Development of amplified fragment length polymorphism (AFLP) markers for the identification of Cholistani cattle

Abstract

Abstract. The identification issue of livestock can be resolved by using molecular identification tools that are acceptable to preserve and maintain pure breeds worldwide. The application of a molecular identification methodology is more important for developing nations, e.g., Pakistan, where uncontrolled crossbreeding has become a common practice and the import of exotic animals and germplasm is ever increasing. This presents a risk to local breeds as also stated by the FAO. Therefore, the current study was designed to develop standard molecular markers for Cholistani cattle to ascertain their purity for breeding purpose. In this study 50 and 48 unrelated males were sampled for Cholistani and each crossbred cattle, respectively. Candidate molecular markers present in Cholistani but absent in crossbred cattle and vice versa were detected using the amplified fragment length polymorphism (AFLP) method. Eleven markers were developed and were converted to single nucleotide polymorphism (SNP) markers for genotyping. The allele frequencies in both breeds were determined for discrimination ability using polymerase-chain-reaction–restriction-fragment-polymorphism (PCR-AFLP). The probability of identifying the Cholistani breed was 0.905 and the probability of misjudgment was 0.073 using a panel of markers. The identified markers can ascertain the breed purity and are likely to extend the facility for breed purity testing before entering into a genetic improvement program in the country.

Advancements in DNA analysis for distinguishing wild boar and domestic pig: a mini-review on research developments for food authentication studies

“…Researchers could produce a high amount of DNA for biology exploration. Several DNA-based methods were used for species identification in food or raw meat, such as singleplex PCR, multiplex PCR, Sequence-specific PCR, DNA Barcoding, Next Generation Sequencing (NGS), qPCR/Real-Time PCR, PCR-RAPD, PCR-RFLP, Digital PCR, Amplified Fragment Length Polymorphism (AFLP), Loop-mediated Isothermal Amplification (LAMP), Random Amplification Polymorphic (RPA), PCR-SSCP, and HRM technique [12,47]. Until now, several studies only used the PCR technique to distinguish domestic pigs and wild boar, such as PCR-RFLP [39, 29, 28; 36], Real-Time PCR [35], PCR-direct sequencing [31], STRs [48,4], and SSCP [37].…”

“…Sasazaki et al [65] used this technique to distinguish domestic and imported beef. Malik et al [47] identify Cholastani cattle with their hybrids using the AFLP technique with a probability of 0.905 (90.5%). The advantages of AFLP are higher reproducibility and sensibility.…”

An overview of remote monitoring methods in biodiversity conservation

“…A Restriction fragment length polymorphism; the genomic DNA extracted from different organisms is PCR amplified and subjected to restriction digestion using specific restriction enzymes, then DNA fragments are separated by electrophoresis and hybridized with radiolabeled probes (Özdil et al 2018 ; Chaudhary and Maurya 2019 ; Panigrahi et al 2019 ; Haider and Nabulsi 2020 ). B Amplified fragment length polymorphism; the restriction fragments of genomic DNA was ligated with compatible adapters and PCR amplified using selective primers against adapters, and the amplified fragments were separated by electrophoresis for DNA fingerprint analysis (Blears et al 1998 ; Malik et al 2018 ; Wu et al 2019b ; Zimmermann et al 2019 ; Neiber et al 2020 ). C Random amplified polymorphic DNA; random PCR fragments were amplified from the genome of different species using primers with random sequences and separated by gel electrophoresis to determine the difference between species based on RAPD markers (Panigrahi et al 2019 ; Saikia et al 2019 ).…”

“…Being a dominant marker, it can analyze multiple loci through amplification of DNA performing PCR reaction (Bryan et al 2017 ). The method employs restriction digestion of DNA and amplification of fragments through ligation of adapters on both ends and using primers specific to adapters (Malik et al 2018 ). Genetic differences can be identified from the disparity in the number and length of bands on electrophoretic separation.…”