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Indian Researchers Develop DArT Arrays to Profile Legume Fungal Pathogens, Overcome Crop Loss

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NEW YORK (GenomeWeb) — Eschewing more well-established platforms, a team of Indian researchers has successfully adopted genotyping technology offered by Diversity Arrays Technology (DArT) to study a highly variable pathogen that causes considerable losses in legume production worldwide, with the intention of using the approach to cultivate disease-resistant crops.

The researchers described the use of the technology in a BMC Genomics paper earlier this month.

Fusarium wilt can cause chickpea yield losses of between 10 and 100 percent, making the disease an economic problem in India, the world's largest producer of chickpeas, where more than 8 million metric tons of the protein-rich legume are cultivated per year.

Better understanding the genetic diversity of Fusarium oxysporum f. sp. Ciceris (Foc), the causal organism of Fusarium wilt, could lead to the cultivation of resistant chickpea varieties, "one of the most durable and economic practices for the management of this disease," co-author Mamta Sharma told BioArray News.

Sharma is a legumes pathology scientist at the International Crops Research Institute for the Semi-Arid Tropics in Pantancheru, India. ICRISAT is a multinational nonprofit agricultural research organization, and chickpea is one of its "mandate crops" for crop improvement research, he noted.

According to Sharma, Fusarium wilt typically causes a yield loss of between 10 and 15 percent annually in India, though it can result in total losses in some particularly susceptible cultivars. While ICRISAT is devoted to generating disease-resistant chickpeas, its work has been complicated by the diversity of Foc, with four varieties of Foc reported in India alone. To overcome this, Sharma and his colleagues collected Foc isolates representing all of the chickpea growing regions in India to get a clearer understanding of the diversity existing in Foc isolates, with the ultimate goal of devising a management strategy for the disease by identifying location- or race-specific resistant varieties.

The genotyping of the isolates required a new technology platform, however. While the researchers had used various approaches in the past, including microsatellite genotyping and randomly amplified polymorphic DNA genotyping, these methods were "put to limited use because of various limitations," according to Rajeev Varshney, principal scientist in applied genomics at ICRISAT and a co-author on the new paper.

RAPD, he noted, "lacks in reliability and resolution," while microsatellite genotyping has "high initial development costs," and other approaches are too labor intensive and not amenable for high-throughput genotyping.

After looking into a variety of other genotyping platforms that are used extensively in agricultural research, including Affymetrix and Illumina SNP arrays, Fluidigm integrated fluidic circuits, and LGC Genomics' KASP genotyping assays, the ICRISAT scientists settled on Diversity Arrays Technology's DArT microarrays.

Based near Canberra, Australia, Diversity Arrays Technology offers both array- and sequencing-based applications of its DArT genotyping approach. At the heart of Diverse Arrays Technology's approach is a process called complexity reduction, which relies on a combination of restriction enzyme digestion and adapter ligation followed by amplification to generate a library of DNA fragments from multiple DNA samples considered to be representative of a particular species. The resulting fragments, including variable fragments found in some samples and referred to by the company as DArT markers, are subsequently arrayed on glass slides and hybridized with labeled DNA fragments from the sample of interest, resulting in informative hybridization patterns and SNP detection.

"The DArT technology combines a reduction of genome complexity with high throughput and cost-effective hybridization-based polymorphism detection," said Varshney. "The advantage of using these DArT markers is that they can simultaneously type several thousand loci in a single array and provide a cost-effective and sequence-independent tool for whole-genome fingerprinting," he said.

The main difference between DArT and other genotyping methods is how complexity reduction is achieved, according to Varshney. "DArT complexity reduction methods are not only very simple, therefore high throughput and inexpensive, but also efficiently target low copy numbers," he said. The approach also "ensures that the quality of extracted markers, both call rates and reproducibility, is very high" and "enables reliable calling of heterozygotes in large proportion of DArT markers, even in polyploidy species."

A first in studying plant pathogens

While DArT arrays have previously been used in agricultural research to characterize germplasm, trait mapping, and to establish marker-trait associations, the ICRISAT researchers maintained in the paper that their study is the first to adopt the technology to study a plant pathogen.

"Since no information is available on use of DArT in any plant pathogen to date, we decided to use this technology, as DArT is the best platform to generate thousands of markers in species like Foc with meager genomic resources," said Varshney.

According to the paper, the researchers generated 4,991 DArT markers as part of the study. After screening a Foc collection of 110 isolates, 1,813 of the markers were found to be useful in providing a picture of genetic diversity on the subcontinent, and the researchers identified multiple new varieties of Foc local to specific regions within India.

Varshney credited the "high number of polymorphic DArT markers" with providing a "greater resolution of genetic differences among Foc isolates and enabling us to examine the extent of variability in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population."

Given the apparent evolution in varieties of Foc in India, the ICRISAT envisions using the DArT markers as "diagnostic tools" going forward to not only better understand regional diversity, leading to the cultivation of region-specific resistant crops, but also to monitor the behavior of cultivars that are widely grown and are resistant to existing varieties of Foc but may be susceptible to newer varieties of Foc or Foc races from other regions.

"These markers will assist in the early detection of introduced races, as well as of changes in the relative frequencies of different races that might occur in response to the use of resistant chickpea cultivars," said Varshney.

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