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Among them, the sequence depth of parents had more impact on alleles calling, since they were the basis for genotyping for each marker. The parents obtained 43.09 M reads, a rate of 9.31%, which was higher than average. A total of 230,584 SLAF loci were detected after reads clustering. The average sequence depths of these SLAFs were 35.15-fold for parents and 4.00-fold for each individual. The extreme depths of the locus-specific sequences suggested the accuracy of the SLAF marker discovery. Repetitive SLAFs were then disregarded, and polymorphic SLAFs accounted for 40.35% of all SLAFs (Table?1). All polymorphic SLAFs were then genotyped separately [http://www.selleckchem.com/products/a-1210477.html A-1210477 cost] for both parents and all individuals. Table?1. SLAF-seq data summary for mei F1 population Sequencing depth alone cannot ensure the genotyping quality; thus, genotyping quality scores (details see methods) were used to select qualified markers. The worst low-quality marker was discarded during each cycle. This dynamic process was repeated until the average genotype quality score of all SLAF markers reached the cut-off value of 30. Finally, 9,412 SLAFs were defined as high-quality SLAF markers with over 80-fold parental sequence depth, over 7-fold progeny sequence depth and >70% integrity among the F1 population. 3.2. High-density [http://en.wikipedia.org/wiki/Diglyceride diglyceride] linkage map construction The 9,412 high-quality markers were distributed into eight LGs according to their locations on mei genome and the MLOD scores with other markers (at least one MLOD score >5). A total of 8,007 markers containing skewed markers (segregation and genotype sequence data are listed in Supplementary Tables S1 and S2) were used to construct the final linkage map (Fig.?1). The information of all markers on the map is organized in Supplementary Table S3, which includes [http://www.selleckchem.com/products/ldn193189.html LDN-193189 datasheet] marker names, LGs, genetic position and physical location in mei genome. Following linkage analysis, coverage of the markers was 155.77-fold in the male parent, 98.21-fold in the female parent and 8.18-fold in each F1 individual (on average). The integrity of each marker among the 387 F1 individuals was also a key parameter to control map quality. All markers on the map demonstrated 96% integrity (on average). The final map was 1,550.62 cM in length with an average distance of 0.195 cM between adjacent markers. As shown in Table?2, the largest LG was LG2, with 1,722 SLAF markers and a 0.15-cM marker interval, while LG6 was the smallest with 698 SLAF markers and a 0.20-cM marker interval (Supplementary Fig. S2). The uneven length of LGs is identical with the karyotypic parameters of mei chromosomes,30 and the distribution of SLAF markers on LGs is shown in Fig.?2. The largest gap was 24.13 cM in LG1, followed by 12.79 cM in LG6. 3.50% skewed markers with P