Search

Swamp buffalo (Bubalus bubalis) is one of the Indonesian germplasm that adapts well to Borneo Island. This study aimed to determine the genetic diversity, phylogenetic tree, and phylogeographic structure of Bornean swamp buffalo based on control region (D-loop) mitochondrial DNA sequences. A total of 120 animals were collected from three populations in Indonesia, i.e., the North, East, and South of Kalimantan Provinces. Along 1140 bp of D-loop mtDNA gene of Bornean swamp buffaloes was amplified with a design primer of F: 5'-CAA CAC CCA AAG CTG AAG TT-3' and R: 5'-CGC TCC TCT TAG TCT CGT TG-3'. Therefore, the forward and reverse sequencing was performed to visualize the full length of D-loop mtDNA gene sequence (1140 bp). Results showed that a total of 47 haplotypes were detected in the animal study, with haplotype and nucleotide diversities of 0.936 and 0.005, respectively. Consequently, two haplogroups were observed in the animal study, i.e., Haplogroup A (North) and Haplogroup B (East and South). Based on structure analysis, Bornean swamp buffaloes were comparable to filing swamp buffaloes of China based on structure. According to the analysis of molecular (AMOVA), the geographical component contributed over 56.44% of the total mtDNA sequence variations. In conclusion, it was discovered that the haplogroups of buffalo from the East and South populations were identical. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the diversity of the complete sequence of D-loop mitochondrial DNA (mtDNA) in Bali and Sumba Ongole (SO) cattlebreeds. A total of 24 blood samples were collected from Bali cattle (19 heads) and SO cattle (5 heads), and were extracted and then analyzed to obtain the sequence of D-loop mt DNA.Multiple alignments of the whole sequence of D-loop mtDNA were determined using clustal W. Genetic distance was calculated using a p-distance method, while the genetic tree was constructed using neighbor-joining (NJ) based on MEGA 6. Haplotype number, haplotype diversity (Hd) and nucleotide diversity (Pi) were analyzed using DnaSP version 6. As a result, the sequence of D-loop mtDNA in Bali cattle (921-1119 bp) and SO cattle (913 bp) was reported to have 8 and 4 haplotypes. Hd and Pi of Bali cattle reached 0.625±0.139 and 0.0266±0.0145, respectively, which wwere different from that of SO cattle, namely 0.900±0.1610 and 0.0064±0.0015, respectively. Specifically, we found 22 bp-repetitive nucleotide in Bali cattle, existing 3-9 times with a length of 66-198 bp present in D-loop mtDNA. This unique feature did not exist in SO cattle. Genetic distance and genetic tree determined according to sequence in hypervariability (HV-1) region of D-loop mtDNA (166 bp) resulted in satisfied separation, successfully classifying Bos javanicus, Bos indicus, and Bos taurus cluster.

This study aimed to investigate the diversity of the complete sequence of D-loop mitochondrial DNA (mtDNA) in Bali and Sumba Ongole (SO) cattle breeds. A total of 24 blood samples were collected from Bali cattle (19 heads) and SO cattle (5 heads), and were extracted and then analyzed to obtain the sequence of D-loop mt DNA. Multiple alignments of the whole sequence of D-loop mtDNA were determined using clustal W. Genetic distance was calculated using a p-distance method, while the genetic tree was constructed using neighbor-joining (NJ) based on MEGA 6. Haplotype number, haplotype diversity (Hd) and nucleotide diversity (Pi) were analyzed using DnaSP version 6. As a result, the sequence of D-loop mtDNA in Bali cattle (921-1119 bp) and SO cattle (913 bp) was reported to have 8 and 4 haplotypes. Hd and Pi of Bali cattle reached 0.625±0.139 and 0.0266±0.0145, respectively, which wwere different from that of SO cattle, namely 0.900±0.1610 and 0.0064±0.0015, respectively. Specifically, we found 22 bp-repetitive nucleotide in Bali cattle, existing 3-9 times with a length of 66- 198 bp present in D-loop mtDNA. This unique feature did not exist in SO cattle. Genetic distance and genetic tree determined according to sequence in hypervariability (HV-1) region of D-loop mtDNA (166 bp) resulted in satisfied separation, successfully classifying Bos javanicus, Bos indicus, and Bos taurus cluster. [ABSTRACT FROM AUTHOR]

Daerah DNA mitokondria (mtDNA) manusia yang mempunyai tingkat polimorfisme tertinggi adalah D-loop. Urutan nukleotida D-loop mtDNA dapat dijadikan alat untuk menentukan identitas genetik seseorang. Saat ini aplikasinya banyak digunakan untuk memecahkan kasus-kasus di bidang forensik. Isolasi fragmen 0,4 kilobasa (kb) D-loop mtDNA dapat dilakukan dengan mengekstrak mtDNA dari dalam sel, kemudian menggandakan (amplifikasi) mtDNA secara in vitro dengan metoda Polymerase Chain Reaction (PCR) dan secara in vivo dengan metoda Kloning. Pada penelitian ini telah berhasil diamplifikasi secara in vitro fragmen 0,4 kb mtDNA sampel forensik dengan metoda PCR menggunakan primer M1 (5’CACCATTAGCACCCAAAGCT-3’) dan M2 (5’GATTTCAC GGAGGATGGTG-3’) dan secara in vivo dengan metoda kloning menggunakan vektor pGEM-T dalam sel inang Eschericia coli JM 109. Identifikasi hasil PCR dilakukan dengan metoda elektroforesis gel agarosa 1% menggunakan pewarna fluorisen Etidium Bromida. Sedangkan sel rekombinan hasil kloning diseleksi menggunakan media selektif mengandung antibiotik ampisilin dan zat pewarna kultur rekombinan yaitu X-gal dan IPTG sebagai indusernya. Identifikasi plasmid rekombinan yang mengandung fragmen 0,4 kb D-loop mtDNA dilakukan menggunakan enzim restriksi Pst I. Hasil kloning diperoleh jumlah koloni putih (sel rekombinan) : biru (sel non rekombinan) adalah 2 : 1. Hasil analisis restriksi enzim PstI pada plasmid rekombinan diperoleh fragmen berukuran 3,4 kb, hal ini menunjukkan bahwa DNA insert berukuran 0,4 kb sesuai dengan fragmen mtDNA hasil PCR. Plasmid rekombinan selanjutnya digunakan pada penelitian berikutnya, yaitu penentuan urutan nukleotida dengan metode sekuensing sehingga dapat diketahui identitas genetika dari sampel forensik yang dianalisis.

The aim of this study was to investigate the different of plumage colors on morphological diversity of Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelang duck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck) in Indonesia. Blood sample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristics of body measurement, production ability and egg quality of Magelang duck were analyzed using Completely Randomized Design with 11 plumage colors as treatments. PCR technique was administered to amplify fragments in mtDNA D-loop region and PCR products were digested with endonuclease restriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck was statistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restriction enzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D, E and F). Haplotype difference showed genetic diversity in the population of Magelang duck and the other native ducks. In conclusion, the different plumage colors affected morphology diversity of Magelang duck. Genetic diversity of Indonesian native duck population could be identified by using PCR-RFLP analysis on mtDNA D-loop region. [ABSTRACT FROM AUTHOR]