Computational protocol: Extensive Variation and Sub-Structuring in Lineage A mtDNA in Indian Sheep: Genetic Evidence for Domestication of Sheep in India

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Protocol publication

[…] The sequences were edited using AUTOASSEMBLER (Perkin Elmer) program and 721 bp of control region mtDNA were obtained with respect to the reference sequence. Alignment showed that control region of the majority of the sequences contained four 75 bp tandem repeats, while two sequences contained three 75 bp & one 76 bp repeats. However, three samples contained one 75 & three 76 bp tandem repeats each. For comparative analysis of our sequences, control region mtDNA sequences of domestic and wild sheep were collated from NCBI as follows: (DQ242050 - DQ242455) [], (DQ491576 - DQ491736) [], (EU019130 - EU019189), (AY829376 - AY829430) [], (DQ903228 - DQ903304) [], (DQ852280 - DQ852289) [], Ovis ammon ammon AF242347 [], Ovis vignei arkal AY091489 [], Ovis ammon nigrimontana AY091494 [], Ovis vignei bochariensis AY091491 [], Ovis ammon collium AY091492 [] and Ovis Vignei arkal AY091489 []. The cytochrome b gene sequences of 326 sheep from the present study were analyzed along with following sequences available from the data bases: (FJ218019 - FJ218150) [], (DQ903208 - DQ903227), (AY879464 - AY879584) [], (DQ097407 - DQ097430) [] (DQ851886 - DQ852082) []. In order to avoid tandem repeats present in sheep control region mtDNA, we considered 432 bp comprising two fragment (I) 103 bp spanning from 15541 to 15643 and (II) 329 bp from 15933 to 16261 with respect to the reference sequence.ClustalX version 65534.0.10.0 program [] was used for the alignment of sequences. GeneDoc version 2.7.0.0 package was used for formatting the aligned sequences to make them compatible for the desired software. MEGA 5 version 5.0.1.102 [] was used to construct Neighbor-joining tree using Tamura-Nei model [] with 10,000 replicates. Maximum Likelihood tree was also constructed with Hasegawa-Kishino-Yano (HKY) [] model (+G = 0.37, I = 0.62) with 5 categories. DNA alignment software version 1.3.1.1 (www.fluxus-engineering.com) was used to convert the aligned sequence into RDF binary format. Reduced median network [,,] was drawn using Network software version 4.6.1.0 (www.fluxus-engineering.com) with parameters set to a weight of 2 and threshold value of 1. Haplotype, nucleotide diversity and its standard error, Fu’s Fs statistics [], mismatch distribution [], AMOVA (analysis of molecular variance), and the population pairwise differences (FST) values were calculated using Arlequin version 3.5.1.2 software []. Pairwise FST values were calculated with 10,000 bootstrap and values were displayed as a multidimensional scaling (MDS) plot with a stress value = 0.12 using SPSS 11.0 software. […]

Pipeline specifications

Software tools Clustal W, MEGA, Arlequin
Applications Population genetic analysis, Nucleotide sequence alignment
Organisms Ovis aries