A pilot-scale comparison between single and double-digest RAD markers generated using GBS strategy in sesame (Sesamum indicum L.)

Abstract

To reduce the genome sequence representation, restriction site-associated DNA sequencing (RAD-seq) protocols is being widely used either with single-digest or double-digest methods. In this study, we genotyped the sesame population (48 sample size) in a pilot scale to compare single and double-digest RAD-seq (sd and ddRAD-seq) methods. We analysed the resulting short-read data generated from both protocols and assessed their performance impacting the downstream analysis using various parameters. The distinct k-mer count and gene presence absence variation (PAV) showed a significant difference between the sesame samples studied. Additionally, the variant calling from both datasets (sdRAD-seq and ddRAD-seq) exhibits a significant difference between them. The combined variants from both datasets helped in identifying the most diverse samples and possible sub-groups in the sesame population. The most diverse samples identified from each analysis (k-mer, gene PAV, SNP count, Heterozygosity, NJ and PCA) can possibly be representative samples holding major diversity of the small sesame population used in this study. The best possible strategies with suggested inputs for modifications to utilize the RAD-seq strategy efficiently on a large dataset containing thousands of samples to be subjected to molecular analysis like diversity, population structure and core development studies were discussed.

Double‐digest restriction‐associated DNA sequencing‐based genotyping and its applications in sesame germplasm management

“…A total of 2496 sesame accessions were genotyped using ddRAD-seq approaches, as reported by us recently with a pilot-scale of 48 sesame accessions comparing ddRAD-and sdRAD-seq methods (Ruperao et al, 2023). Most accessions constituting the 2496 set were collected from India (1081), Singapore (1025), and Bangladesh (62) (Table S1).…”

“…The 1 µg of genomic DNA was digested with Sph1 and MluC1 restriction enzymes (Peterson et al, 2012), and the digested product was cleaned with Ampure beads. The ligation, pooling, size selection, PCR amplification, and QC check were done as reported by us earlier (Ruperao et al, 2023). The final pooling and sequencing were performed on a HiSeqX or NovaSeq6000 platform.…”

“…The ddRAD-Seq data for 2496 sesame accessions were subjected to k-mer analysis (Ruperao et al, 2023), resulting in 19.42 billion k-mer sequences with an average of 7.7 million distinct k-mers per accession. On the k-mer sequence comparison between the 2496 accessions, 189 million k-mers were commonly shared between 2494 (maximum) accessions indicating the conserved k-mers (possibly belonging to the core-part of the genome), 6 million k-mers are shared among 70% (1747) of accessions as a softcore k-mer sequence, and 562 million k-mers were present only in 30% of sesame accessions.…”

“…The cultivation of sesame across the globe, besides Indian subcontinent, had led to the crop diversification in array pattern during its domestication process (Mondal et al, 2016;Pathak et al, 2015;. Variability for seed coat color, capsule length, plant type, carpel number, monostem nature, flower color, determinate nature, leaf shape, oil content, sesamin content, and other biochemical traits, abiotic and biotic stress tolerance traits, is reflected in the genebank holdings for sesame (Bhunia et al, 2015;Mondal et al, 2016;Ruperao et al, 2023;Uzun et al, 2008;Yadav et al, 2022;Yol & Uzun, 2012). Handling these collections for efficient utilization and management requires a large-scale drive to identify the most diverse and promising accessions.…”

“…Although these were promising output at those time, those strategies cannot support handling the larger germplasm sets, say in thousands, that is, being conserved in the genebanks. In this scenario, the genomicsassisted approaches like double-digest restriction-associated DNA sequencing (ddRAD-seq) for genotyping in sesame would be most appropriate to handle 1000s of germplasm (Ruperao et al, 2023). The present study reports generation of sequence data at low coverage and reduced representation strategy, ddRAD-seq, for 2496 sesame germplasm and demonstrate its application in efficient management of sesame genetic resources through development of a CC set and a mid-density panel (MDP) of markers for its future use in accelerated sesame improvement program.…”