Next generation sequencing technologies for next generation plant breeding

As a term, “next generation plant breeding” is increasingly becoming popular in crop breeding programmes, conferences, scientific fora and social media (Schnable, 2013). Being a frontier area of crop science and business, it is gaining considerable interest among scientific community and policymakers and funds flow from entrepreneurs and research funding agencies. Plant breeding is a continuous attempt to alter genetic architecture of crop plants for efficient utilization as food, fodder, fiber, fuel or other end uses. Although the scientific concepts in plant breeding originated about 100 years ago, domestication and selection of desirable plants from prehistoric periods have contributed tremendously to ensure human food security (Gepts, 2004). During the past few decades, well supported crop improvement programmes for major crops started reaping benefits from cutting edge technologies of biological sciences, particularly in the form of molecular markers and transgenic crop development, which in combination with conventional phenotype based selection, defines the current generation plant breeding practices. Different types of molecular markers have been developed and extensively used during the last three decades for identifying linkage between genes and markers, discovering quantitative trait loci (QTLs), pyramiding desired genes and performing marker assisted foreground and background selections for introgression of desired traits (Varshney and Tuberosa, 2007). However, these markers are based mostly on electrophoretic separation of DNA fragments, which limits detection of genetic polymorphism. In large plant breeding populations, genotyping may take up several months depending on marker system, adding more cost to genotyping. The next generation plant breeding would thus demand more efficient technologies to develop low cost, high-throughput genotyping for screening large populations within a smaller time frame. With the availability of whole genome sequences (WGS), the perspective of identification of DNAmarkers has shifted from fragment based polymorphism identification to sequence based single nucleotide polymorphism (SNP) identification to expedite the marker identification process and to increase the number of informative markers. But the WGS technologies based on Sanger sequencing are time consuming, costly and provide information only on the target individual, which have limited its use in specific gene discovery. Its direct use in large breeding populations is limited by time and cost factors. The advent of next generation sequencing (NGS) technologies and powerful computational pipelines has reduced the cost of whole genome sequencing by many folds allowing discovery, sequencing and genotyping of thousands of markers in a single step (Stapley et al., 2010). NGS has emerged as a powerful tool to detect numerous DNA sequence polymorphism based markers within a short timeframe (Figure S1), growing as a powerful tool for next generation plant breeding. The initial steps of NGS based marker development involve library construction prior to sequencing. Several targeted marker discovery techniques have been devised using NGS platforms which involve partial representation of the genome and those can be utilized even in absence of prior knowledge on WGS (Figure 1). Based on the approaches, partial genome representation libraries are either (i) complexity reduced representation libraries constructed by using restriction enzymes, or (ii) sequence capture libraries without involving restriction digestion. The first group includes reduced-representation libraries (Gore et al., 2009), complexity reduction of polymorphic sequences (Mammadov et al., 2010), restriction-site associated DNA sequencing (RAD-seq) (Pfender et al., 2011), sequence based polymorphic marker technology (Sahu et al., 2012), multiplexed shotgun genotyping (Andolfatto et al., 2011), and genotypingby-sequencing (GBS) (Elshire et al., 2011). The second group includes technologies like molecular inversion probe (Porreca et al., 2007), solution hybrid selection (Gnirke et al., 2009) and microarraybased genomic selection (Albert et al., 2007). Sequence capture can also be performed for broad or specific targets in the genome such as exome sequencing (Teer and Mullikin, 2010) and sequencing of the genomic region associated with particular trait (Teer et al., 2010). NGS technologies are already gaining widespread acceptability in the field of crop breeding. Many of the NGS based