A team of Indian researchers has undertaken the sequencing of thousands of chickpea genomes as part of an international project to make it resistant to climate change and pests and thereby increase yields of a key crop across the developing world.
The accessions have been resequenced at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, as part of the ‘3,000 Chickpea Genome Sequencing Initiative’ —  an international effort to sequence and phenotype the chickpea global composite collection.
The scientists are also preparing to phenotype the chickpea accessions at different sites across India for a second year. Combined with the genomic data, they hope to identify superior, stress- resistant varieties of chickpea, with the first results of the project expected by the end of 2016.
Rajeev Varshney, principal scientist, applied genomics, ICRISAT, said that the centre has been resequencing the global composite collection on the ‘Illumina HiSeq2500’ platform. The collection includes ICRISAT’s 1,956 accessions, with an additional 709 accessions contributed by the International Centre for Agriculture Research in the Dry Areas in Beirut, Lebanon.
The initiative has also sequenced dozens of advanced breeding lines and cultivars, he said, as well as morphological variants, wild species, and more than 200 accessions that are known to carry specific traits such as resistance to different kinds of stress.
About 2,500 accessions have been sequenced to date, says Varshney, with the data for the remaining lines expected within the next month. Ultimately, the project plans to generate around 10 gigabases of raw sequence data for each line to get a minimum of 10X genome coverage.
This genomic data, combined with the new phenotyping information, should provide superior alleles for use in breeding programmes, Varshney said. “In addition, the genome architecture as well as millions of variations will be catalogued in the chickpea global composite collection,” he noted.

Orphan crops

ICRISAT’s leadership of the initiative falls in line with its long-term goal of generating genomic resources for legume crops using next-generation sequencing. Nicknamed ‘orphan crops’ —because they have largely been passed aside by Western researchers — legumes such as chickpea, pigeonpea, and groundnut, are a major source of nutrition in Asia and Sub-Saharan Africa, but are susceptible to pests, drought, and other environmental stressors.

Varshney and his colleagues at ICRISAT authored an overview of their decade’s worth of work on orphan crops in Plant Science in January.

“ICRISAT has taken initiative to generate genomic resources for these orphan crops by decoding their genome sequences using NGS,” Varshney commented.

In addition to using NGS (next generation sequencing) to explore the genomic diversity contained within global gene bank holdings for these crops, ICRISAT has also initiated efforts to resequence germplasm for the identification of new alleles, data that will be aligned to the crops’ reference genomes for the identification of millions of SNPs (single nucleotide polymorphisms), he said. SNPs detect the smallest unit of genetic variation present in genomes.

The chickpea initiative is therefore a natural extension of ICRISAT’s continued work on orphan crops, and will contribute towards its objective of “unravelling the genome architecture as well as to address the issues of low productivity and genetic bottlenecks in chickpea,” said Varshney.

Sequence data for all the samples are currently being de-multiplexed and stored, Varshney said. In parallel, raw sequence data are being mapped to the reference genome. The project also aims to phenotype the 3,000 lines at six different locations in India. During the 2014—2015 growing season, all the lines were sown and data on yield and yield related traits were recorded, Varshney said. The lines are being sown this year again and data recording will be compiled by the end of June, he said.

“Analysis of phenotyping data will help identify the superior lines with better stress tolerance and higher yield for each region,” noted Varshney. “The analysis of phenotyping data along with sequencing data will provide the information about the haplotypes that are associated with trait of interest,” he said. “These haplotypes can be used for developing improved varieties using modern breeding approaches.”
According to Varshney, once all of this data has been collected, ICRISAT’s scientists will commence variant calling. Other analyses related to genome-wide structural variation will be followed by variant detection, he said. This will entail genome-wide association studies for the identification of markers and haplotypes associated with traits of interest.
The chickpea project is also being encouraged by Diversity Seek (DivSeek), a new initiative that supports the genomic characterisation of the roughly seven million crop accessions stored at gene banks worldwide.
Started last year at the instigation of the Global Crop Diversity Trust, DivSeek brings together gene banks, researchers, plant breeders, and big data experts to partner on sequencing projects and share information.
Peter Wenzl, DivSeek liaison for the Bonn, Germany-based Crop Trust, said that in addition to the ICRISAT-led chickpea project, similar genome sequencing projects have been undertaken for rice at the International Rice Research Institute in the Philippines as well as for maize and wheat at Mexico’s CIMMYT International Maize and Wheat Improvement Center. He also noted that other sequencing projects on common bean, barley, bambara groundnut, and many others, are currently underway.
All of these efforts should eventually lead to an improvement in agricultural output in developing countries, he claimed.

“Most international gene banks are hosted at international agricultural research centres such as ICRISAT, CIMMYT, or IRRI, all of which are located in developing countries,” noted Wenzl. “Breeding programmes at these centres and other organisations in developing countries are developing improved crop varieties using gene bank samples as raw materials.”

 “Projects like the 3,000 Chickpea Genome Project will enable breeding programmes to more effectively identify and utilise genetic diversity for the development of high-yielding, climate-ready crop varieties,” Wenzl added.