Computational protocol: Heritability for body colour and its genetic association with morphometric traits in Banana shrimp (Fenneropenaeus merguiensis)

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Protocol publication

[…] Ten microsatellite loci (GenBank Accession No’s: KM213743-KM213752) with consistent PCR amplification, clear allelic variation, and clarity of electrophoretic signatures were used to construct the pedigree in the present study (Additional file ). A detailed description of marker development from the pooled genomic DNA of 20 F. merguiensis individuals using GS-FLX Titanium chemistry (Roche Applied Science; Mannheim, Germany) is given in Knibb et al. [,].Once validated in simplex, two multiplex PCR pools, each containing 5 microsatellite primer pairs (Pool 1: FM002, FM004, FM011, FM047, FM057, and Pool 2: FM001, FM005, FM014, FM052, FM056) were amplified using Qiagen Multiplex PCR Plus Kits (Qiagen, Germany). Forward and reverse primers for each multiplex pool were combined in a 10× primer mix using 1–3 μM of each primer, dependent upon PCR product fluorescence intensities. Reactions, with volumes adjusted to 10 μL, each contained 1 μL of 10× primer premix, 3.0 μL of Qiagen Multiplex Buffer (2×) buffer, 3.5 μL of DH2O, and 2.5 μL of template gDNA (10 ng/μL). Amplification was performed using an Eppendorf Mastercycler (Hamburg, Germany) with cycling conditions as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 57°C for 90 s, and 72°C for 30 s; with a final extension at 68°C for 10 min. PCR products were separated by capillary electrophoresis on an AB 3500 Genetic Analyser (Applied Biosystems). Fragment sizes were determined relative to an internal lane standard (GS-600 LIZ; Applied Biosystems) using GENEMARKER v1.95 software (SoftGenetics; State College, USA) and double-checked manually. Individuals with low or missing peaks were amplified and genotyped a second time. MICRO-CHECKER v2.2.3 [] was used to look for evidence of large allele dropout, extreme stuttering and null alleles, based on 1000 bootstraps and a 95% confidence interval. Tests for HWE at each locus and genotypic linkage equilibrium among pairs of loci were conducted in FSTAT v2.9.3 []. Numbers of alleles and the observed and expected heterozygosities of each locus were determined using GENALEX v6.5 [], while polymorphic information content (PIC) was computed in CERVUS v3.0 []. Parentage assignment was completed using COLONY software [] with confidence scores of above 95%. Our earlier study using both mtDNA and microsatellite markers [] showed the evidence of monogamy in this banana prawn population. Thus the monogamy model was assumed to construct the pedigree that included 60 full-sib groups, with the family size of 3 to 108 offspring. A total of 1957 offspring out of 1998 were assigned to full sib families. This previous study [] also reported pedigrees constructed using these microsatellite loci contained very few errors when cross checked with independent mtDNA sequence data. The number of offspring per family is given in Figure . The pedigree data file with phenotypes is available on request.Figure 1 […]

Pipeline specifications

Software tools GeneMarker, GenAlEx, Cervus
Application Population genetic analysis
Organisms Musa acuminata, Fenneropenaeus merguiensis