Identification of the e allele at the Extension locus (MC1R) in Brazilian Creole sheep and its role in wool color variation

The melanocortin 1 receptor (MC1R) gene has been described as responsible for the black color in some breeds of sheep, but little is known about its function in many colored breeds, particularly those with a wide range of pigmentation phenotypes. The Brazilian Creole is a local breed of sheep from southern Brazil that has a wide variety of wool colors. We examined the MC1R gene (Extension locus) to search for the e allele and determine its role in controlling wool color variation in this breed. One hundred and twenty-five animals, covering the most common Creole sheep phenotypes (black, brown, dark gray, light gray, and white), were sequenced to detect the mutations p.M73K and p.D121N. Besides these two mutations, three other synonymous sites (429, 600, and 725) were found. The dominant allele (E(D): p.73K, and p.121N) was found only in colored animals, whereas the recessive allele (E(+): p.73M, and p.121D) was homozygous only in white individuals. We concluded that MC1R is involved in the control of wool color in Brazilian Creole sheep, particularly the dark phenotypes, although a second gene may be involved in the expression of the white phenotype in this breed.     

Allozyme variation among Brazilian and U.S. populations ofRhyzopertha dominicaresistant to insecticides

The lesser grain borer (Rhyzopertha dominica (F.)) is an important pest of stored grain in many parts of the world (Paleartic, Ethiopian, Oriental, Australian, Neotropical, and Neartic regions) with the ability to fly long distances. These insects have been shown to be resistant to organophosphorus insecticides in several studies. Polyacrylamide gel electrophoresis was used to assess the genetic variability within and among eight Brazilian and seven United States populations of R. dominica and to determine how insecticide resistance may be spreading within both countries. Significant variation in allele frequency among populations was observed at all six polymorphic enzyme loci that were examined. The Brazilian and U.S. populations were genetically differentiated from one another; populations within the U.S. and those within Brazil were also differentiated from one another. The mean genetic similarity among the seven U.S. populations collected in a small region in northeast Kansas was smaller than that among eight Brazilian populations collected in a relatively large geographical area. These results are consistent with the resistance ratios to chlorpyriphos-methyl in R. dominica populations from Brazil and the U.S. and the information available concerning patterns of flight activity in this insect.     

Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat.

We used PCR to obtain phylogenetically informative sequences from the high molecular weight glutenin genes of wheat. The validity of partial sequence comparisons as a means of studying glutenin phylogenetics was established by constructing neighbour-joining trees from partial alignments of 12 published glutenin allele sequences. PCR was then used to obtain 20 novel glutenin allele sequences from various Triticum and Aegilops species, including a 3000 year old preserved wheat. A neighbour-joining tree derived from all known glutenin allele sequences had eight clades, representing the eight loci from which the allele sequences were derived, and was split into two halves, one comprising alleles from the Glu-1-1 loci and the other comprising Glu-1-2 alleles. The topology was compatible with the postulated relationships between the A, B, D, and G genomes. The Glu gene duplication event was tentatively dated at 7.2-10.0 million years ago (MYA), the origin of the four genomes at 5.0-6.9 MYA, and the split between the B and G genomes at 2.5-3.5 MYA. The Glu-B1-1 alleles in cultivated wheats fell into two subgroups that diverged 1.4-2.0 MYA, suggesting that emmer was domesticated twice. The D allele sequences were relatively diverse, indicating that the hybridization event that resulted in the hexaploid bread wheats might have occurred more than once.     

Enzyme Variability in the DROSOPHILA WILLISTONI Group. IV. Genic Variation in Natural Populations of DROSOPHILA WILLISTONI

We describe allelic variation at 28 gene loci in natural populations of D. willistoni. Seventy samples were studied from localities extending from Mexico and Florida, through Central America, the West Indies, and tropical South America, down to South Brazil. At least several hundred, and often several thousand, genomes were sampled for each locus. We have discovered a great deal of genetic variation. On the average, 58% loci are polymorphic in a given population. (A locus is considered polymorphic when the frequency of the most common allele is no greater than 0.95). An individual fly is heterozygous, on the average, at 18.4% loci.-Concerning the pattern of the variation, the most remarkable finding is the similarity of the configuration of allelic frequencies from locality to locality throughout the distribution of the species. Our observations support the conclusion that balancing natural selection is the major factor responsible for the considerable genetic variation observed in D. willistoni.     

Characterization of a novel D1S80 pseudoallele

During a D1S80 population study conducted for databasing purposes in the New York City Ashkenazi Jewish population, eight out of 96 samples were typed with a band corresponding to the position of a #15 allele. In seven of the eight samples, three bands appeared. Further investigation was needed to explain the high frequency of an allele considered so rare that it is not included in the commercially provided allelic ladder. After extraction of the putative D1S80 15-repeat amplicon band from the 6% polyacrylamide genotyping gel, the amplicon bands were reamplified with D1S80 primers. After retyping as putative 15 alleles, these samples underwent Southern hybridization with a D1S80 locus-specific probe followed by DNA sequence analysis. Sequence analysis revealed that these bands did not arise from true D1S80 15 alleles. However, the PCR product was of a size that fell within the allelic ladder region corresponding to the 15 band and contained end sequences with strong homology to the D1S80 primers. An alignment search of the sequenced product revealed that a portion of the amplicons contained 72% identity to a known gene. These results emphasize the importance of sequencing analysis when questions arise about the authenticity of an allele.     

