize lines (Lai et al. 2011). This study also identified a large number of presence/absence variations (PAVs), which may be associated with heterosis in this species. More recently, Allen et al. (2011) identified 14,078 putative SNPs in 6,255 distinct wheat reference sequences from Illumina GAIIx data. The validation rate from a subset of 1,659 was 67 % (data accessible at http://www.cerealsdb.uk.net/NGSdata/AllenSupplement). Around 3.6 million SNPs were identified by sequencing 517 rice landraces (Huang et al. 2010). This study allowed the association of genome variation with complex traits in rice and is a model for future studies in more complex species, including wheat and canola. A pipeline package called AGSNP has been applied to identify SNPs between two accessions of the diploid wheat, Ae. tauschii (Luo et al. 2009). Roche 454 sequencing of Ae. tauschii accession AL8/78 has since been combined with Applied Biosystems SOLiD sequencing of genomic DNA and cDNA from Ae. tauschii accession AS75 using AGSNP to identify a total of 497,118 candidate SNPs (You et al. 2011). SGSautoSNP (Second-Generation Sequencing autoSNP) was designed specifically to predict SNPs from whole-genome Illumina shotgun sequence data and has been successfully applied to identify more than 1.5 million SNPs in canola, with accuracy greater than 95 % (D. Edwards, unpublished data), as well as more than 900,000 SNPs across the wheat group 7 chromosomes (Edwards et al. 2012; Lai et al. 2012a; Lorenc et al. 2012) (http://www.wheatgenome.info). The application of this approach to identify large numbers of genome-wide SNPs even in complex genomes has the potential to be a significant driver in genomics-assisted improvement of wheat, canola and other polyploid crops in coming years (Hayward et al. 2012a).

Several complexity-reduction resequencing approaches have been successfully applied for SNP discovery in complex crop genomes, including B. napus and wheat (Bundock et al. 2012; Gholami et al. 2012; Liu et al. 2012; Winfield et al. 2012). These range from sequencing the transcriptome using Illumina technology (Allen et al. 2011; Bancroft et al. 2009; Trick et al. 2009); anchored EST sequencing (Parkin et al. 2005); sequence capture using oligonucleotide probes (Fu et al. 2010; Pichon et al. 2010; Saintenac et al. 2011), or restriction-based complexity reduction (Elshire et al. 2011). While these approaches can reduce the cost of SNP discovery and genotyping by sequencing, the continued increase in data volumes at an ever-reducing cost may make whole-genome sequencing more cost-effective in the future.

Sequence capture is a common method utilised for SNP discovery from polyploid species. Pichon et al. (2010) demonstrated the usefulness of sequence capture in B. napus for SNP discovery between the distinct cultivars Aviso and Montego. Over 7,000 SNPs were identified, of which 266/398 validated SNPs (66.8 %) were suitable for an Illumina GoldenGate™ assay, genotyped across 480 lines. This approach was also utilised in wheat (Saintenac et al. 2011) where the re-sequencing of a 3.5 Mb exonic target covering 3,497 genes in allotetraploid wheat allowed the identification of over 4,000 SNPs. Snowdon and Iniguez Luy (2012), describe the use of different sequence capture techniques in B. napus, on the one hand to capture meta-QTL for yield-related traits and on the other hand for targeted resequencing of regulatory genes potentially involved in expression of developmental and yield-related traits.

Transcriptome sequencing has been utilised widely in B. napus for SNP discovery (Trick et al. 2009; Parkin et al. 2005). The first of these studies used Illumina RNAseq data in doubled haploid (DH) lines from the Tapidor × Ningyou 7 mapping population. Depending on the stringency applied a total of 23,000 to 41,000 SNPs were identified between the cultivars, using 20 million ESTs from each cultivar. One disadvantage of using transcriptome sequencing for SNP discovery in a polyploid species was demonstrated in this research, however, with over 87 % of SNPs identified as being between homeologous genes.

A public, high-density B. napus SNP array

High-density SNP arrays can be a cost-effective alternative for genome-wide polymorphism screens not only for genome-wide association studies, but also by breeders as a tool for comprehensive genome-wide screens of elite germplasm and breeding pools (Batley and Edwards 2007). A major problem in the development of effective SNP arrays for polyploid species is the fact that inter-homoeologue SNPs within one genotype can mask polymorphisms at the same locus between individuals. Large-scale next-generation re-sequencing efforts have facilitated the identification of vast numbers of true, locus-specific SNPs even for complex polyploids, so that development of high-density genome-wide SNP screening arrays has recently become a viable option for oilseed rape and wheat. In 2011, an international Brassica SNP consortium was established in cooperation with Illumina Inc. (San Diego, CA, USA) to generate a public, high-density B. napus Infinium SNP array. The International Brassica SNP Consortium array, containing over 50,000 SNPs derived from sequence data contributed by 16 academic and commercial partners from Australia, China, Europe, North and South America, was released in 2012 with a per-sample cost of under US$70. A similar array is also currently under development for wheat (http://www.tinyurl.com/wheatSNP). These resources will open the way for high-throughput germplasm screening and genome-wide association studies in established diversity collections, new “nested” association mapping (NAM) populations and breeding pools. Ultimately, cheap genome-wide SNP screening tools, in tandem with knowledge of global gene expression patterns and relevant expression networks, can be expected to lay the foundation for predictive breeding (Bancroft et al. 2009; Stokes et al. 2010) and genomic selection (Heffner et al. 2009; Jannink et al. 2010; Duran et al. 2010).

