Optimizing factors influencing DNA extraction from fresh whole avian blood

A study was conducted to optimize the efficient combination of lysis buffer, proteinase K, incubation time, phenol-chloroform-isoamyl alcohol (PCI) volume, spinning rate (rpm), and precipitation agent onquantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and swiftly transferred to a laboratory for DNA extraction. The lysis buffer used hadcomposition 5 M NaCl, 1 M Tris (pH=8.0), 0.5 M EDTA and20% SDS. The effect of various levels for each factor concerned was examined using General Linear Models or t-test procedures of SAS® software. The volume removed from the top aqueous part following the first and the second PCI washings was included in the models as a continuous variable; the variables of interest were OD280, OD260, OD260/OD280 (as quality criterion), total extracted DNA, extraction efficiency (ìg DNA/ìl blood), assay scores for easiness of removing the top aqueous phase after the first (assay 1) and the second (assay 2) spinning. The optimum level of factors significant for DNA extraction from fresh avian blood was found to be lysis buffer : blood sample ratio of 31:36 (ìl : ìl), incubation time of 60-70 min at 58oC, two washings with PCI at 1.2:1.3 PCI : top aqueous phase (ìl : ìl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 2000 - 2500 rpm for 20 min, precipitation of DNA with 1.5 – 2.0 volume ofabsolute ethanol

Characterizing Genetic Diversity of Contemporary Pacific Chickens Using Mitochondrial DNA Analyses

Background\ud Mitochondrial DNA (mtDNA) hypervariable region (HVR) sequences of prehistoric Polynesian chicken samples reflect dispersal of two haplogroups—D and E—by the settlers of the Pacific. The distribution of these chicken haplogroups has been used as an indicator of human movement. Recent analyses suggested similarities between prehistoric Pacific and South American chicken samples, perhaps reflecting prehistoric Polynesian introduction of the chicken into South America. These analyses have been heavily debated. The current distribution of the D and E lineages among contemporary chicken populations in the Western Pacific is unclear, but might ultimately help to inform debates about the movements of humans that carried them.\ud \ud Objectives\ud We sought to characterize contemporary mtDNA diversity among chickens in two of the earliest settled archipelagoes of Remote Oceania, the Marianas and Vanuatu.\ud \ud Methods\ud We generated HVR sequences for 43 chickens from four islands in Vanuatu, and for 5 chickens from Guam in the Marianas.\ud \ud Results\ud Forty samples from Vanuatu and three from Guam were assigned to haplogroup D, supporting this as a Pacific chicken haplogroup that persists in the Western Pacific. Two haplogroup E lineages were observed in Guam and two in Vanuatu. Of the E lineages in Vanuatu, one was identical to prehistoric Vanuatu and Polynesian samples and the other differed by one polymorphism. Contrary to our expectations, we observed few globally distributed domesticate lineages not associated with Pacific chicken dispersal. This might suggest less European introgression of chickens into Vanuatu than expected. If so, the E lineages might represent lineages maintained from ancient Pacific chicken introductions. The Vanuatu sample might thus provide an opportunity to distinguish between maintained ancestral Pacific chicken lineages and replacement by global domesticates through genomic analyses, which could resolve questions of contemporary haplogroup E chicken relationships and inform interpretations of debated sequences from archaeological samples

Microsatellite analysis of genetic diversity in Indian chicken populations [Анализ генетической изменчивости внутри и между шестью индийскими популяциями кур с помощью микросателлитных маркеров. (Иран. Индия)]

Characterization of genetic diversity by employing molecular tools is a prerequisite in developing strategies for conservation and utilization of poultry genetic resources. We assessed genetic variation within and between six Indian chicken populations (Naked Neck, White Silkies, Commercial Layer and Broiler, Giriraja and Desi) using nine microsatellite markers. Seven loci were polymorphic in all populations, with the number of alleles varying from 3 to 27 per locus and 4.1 to 8.6 per population. Average heterozygosity and gene diversity in the populations were 0.68 and 0.66, respectively. Some populations and loci deviated from Hardy-Weinberg equilibrium. Except Naked Neck, none of other populations was in genotypic disequilibrium for a single pairwise locus combination. F-statistics indicated that populations are genetically structured, ∼85% of the total genetic variation being due to genetic differentiation within the populations. Estimates of Latter’s FST and Nei’s DS genetic distances revealed relatively close genetic similarity between Naked Neck and Giriraja, while White Silkies was the most distant. An estimated divergence time between Naked Neck and Giriraja was over 100 years, with a comparatively longer (over 450 years) period of separation between Giriraja and White Silkies. We suggest that there is an essential room left for genetic improvement of Indian native breeds and commercial stocks

Identification of molecular markers associated with mite resistance in coconut (Cocos nucifera L.)

Coconut mite (Aceria guerreronis 'Keifer') has become a major threat to Indian coconut (Coçcos nucifera L.) cultivators and the processing industry. Chemical and biological control measures have proved to be costly, ineffective, and ecologically undesirable. Planting mite-resistant coconut cultivars is the most effective method of preventing yield loss and should form a major component of any integrated pest management stratagem. Coconut genotypes, and mite-resistant and -susceptible accessions were collected from different parts of South India. Thirty-two simple sequence repeat (SSR) and 7 RAPD primers were used for molecular analyses. In single-marker analysis, 9 SSR and 4 RAPD markers associated with mite resistance were identified. In stepwise multiple regression analysis of SSRs, a combination of 6 markers showed 100 association with mite infestation. Stepwise multiple regression analysis for RAPD data revealed that a combination of 3 markers accounted for 83.86 of mite resistance in the selected materials. Combined stepwise multiple regression analysis of RAPD and SSR data showed that a combination of 5 markers explained 100 of the association with mite resistance in coconut. Markers associated with mite resistance are important in coconut breeding programs and will facilitate the selection of mite-resistant plants at an early stage as well as mother plants for breeding programs. © 2007 NRC