Phenotypic studies regarding revolution seed and you can strict-placenta fruit qualities was basically compiled out of P
step step step one, Pdos, F1, and BC1P1 in 2013. The phenotypic data of F2, BC1P1, and BC1P2 for wave seed and tight-placenta fruit traits were investigated in 2015. Data of wave seed and tight-placenta fruit traits were categorized as nonmetric data. Single-melon seeds were classified by visual inspection as wave seeds or glossy seeds. The surface of the wave seed is wrinkled, whereas the surface of glossy seed is flattened (Fig. 1). Similarly, single-melon fruits were scored for tight-placenta fruit trait by visual inspection. Tight-placenta fruit means that the placenta is closely integrated with mesocarp as the ‘Queen’. The segregation ratios of the F2 and BC1 populations were analyzed with a ? 2 test using SAS software.
Data of SW, SD, SL, and SS were categorized as quantitative data. Data for each trait in the mapping population were measured using three replications. One hundred and twenty-eight BC1P1 individuals were used to measure SW, SL, and SD. For SW (g), a random sample of 30 seeds was taken for each individual plant. For average SL and SD (cm), a random sample of 10 seeds was quantitatively evaluated for each individual plant using ImageJ software. The ratio of SS is defined as SL divided by SD. The average value was used for the analysis. Statistical analysis of the data was performed using GraphPad Prism 6 ( The phenotypic correlation for seed size traits was obtained using the Pearson’s correlation coefficient.
DNA separation and molecular ent.
Genomic DNA is actually extracted from new melon actually leaves because of the cetyltrimethylammonium bromide means (Porebski mais aussi al., 1997) having minor changes. The filtered DNA try toned down to a hundred ng·?L ?step one . A maximum of 499 SSR markers was indeed picked away from wrote source (Chiba ainsi que al., 2003; Danin-Poleg mais aussi al., 2000; Diaz ainsi que al., 2011, 2015; Fernandezsilva et al., 2008; Fukino ainsi que al., 2007; Gonzalo mais aussi al., 2005; Harel-Beja ainsi que al., 2010; Ritschel mais aussi al., 2004) otherwise regarding Several new melon SSR and you will InDel primers was in fact designed on address area with respect to the resequencing research from two parent lines (Supplemental Tables step 1 and you will 3). The genome of the melon accession DHL92 was installed from (version CM_3.5.1) and you can used just like the a research genome. Resequencing away from ‘Queen’ and you will MR-step 1 are did during the ?20? coverage over the entire genome with the Illumina HisEq 2000 program to your built matched-stop sequence libraries in the Bioplification are performed in a good thermal cycler (BIO-RAD C1000) to your pursuing the protocol: 94 °C for 3 minute; thirty five schedules regarding 94 °C to own 30 s, 55 °C getting 29 s, 72 °C to own 31 s; and you will 72 °C 10 min. In the long run, the latest PCR affairs have been split on the thirty two ? 38 ? 0.04-cm 3 six% denaturing polyacrylamide ties in and you may tarnished with gold.
Linkage map framework.
According to Lander et al. (1987), the heterozygous bands or the glossy seed/untight-placenta fruit of BC1P1 plants were recorded as “H,” the homozygous bands or the wave seed/tight-placenta fruit plants as “‘A,” and unclear or missing data as “–.” For F2 population, the band pattern of the maternal parent (‘Queen’) was denoted as “A,” that of the paternal parent (MR-1) as “‘B,” and the heterozygous band as “H.” The F2 population with wave seed/tight-placenta fruit as the ‘Queen’ was denoted as “A” and that with glossy seed/untight-placenta individual as the MR-1 was denoted as “HB.”
Phenotypic research, and additionally SSR and InDel analyses, was shared having linkage analysis using QTL IciMapping V4 Land und Single-Dating-Seite.step 1 ( Recombination units was converted into genetic distances according to Kosambi function (Kosambi, 1944). Indicators have been for the minimum LODs ?step three.0 and you may limit range ?fifty.0 centimorgan (cM). Fundamentally, linkage charts was indeed visualized playing with MapChart 2.2 (Voorrips, 2002). Gene prediction is performed depending on the melon genome automatic annotation database ( Gene annotation and research was indeed conducted into the Great time browse new published NR (nr database), SwissProt (Deng ainsi que al., 2006), Wade (Gene ontology; Ashburner et al., 2000), COG (Groups from orthologous groups of protein; Tatusov mais aussi al., 2000), and you may KEGG database (Kyoto encyclopedia out of genetics and you may genomes; Kanehisa mais aussi al., 2004).