Enabling technology and training is providing a step change for chickpea breeding.

Agriculture Victoria Research (AVR), with investment from GRDC, has developed a cutting-edge, low-cost DNA genotyping platform for chickpeas, field peas, lentils and lupins.

The platform seamlessly connects research to breeding, heralding a significant efficiency boost and accelerating both pre-breeding research and breeding for the crops.

Data generated using this platform can be more easily combined with legacy, current and future  datasets from GRDC projects and the public domain. The knowledge generated by combining datasets accelerates research outcomes and the translation of pre-breeding outputs into breeding programs.

The utility of the genotyping platform has been enhanced through Pretzel, a web-based tool developed by AVR that enables the data generated to be visualised and interrogated interactively in the context of other research and breeding information. This allows researchers and breeders without any specialised bioinformatic skills to combine data generated across projects, time and different technology platforms to objectively answer specific questions that are otherwise challenging to address.

The genotyping platform is delivered to the Australian pulse pre-breeding and breeding sectors through AVR and has been widely adopted, with more than 108,000 samples genotyped over the past two years.

A low-cost DNA chip for genotyping pulses has been developed by a team at Agriculture Victoria Research. From left to right (back row): Kirushanthy Kajendran, Dhriti Sharma, Sukhjiwan Kaur; front (left to right) Thanjua De Alwis, Shimna Sudheesh. Photo: Adam Dimech

Uniqueness of chip design

The genotyping platform assays specific positions in the genome of each pulse crop known as Single Nucleotide Polymorphisms (SNPs). These positions reveal differences between individuals at the DNA sequence level. Across the four pulse crops, a total of 30,000 SNP are assayed on the DNA chip.

The SNP content was carefully selected for two purposes.

The first set – called imputation SNP – were selected using a custom algorithm that chooses the minimum set of SNP needed to unambiguously track the inheritance of DNA at the whole genome level during breeding. In other words, the imputation SNP are selected to provide almost the same amount of information as whole genome sequence, where every DNA difference between individuals is known. The selection of the imputation SNP required a detailed understanding of the genetic variation that exists in globally diverse germplasm for each pulse crop. To generate this information, 1000+ accessions for each pulse crop were genotyped and 250 of the most genetically diverse accessions within each pulse crop group was whole genome sequenced. The accessions used included landraces, introgression lines, historical and released varieties and wild species, most of which were obtained from the Australian Grains Genebank.

The availability of whole genome sequence information for each pulse crop also made it possible to design assay probes for the selected SNP that only work in the pulse crop for which they were designed. For example, an assay probe designed for chickpea gives no signal in field pea, lentil and lupin. This design feature enables up to four DNA samples, one from each of the four pulse crops, to be jointly assayed to the same DNA array, effectively reducing the assay cost by up to four-fold.

The second type of SNP on the array – called knowledge-linked SNP – were selected to provide a strong connection between research and breeding knowledge. These SNP include trait-linked markers for each pulse crop that have been identified in national and international research and allow breeders and researchers to immediately know what trait alleles a genotyped accession possesses. The knowledge-linked SNP also provide a direct connection to important germplasm and genomic resources that have been developed by the international research community. For example, in chickpea there are SNP that provide a direct connection to a vast array of genomic resources developed by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). The third set of knowledge-linked SNP enable DNA introduced from wild species to be accurately tracked in domestic germplasm during breeding. Collectively, the knowledge-linked SNP work to accelerate genetic gain for pulse crop improvement by enabling research outputs to be more seamlessly translated into breeding programs.

Chip deployed widely

The utility of the genotyping platform has been demonstrated across numerous GRDC pre-breeding investments including in chickpeas for improving Ascochyta blight resistance, reproductive-stage chilling tolerance and acid soil tolerance.

In the program for increasing Ascochyta blight resistance, the genotyping of more than 5500 diverse chickpea lines (including varieties, wild species, imported germplasm and breeding material) allowed the characterisation of existing genetic variation for resistance in breeding germplasm pools, as well as the identification of new sources of resistance.

The genotyping platform was also used to accelerate the crossing of the new resistance sources into domesticated backgrounds to generate improved seed stocks for breeder evaluation.

In the chilling tolerance project, the genotyping platform was used to genotype a population of wild chickpea crosses that were known to carry novel sources of cold tolerance during the reproductive phase. The 1700 markers for tracking wild chickpea genomic segments in crosses with commercial lines allowed the identification of strong marker-trait associations and helped identify lines that can withstand cold temperatures during flowering.

Similarly, in the acid tolerance project the genotyping of more than 1500 lines (including wild crosses) led to the identification of germplasm with novel sources of tolerance, which are currently being crossed into elite backgrounds and will be ready for handover to breeders in the near future.

The genotyping platform has also been widely adopted by breeding programs, including Chickpea Breeding Australia, the National lentil and field pea breeding programs and Australian Grain Technologies (AGT) lupin breeding, to support the implementation of genomic selection, a technique used to accelerate the breeding of new improved varieties.

Breeders are using the ability to seamlessly link the genotype data generated to research knowledge to accelerate the adoption of pre-breeding outputs into their breeding programs.

Pretzel training

A Community of Practice for Pretzel is currently being established and will be supported by extensive online training resources and tutorials. Researchers and breeders interested in taking part should contact [email protected]

More information: Dr Sukhjiwan Kaur, [email protected]