Genome-wide SNPs from multiple samples and assessment of filtering parameters
We identified the following numbers of SNPs: 173,375 for F0 alone (15 individuals); 291,546 for F2 alone (47 individuals); 317,193 for F0 (15 individuals) + F2 (47 individuals); 310,249 for F1 (8 individuals) + F2 (47 individuals); and 335,104 for F0(15 individuals) + F1 (8 individuals) + F2 (47 individuals). Approximately 54% of the SNPs were singletons, occurring only once in the entire population, as calculated for 20 individuals including F0 and F1.
The parameters investigated for SNP filtering are shown in Table S4, and filtering condition set no. 20, which accurately reproduced the known kinships, was adopted as the filtering combination for estimating unknown kinships. First, we identified eight sets of conditions (numbers 13, 18–23, and 25 in Table S4) that accurately reproduced all of the known kinships using COLONY 2.0 for the dataset from 20 individuals. The higher the sequential number among these eight sets of conditions, the “looser” they are; i.e., the greater the number of loci left after filtering. Filtering condition set no. 20 was selected as the filtering conditions for estimating unknown relationships (MIN_DP = 5, MAX_DP = 50, MIN_MEAN_DP = 15, MIN_GQ ≥ 30, CR > 0.9, MAF ≥ 0.03, HWE < 0.00001, HET ≤ 0.7, LD ≤ 0.2) because these yielded a probability of 1.000 (=maximum) for all known kinships, and the number of loci left was the largest among those with the same results. However, the MAF value was adjusted based on the number of individuals analyzed simultaneously, so that only heterozygous SNP loci that occurred in a single individual would be filtered out (the threshold value of MAF ≥ 0.03 shown here is for the case of 20 individuals).
The number of loci in each combination of generations after filtering was 2479 for F0 alone (15 individuals), 2230 for F2 alone (47 individuals), 2541 for F0(15 individuals) + F2 (47 individuals), 5674 for F1 (8 individuals) + F2 (47 individuals), and 3045 loci for F0 (15 individuals) + F1 (8 individuals) + F2 (47 individuals).