Genomic selection and predictive breeding

Since DNA markers were first implemented into practical breeding programs over two decades ago it has been expected that they would significantly improve the efficiency and speed of cultivar development (Beckmann and Soller 1986; Tanksley et al. 1989), by improving the breeder’s ability to select for traits that are difficult to assess phenotypically. Although many breeders indeed implement marker-assisted selection for simple monogenic traits or major-effect QTL, there are still very few examples of successful application of molecular markers for polygenic trait selection (Bernardo 2008; Xu and Crouch 2008). The primary reason for this is that classical QTL analyses tend to identify only small numbers of markers associated with large phenotypic effects, and restrictive costs of single-marker analyses in large breeding populations meant that marker-assisted selection strategies were only cost-effective for traits with high economic return. Recently, this situation has changed dramatically. Using high-throughput SNP array technologies or genotyping-by-sequencing (see above) it is today possible to survey tens of thousands or even hundreds of thousands of markers at a cost that is already not considerably greater than that which a breeder may have previously spent on a small number of major-gene selection markers. By estimating the individual phenotypic effects of large numbers of genome-wide SNPs in a large and diverse “training” population, it becomes possible using appropriate statistical models to calculate genomic estimated breeding values (GEBVs) that can predict the expected performance of an non-phenotyped individual (Meuwissen et al. 2001). Even without knowing anything about the functional contribution of the SNPs to any given trait of interest, the GEBV represents a fundamentally ideal selection tool because it can theoretically eliminate the need for expensive and time-consuming phenotyping of new breeding lines. This concept, known as “genomic selection”, has revolutionised livestock breeding (Hayes et al. 2009; Bagnato and Rosati 2012). As genotyping and sequencing costs continue to fall there is little doubt that genomic selection will also play an ever-increasing role in plant breeding (Jannink et al. 2010).

Predictive breeding strategies based on the genome-wide SNP markers might also play an important role in selection of optimal crossing partners for the development of high-performing F1 combinations. In crops like maize and canola, F1 hybrid cultivars play a major role, however neither the parental line performance nor genetic distance estimates are good predictors of hybrid performance. Instead breeders are faced with time-consuming and expensive evaluation of general combining abilities (GCA) in large numbers of homozygous inbred parental lines from their breeding pools. In a recent study in maize, Riedelsheimer et al. (2012) reported that accurate statistical prediction of GCA could be achieved using genome-wide SNP markers, raising the possibility of selecting cross parents based on genomic predictors rather than phenotypic or population genetic parameters. The use of NGS techniques to survey sequence variation on a whole-genome scale, or to additionally include transcriptome sequence data into prediction models (Bancroft et al. 2009; Stokes et al. 2010), will potentially add further power to genomic selection techniques for breeding of major crops.

Conclusions

The use of NGS technologies has enormous power and potential to access the complex polyploid genomes of many major crops. Discovery and screening of genome-wide SNPs is no longer a bottleneck even in large polyploid genomes like those of oilseed rape and wheat, opening the way for the application of high-resolution association genetics and genomic selection approaches for genetic analysis and improvement of these important crops. As sequencing costs continue to fall, it is feasible that reduced-representation or even whole-genome resequencing could become the method of choice for genotyping in germplasm diversity collections and breeding populations alike. A greater focus on sequence-based global gene expression profiling can also be expected, adding another systemic level of “phenotype” data for genotype-trait associations and predictive breeding strategies. As such developments progress a major bottleneck in our understanding of complex traits in complex crop genomes will not be our ability to characterise their genetic makeup, but rather a lack of comprehensive and detailed phenotypic characterisation under variable environments.

Acknowledgments

DE and JB acknowledge funding support from the Grains Research and Development Corporation (Project DAN00117) and the Australian Research Council (Projects LP0882095, LP0883462, LP110100200 and DP0985953), along with further support from the Australian Genome Research Facility (AGRF) and the Queensland Cyber Infrastructure Foundation (QCIF). RS was supported by a fellowship from the OECD Cooperative Research Programme “Biological Resource Management for Sustainable Agricultural Systems” and by DFG grants SN14/11-1 and SN14/12-1.

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