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Resistance Gene Cloning from a Wild Crop Relative by Sequence Capture and Association Genetics

Year : 2020 | Category : Target Region Sequencing

During wheat development, its quality and yield will be affected significantly by pathogens and diseases. The study of wheat resistant genes helps us understand the resistant mechanism, which could be potentially used for wheat quality improvement and disease control.

By combining association genetics with R gene enrichment sequencing (AgRenSeq) method and K-mer analysis, the resistant genes are selected and identified.

Samples Selection:
In this study, 195 Aegilops tauschii accessions were selected. They are phenotyped with races of the wheat stem rus pathogen Puccinia graminis f. sp. tritici (PGT). Illumina based library was prepared. The library was sequenced on an Illumina HiSeq with 250 bp
PE reads at Novogene.

Target region sequecing premade library

Experimental design and analysis:
Majority of resistant genes encode nucleotide binding/leucine rich repeat (NLR) immune receptor proteins. A customized bait library was designed to capture NLR repertoire across selected species, including 2,110 SNP markers.

Averagely, 1,437 contigs were obtained from each accession, among which, 299 encoded full-length NLR, while, 1,137 encoded partial NLRs. Through SNP analysis, 13 lines were identified with resistance based on the PGT race. K-mer sequences were firstly filtered based on the correlation of their presence/absence to the level or resistance/susceptibility. Then the phenotype was predicted using the linear regression analysis model and filtered k-mer results.

Figure 1. Workflow from experiment design to analysis

Figure 2. K-mer analysis pipeline

By mapping the k-mers to Sr33 genes, the author found that the assembled contigs from AgRenSeq covered the resistant genes well.

In addition, three non-redundant and high confidence genes, sr45, sr46 and srta1662, were identified. Sr46 and srta1662 were the most widely distributed resistant genes within samples, while sr33 and sr45 were with lower frequency (5% and 7%, respectively).

Figure 3. Identification of Sr33, Sr45, Sr46, and SrTA1662 by AgRenSeq

The association genetics combined with the R gene enrichment sequencing method can be used for rapid screening of plant resistance genes, but it is not suitable for the sequencing of atypical resistance genes.

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