Application of PCR-HRM method for microsatellite polymorphism genotyping in the LDHA gene of pigeons (Columba livia)

High-resolution melting (HRM) is a post-PCR method that allows to discriminate genotypes based on fluorescence changes during the melting phase. HRM is used to detect mutations or polymorphisms (e.g. microsatellites, SNPs, indels). Here, the (TTTAT)3-5 microsatellite polymorphism within intron 6 of the LDHA gene in pigeons was analysed using the HRM method. Individuals (123 homing pigeons) were genotyped using conventional PCR. Birds were classified into groups based on genotype type and the results were tested by qPCR-HRM and verified using sequencing. Based on the evaluated protocol, five genotypes were identified that vary in the number of TTTAT repeat units (3/3, 4/4, 3/4, 4/5, and 5/5). Sequencing have confirmed the results obtained with qPCR-HRM and verified that HRM is a suitable method for identification of three-allele microsatellite polymorphisms. It can be concluded that the high-resolution melting (HRM) method can be effectively used for rapid (one-step) discrimination of the (TTTAT)3-5 microsatellite polymorphism in the pigeon’s LDHA gene.

Microsatellite polymorphism for molecular characterization of pomelo (Citrus maxima) accessions from Indonesia

Susandarini R, Subandiyah S, Daryono BS, Rugayah. 2020. Microsatellite polymorphism for molecular characterization of pomelo (Citrus maxima) accessions from Indonesia. Biodiversitas 21: 2390-2395. Citrus maxima (Burm.) Merr. (pomelo) as a minor fruit crop deserve attention on its phenotypic and genotypic variability to avoid the risk of extinction. Previous study showed that pomelo from Indonesia has high morphological variability, and thus it is interesting to explore its genotypic variability using molecular markers. Microsatellite is a molecular marker widely used in Citrus taxonomy studies. This study aimed at revealing microsatellite polymorphism and its potential application in cultivar characterization of C .maxima. Eighty accessions of C. maxima consisting of registered cultivars and landraces from Indonesia were used in this study. Analysis of microsatellite sequences from genomic DNA amplified using DY296883 primer showed that C. maxima microsatellite has high polymorphism in the form of repeat length variation of (GA)n, ranging from (GA)7 to (GA)19. This study proved the existence of high genotypic variability in C. maxima, and confirmed the role of microsatellite as a useful molecular marker for uncovering variability at intraspecific level. Observation of the microsatellite polymorphisms indicated that variability of (GA)n can be used to distinguish some pomelo cultivars.