Development and validation of a mid-density DArTag-based genotyping platform for lettuce (Lactuca sativa)

Authors: LING, MENG - Cornell University; SAPKOTA, MANOJ - Cornell University; TANG, XUEMEI - Cornell University; Eriksen, Renee; Mou, Beiquan; Richardson, Kelley; Simko, Ivan; BEIL, CRAIG - Cornell University; SHEEHAN, MOIRA - Cornell University

Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: 11/14/2023
Publication Date: 1/16/2024
Citation: Ling, M., Sapkota, M., Tang, X., Eriksen, R.L., Mou, B., Richardson, K.L., Simko, I., Beil, C., Sheehan, M. 2024. Development and validation of a mid-density DArTag-based genotyping platform for lettuce (Lactuca sativa). Plant and Animal Genome Conference, January 16, 2024, San Diego, California.

Interpretive Summary:

Technical Abstract: Lettuce is one of the most important leafy vegetable crops produced and consumed in the United States. Lettuces are widely used in the preparation of salad, soup, and vegetable curries, and it is a rich source of Vitamins A, C, and K, fiber, potassium, iron, and folate. Compared with array-based genotyping technologies and genotyping-by-sequencing (GBS) platform, targeted amplicon sequencing techniques can provide cost-effective genotyping profiles of consistent loci across the genome. To integrate genomic information into the USDA-ARS lettuce breeding programs, Breeding Insight developed a 3K DArTag panel from a set of 115K single nucleotide polymorphisms (SNPs) generated from tunable GBS (tGBS) data of 77 cultivated lettuce lines. These 77 lines were selected from a set of ~500 inbred lines to serve as the core diversity set for marker selection to ensure capture of representative genetic diversity among the North American breeding germplasm. DArTag markers were selected based on even genomic distribution, maximal genetic diversity, and markers associated with previously mapped quantitative trait loci. The lettuce 3K DArTag panel was tested using a diverse collection of 144 inbred lines (including 18 duplicated samples), a bi-parental population of 162 recombinant inbred lines (RILs) and 39 mimic F1 samples by mixing seeds of cultivar ‘Salinas’ with other inbred lines. Due to the large number of duplicated sequences, primarily caused by the whole-genome triplication in lettuce, aligning DArTag reads to the 180 bp flanking sequences of the 3K target loci produced high heterozygosity in genotyping results because of the collapsing of target and paralogous sequences. Therefore, we conduced whole-genome alignment of the DArTag reads, where we discovered 5,436 high quality SNPs, of which ~2K were the target loci of the lettuce DArTag panel. The DArT genotyping results showed high repeatability between duplicated samples. It was also highly consistent with the tGBS genotyping results with the average line-based identical-by-state value of 0.98. The mimic F1 samples showed predictable genotyping results for most of the loci based on their parents’ genotyping profiles. Linkage maps were constructed for the RIL populations using 722 non-redundant markers, which resulted in linkage groups with a total length of 1,342.13 cM. The open accessibility of the lettuce 3K DArTag panel can be used as a resource for genomic studies and genomic-facilitated breeding for both public and private breeding programs.