A high-resolution genetic map derived from RAD sequencing and QTLs analysis of yield-related traits in Vigna unguiculata
Genomic DNA from each individual sample was quantified using spectrophotometry (Qubit Fluorometer, Invitrogen) and checked for genomic integrity by 0.8% agarose gel electrophoresis. Restriction-site Associated DNA (RAD) strategy combined with illumina DNA sequencing was used for fast and effective identification of SNPs markers. The construction of RAD libraries generally followed a protocol from Baird et al (2008)29. The general protocol was as follows: firstly, each sample of genomic DNA£¨~ 0.5¦Ìg per sample£©from the 170 individuals was digested with 20 units (U) of EcoRI or SbfI (New England Biolabs, NEB) for 15 min at 37 ¡ãC in a 50 ¦ÌL reaction and then heat denaturation at 65 ¡ãC. P1 Adapters, each with a unique 4-8 bp molecular-identifying sequence (MID), were ligated to the genomic DNA of each sample. The adapter sequences were as follows: EcoRI digestion, top: 5¡¯-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxxx-3¡¯ [x=barcode], bottom: 5¡¯-Phos-AATTxxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-3¡¯, for SbfI digestion, top: 5¡¯-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxxTGCA-3¡¯, bottom: 5¡¯-Phos-xxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-3¡¯. The ligation reaction was incubated at room temperature (RT) for 20 min in a total volume of 10 ¦ÌL, containing 2.5 ¦ÌL P1Adapter (100 nM), 1 ¦ÌL rATP (100 mM) (Promega), 1 ¦ÌL 10 ¡Á EcoRI buffer, 0.5 ¦ÌL T4 DNA Ligase (1000 U) (NEB), and 5 ¦ÌL H2O. Samples treated with heat denaturation at 65 ¡ãC for 20 minutes again. The samples within each library were pooled and randomly sheared to DNA fragments of 400-700 bp. Size of sheared DNA were isolated through 1% agarose gel electrophoresis using a DNA gel extraction kit (Tiangen, China). The ends of the DNA were polished by using the Quick Blunting Kit (NEB). After purifying the samples using a DNA purification kit (Tiangen), 15U of Klenow exo- (NEB) was added for adenine (Fermentas) overhangs on the 3¡¯end of the DNA at 37 ¡ãC. 1 ¦ÌL P2 Adapter (10 nM) was ligated to the DNA fragments at RT. Samples were again purified and eluted in 50 ¦ÌL. Five microliter (¦ÌL) of this product was used in a PCR amplification along with 50 ¦ÌL Phusion Master Mix (NEB), 40 ¦ÌL H2O and 5¦ÌL primer mix (10 ¦ÌM) (P1-forward primer: 5¡¯-AATGATACGGCGACCACCGA-3¡¯; P2-reverse primer: 5¡¯-CAAGCAGAAGACGGCATACGA-3¡¯). The PCR enrichment was performed following guidelines (NEB) for a total of 11 cycles. Samples were gel purified, excising DNA 400¨C700 bp, and diluted to 10 nM.Each library of the RAD products from the 170 individuals was sequenced on an individual lane of the Illumina HiSeq2000 next-generation sequencing platform (BGI-Shenzhen£¬Shenzhen£¬P.R.China). Sequencing data for each individual were then extracted according to the specific MID (molecular-identifying sequence, MID).