Excess Of Heterozygous Genotypes Research Articles

Genetic variability of three local cattle breeds (Calvana, Pontremolese, Garfagnina) by STR analysis

The dramatic size contraction of local cattle breeds due to replacement with cosmopolite improved breeds highlights the need for native genetic resources conservation. In 1985, the Anagraphic Register of local cattle breeds and small-size ethnic groups was established by the Italian Ministry of Agriculture and Forestry. Calvana, Pontremolese and Garfagnina are among the included breeds. They are all native from Tuscany. Present breeding area covers the provinces of Firenze, Prato, Pistoia and Siena for the Calvana breed (around 280 heads), while it is restricted to the province of Lucca for both Garfagnina (around 180 heads) and Pontremolese. This latter breed consists, nowadays, of less than 40 heads, while being around 15000 in 1940s.The characterization of the genetic structure and variability via molecular markers could provide useful information for breed management and conservation. In the present study, a total of 149 animals, evenly distributed among the three breeds, were genetically characterized by using 22 STR markers located on 13 different chromosomes. Among these, ten (BM1818, BM1824, ETH10, ETH152, ETH3, HEL9, ILST006, INRA63, TGLA126, TGLA227) belonged to the panel of 30 microsatellites recommended by the ISAG-FAO Working Group on domestic animal diversity. Mean number of alleles per locus in the total sample was 9.9, ranging from 5 (ETH10) to 14 (BMS690, HEL9, ETH152). Within population mean number of alleles did not vary among the three population samples (range, 6.4 to 6.8).On the other hand, almost all loci showed several low frequency alleles “private” to one breed only. Mean locus heterozygosity in the total sample was 0.426. All loci showed a significant excess of homozygous genotypes in all the three breeds. Only the HEL9 locus (BTA8) was characterized, in all breeds, by a significant excess of heterozygous genotypes. Average FIS over all loci was 0.287 for Garfagnina, 0.378 for Calvana, and 0,479 for Pontremolese.Significant pair-wise linkage disequilibrium (or gametic imbalance, for unlinked loci) was observed for all breeds. In particular, more than 40% of all possible pair-wise comparisons were in linkage disequilibrium in the Pontremolese breed. A breed assignment test based on a log-likelihood approach allowed the correct allocation of 100% of individuals to their true breed of origin.

Co‐occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon

Point mutations in the voltage-gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine-phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine-serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S-form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild-type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.

Role of excess inorganic pyrophosphate in primer-extension genotyping assays.

We have developed and genotyped >15,000 SNP assays by using a primer extension genotyping assay with fluorescence polarization (FP) detection. Although the 80% success rate of this assay is similar to those of other SNP genotyping assays, we wanted to determine the reasons for the failures and find ways to improve the assay. We observed that the failed assays fell into three general patterns: PCR failure, excess of heterozygous genotypes, and loss of FP signal for one of the dye labels. After analyzing several hundred failed assays, we concluded that 5% of the assays had PCR failure and had to be redesigned. We also discovered that the other two categories of failures were due to misincorporation of one of the dye-terminators during the primer extension reaction as a result of primer shortening with a reverse reaction involving inorganic pyrophosphate, and to the quenching of R110-terminator after its incorporation onto the SNP primer. The relatively slow incorporation of R110 acycloterminators by AcycloPol compounds the problem with the R110 label. In this report, we describe the source of the problems and simple ways to correct these problems by adding pyrophosphatase, using quenching as part of the analysis, and replacing R110 by Texas red as one of the dye labels. With this new protocol, we have achieved approximately 95% success rate in assay development without the need for optimization.

Conservation genetics of the endemic species Myricaria laxiflora (Tamaricaceae) in the Three Gorges Reservoir area, Hubei

Allozymic variation of 13 natural populations and one ex situ population of Myricaria laxiflora were analyzed using isoelectric focusing in thin-layer polyacrylamide slab gels. Thirteen loci of five enzyme systems were examined and used for the analysis of population genetic diversity and structure. A high genetic diversity was observed in this species, with mean number of alleles per locus of A=1.8, mean percentage of polymorphic loci P=78.7% and average expected heterozygosity H e=0.317, which were higher than the average value of Chinese endemic plant species. An excess of heterozygous genotypes was found both in natural and ex situ populations, suggesting a slight deviation from the Hardy-Weinberg equilibrium in the populations. The total genetic diversity (H t) is 0.3733 within the species, while the genetic diversity within and among populations (H s vs Dst) were 0.3168 and 0.0565, respectively, indicating that 84.86% of the genetic variation occured within populations and only 15.14% among natural populations. The mean gene flow among populations (Nm) was 1.401 and genetic distances among populations ranges from 0.002 to 0.176. Cluster analysis using UPGMA on Rogers' genetic distance revealed that populations of M. laxiflora growing along riversides in the Three-Gorges Reservior area were separated into upstream and downstream groups at Baishui River, Xietan region in Hubei Province. A level of genetic diversity similar to that of natural populations was observed in the ex situ population at Wuhan Botanical Garden, so the ex situ conservation program was considered to be successful in preserving genetic diversity. Combining analysis of the genetic diversity with available information on breeding system, status of natural habitat, and evolutionary history, the causes for the endangerment of M. laxiflora are discussed. It is likely that the species gradually evolved as a relict species after the Quaternary Glaciation. Based on evaluation of the current ex situ conservation program, we suggest that similar habitats upstream and downstream of the Three-Gorges Reservoir or on nearby tributaries should be selected as ex situ conservation spots for M. laxiflora populations with high genetic diversity before their original habitats are submerged by the Three-Gorges Reservoir.

The use of self-incompatibility in the production of hybrid eucalyptus seed by `Aracruz Celulose' in Brazil

Self-incompatibility found in a Eucalyptus grandis clone was used to promote interspecific hybridization between E. grandis and E. urophylla clones. The interspecific hybridization achieved in an open-pollinated commercial seed orchard planted in Espírito Santo, Brazil was evaluated by the multi-locus outcrossing rate (tm) of the seed producing clone, E. grandis. The percentage of outcrossed seeds reached 95.9%. The outcrossing rate of individual trees was quite variable, but was always above 70.0%. Wright's coefficient was negative (F = -0.30) revealing an excess of heterozygous genotypes in the progeny. Genetic parameters confirmed the high degree of hybridization expected in this orchard. The 800-m wide protection belt of native tropical forest that surrounds this orchard has significantly reduced pollen contamination, in comparison to a 400-m wide belt used in another local seed orchard.