Applications of Next Generation Sequencing Technologies in Crop Improvement
Author: Ganapathy KN | Co-authors: Sunil Gomashe and Sujay Rakshit

Advancement in the science of genome sequencing and subsequent analysis of the DNA sequences led to identification of many novel genes and understanding the genetics of various qualitative and quantitative traits in crop plants. During 1970's DNA sequencing technologies were independently developed by Sanger and Maxam Gilbert. Maxam's method is based on the chemical modification of DNA and involves subsequent cleavage at specific bases while, Sanger method is based on the chain termination method and is popularly known as sanger sequencing. Sanger's method are referred to as simpler and uses less toxic chemicals and lower radio activity making it widely used method for the last three decades. During recent years, several non-sanger ultra high throughput sequencing technologies emerged and commercialized collectively known as second generation sequencing technologies or next generation sequencing (NGS) technologies. NGS technologies has made it possible to resequence large number of plant genomes at much higher speed and relatively lower cost compared to traditional sequencing methods. The commercially available next generation sequencing methodologies include the 454 genome sequencer FLX (Roche applied science), Illumina (solexa) genome analyzer and 'Applied biosystems ABI SOLiD and polonator G-007. Another non-Sanger DNA sequencing technology involves use of single-molecule sequencing (SMS) also known as third generation or "next-next generation sequencing" technology. Third generation sequencing techniques are predicted to be much cheaper and faster than the second generation sequencing technologies (Gupta, 2008).

Applications of NGS technologies

a) Functional diversity of crop genomes
Knowledge about genetic diversity present in the breeding materials is valuable information for crop improvement. Earlier diversity methods are based on morphological observations and more recently done using DNA markers such as AFLP and SSRs. Diversity based on morphological observations are influenced by the environment and do not reveal the complete variation present in the crop species. while the use of DNA markers such as SSRs portray the allelic variation from the genic or non-genic regions and their functional role is less known. Recently use of genic microsatellites or EST-SSRs aid in assaying the variation in transcribed and known-function genes. Using NGS technologies it is now possible to sequence whole genome at a faster rate which then can be subsequently used for comparing crop genomes for studying functional genome diversity using various bioinformatics tools.

b) SNP discovery for genome studies
Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation available in a species and forms the basis for most molecular markers used in plant genomic research. Recently, SNP markers have gained lot of interest in the scientific and breeding community due to high frequency across crop genomes. For example, In maize, the frequency of SNP is reported to be one polymorphism per 31 bp in non-coding regions and 1 polymorphism per 124 bp in coding regions analyzed from 18 genes in 36 maize inbreds. Insertions and deletions were reported to be frequent in non-coding regions (1 per 85 bp), but rare in coding regions (Ching et al. 2002). The high frequency of SNPs discovered among the maize genes reveals the need of the NGS technologies in fast discovery of SNP's throughout the genome of individuals which can be used for genome wide association studies of economically important genes.

c) QTL mapping and marker assisted selection
DNA markers that tightly linked to QTL governing traits of economic importance can be used as a supplement to phenotypic selection. Due to the higher cost involved in sequencing of the genes/genomes, identification of DNA variation at SNP level and subsequent mapping have not been exploited on a larger scale in orphan crop species. In coming years due to development of high throughput NGS technologies SNPs based markers are expected to replace the SSR markers. Due to lesser price of sequencing and more bases per run, NGS technologies can be largely exploited in orphan crop species which are otherwise neglected in terms of genomic research. In particular, NGS technologies will be useful for mining of SNP variation in parental genotypes for its use in gene mapping.

d) Transcriptome analysis and functional genomics
The identification and quantification of mRNA under different stresses in various cell types have long been used by the scientists for gene expression studies on genome wide scale. Different methods are used for the analysis of transcript profiles (transcriptomics) like differential display, cDNA-AFLP, microarray and serial analysis of gene expression (SAGE). With the advent of high throughput NGS technologies, genome wide gene expression analysis and mapping will be cost effective and facilitates wider use of functional genomics. The term RNA seq refers transcriptomics by NGS.

The future plant genomics research will certainly derive benefit from the recent development of new-generation sequencing technologies. The major gap in the genomic approaches to crop improvement is in the application of genomic information for development of improved crop genotypes. The most effective effort to fulfill the gap is to integrate various research disciplines which form core components of molecular plant breeding. The integration of various approaches required knowledge of whole genome organization, strong statistical knowledge to estimate the gene/genetic effects, good experience in molecular biology techniques and traditional breeding methodologies. These integrated approaches will revolutionize the crop improvement in future.

1. Varshney, R. K., Nayak S. N., May G. D., and. Jackson S. A., 2009, Next-generation sequencing and their implications for crop genetics and breeding. Trends Biotechnol., 27 : 522-530.
2. Gupta, P. K., 2008 Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol., 26: 602-611
3. Ching, A, Caldwell, K. S., Jung M., Dolan M., Smith O. S., Tingey S., Morgante M and Rafalski A. J., 2002, SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines. BMC Genetics 3:19

About Author / Additional Info:
I am a scientist currently working on genetic improvement of sorghum crop using conventional breeding methods and molecular tools.