Veriti 384 well thermal cycler

KASP markers were designed and developed from single SNP variant sites in HvAnt1 and InDel variation in HvAnt2. The primer information is provided in in Supplemental Table 10.
The PCR system for KASP genotyping consisted 2× Master Mix 96/384, High Rox, (LGC Genomics, Middlesex, UK). PCR were performed in an Applied Biosystems^® Veriti^® 384-Well Thermal Cycler. The fluorescent signals of PCR products were detected by BMG Omega F (LGC Genomics, UK), and Kluster Caller software was used to perform the statistics.

We extracted DNA from 600 individuals, 100 adults and 500 seedlings, following the Cetyltrimethylammonium Bromide (CTAB) protocol [23] . All individuals were genotyped at four microsatellite loci (Table 1) using primers developed for the related legumes Pithecelobium elegans and I. edulis[18] (link), [24] (link). Simplex PCR reactions were performed using a Veriti 384-well Thermal Cycler (Applied Biosystems) in a total volume of 20 µl. The reaction mix contained ∼ 1.5 ng template DNA, 0.32 mM of each primer (the forward was fluorescently labeled with 6-FAM, NED and PET dyes, Table 1), 4 mM KCL-MgCl₂, 3 mM MgCl₂, 160 µM of dNTPs, 40 µM of Taq DNA polymerase (Fermentas). We used the following PCR profile: 95°C for 2 min followed by 30 cycles of 95°C for 15 s, 56 or 62°C for 30 s, 72°C for 30 s; 72°C for 15 min (Table 1; Cruz Neto et al., unpublished). PCR products were diluted to 10 ng final concentration with HPLC water. PCR products with each fluorescent dye color were mixed in a single reaction containing 1µl of each diluted PCR product and 6µl of formamide Hi-Di (Applied Biosystems) prior to fragment analysis with a 500-Liz size standard.
PCR fragments were run on an ABI3730 genetic analyzer (Applied Biosystems). Alleles were scored manually using Peak Scanner 1.0 (Applied Biosystems) and binned according to their motif length with Flexibin [25] .

Genomic DNA was extracted from Chinese-native Jin Ling Hua (JLH) chickens (n = 384), and its quality and quantity were determined using a NanoDrop spectrometer and agarose gel electrophoresis analysis. MassARRAY detection was performed by Beijing Compass Biotechnology Co., Ltd. (Beijing, China) using the MassARRAY® analyzer (Agena Bioscience, San Diego, CA) to characterize the genotypes of candidate SNPs. Briefly, specific amplification of candidate SNPs was performed in a Veriti® 384-Well Thermal Cycler (Applied Biosystems, Foster City, CA) and the PCR products treated with alkaline phosphatase. A single-base extension reaction and resin purification were carried out, after which the PCR products were hybridized to 384 chips for mass spectrometry detection. Finally, single SNP correlation analysis of 21 SNPs in a total of 384 individuals was performed using a general linear model with PLINK.