Molecular evidence for allopolyploid speciation and a single origin of the western Mediterranean orchid Dactylorhiza insularis (Orchidaceae)

The hybrid origin of the western Mediterranean orchid Dactylorhiza insularis was demonstrated by genetic markers. Allozyme data showed that throughout its range D. insularis has an allotriploid constitution and reproduces apomictically. The parental species of D. insularis were identified as D. romana andD. sambucina; they contributed 2 alleles and 1 allele, respectively, at the allozyme loci studied. The maternal species of D. insularis was D. romana , as inferred from cpDNA ( trn L(UAA) intron). High genetic similarities were found when comparing present populations of D. romana and D. sambucina with their respective genomes 'frozen' in D. insularis. Dactylorhiza insularis showed fixed (or nearly fixed) heterozygosity at 11 out of the 19 loci studied, and poor genetic variation: eight multilocus genotypes were detected at allozyme level. No multilocus genotype differs from the most similar one by more than one allele substitution. All D. insularis individuals showed the same cpDNA haplotype (I) , regardless of their geographic origin and multilocus genotype. The I haplotype is similar, but not identical to that found in D. romana (R). No recurrent formation of D. insularis was observed in hybrid zones between D. romana and D. sambucina , where diploid sexual hybrids (F_1; F_n, backcrosses) were detected. Available data agree with a single origin for D. insularis , which possibly occurred in the present postglacial, when D. romana and D. sambucina , expanding from their glacial refugia, came into contact. The genetic homogeneity found between D. romana and D. markusii , both from their locus classicus , indicates that the latter is a junior synonym of D. romana; on the other hand, D. romana and D. sambucina are well differentiated species ( D_Nei = 0.59).     

Phenotypic plasticity of abdominal pigmentation in Drosophila kikkawai: multiple interactions between a major gene, sex, abdomen segment and growth temperature

Drosophila kikkawai is known to be polymorphic for a single autosomal locus controlling abdomen pigmentation in females. Two strains homozygous at this locus (Abdomen pigmentation, Abp) were established from a polymorphic Indian population: one was homozygous (DD) for the dark allele, the other (LL) for the light allele. A Mendelian analysis of crosses at 25°C confirmed the occurrence of a major locus, with dominance of the D allele. Phenotypic variation of pigmentation according to growth temperature was then analyzed in DD and LL male and female flies, and in reciprocal F1. A slight difference was found between reciprocal F1 females from a dark mother were darker but not at all temperatures. In females, the D allele exhibited an antero‐posterior gradient of increasing expression from segment 27, with dominance over L and an increased expression at low temperatures. In males, abdomen pigmentation was uniformly light in segments 25, the D allele being repressed by the sex genotype. In segment 6, the D allele was expressed but only at low temperatures, and was either recessive to L or codominant. Phenotypic plasticity that is, amount of change induced by different growth temperatures, was variable according to genotype and segment. It always corresponded to a darkening of the fly at lower temperatures, but was generally much less than in D. melanogaster. In D. kikkawai, climatic adaptation might occur more by changing the frequency of the D allele than by phenotypic plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Notes on the esterase variation in Swedish Dactylorhiza incarnata s. l. (Orchidaceae)

Swedish material of Dactylorhiza incarnara s. l. shows little variation at commonly investigated allozyme loci. However, interpretable variation was found at one esterase locus. All plants investigated of D. incarnata var. cruenta from southern Sweden (with spotted leaves) were homozygous for allele a at this locus, whereas all plants investigated of D. incarnata var. ochroleuca were fixed for allele b. In D. incarnata var. incarnata , both alleles were found, although the b allele dominated. In northern Swedish material of D. incarnata s. l., only allele b was found, regardless whether the material had spotted leaves (sometimes referred to as var. cruenta ), or had unspotted leaves.
These results indicate that there is restricted gene flow between var. cruenta and the other varieties in southern Sweden, although they often grow in mixed populations. The northern Swedish material with spotted leaves appears not to be related to the southern var. cruenta .     

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.     

Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil

Allozyme variation in New World screwworm,Cochliomyia hominivorax (Coquerel), populations from Brazil was examined. Variability was observed in 8 of 13 enzyme loci and the frequency of the most common allele was <0.95 for seven loci. Observed and expected heterozygosities were 0.159 and 0.165, respectively. Comparisons of the Brazilian populations with previously studied populations from Costa Rica resulted in Nei's genetic distances of between 0.000 and 0.006, with the greatest distance being between populations within Brazil. Comparisons with Mexican populations using only three loci resulted in genetic distances ≤0.031. Goodness-of-fit statistics for Hardy-Weinberg equilibrium and Wright'sF statistics indicated small deviations from expected equilibrium genotype frequencies and low levels of differentiation between populations within Brazil. Differentiation among screwworm populations from Brazil, Costa Rica, and Mexico was minimal.     

Genetic variation at nine short tandem repeat loci among islanders of the eastern Adriatic coast of Croatia

We have analyzed the extent of genetic variation at nine autosomal short tandem repeat loci (D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820) among six populations from Croatia: five distributed in the islands of the eastern Adriatic coast and one from the mainland. The purpose is to investigate the usefulness of these loci in detecting regional genetic differentiation in the studied populations. Significant heterogeneity among the island and mainland populations is revealed in the distributions of allele frequencies; however, the absolute magnitude of the coefficient of gene differentiation is small but significant. The summary measures of genetic variation, namely, heterozygosity, number of alleles, and allele size variance, do not indicate reduced genetic variation in the island populations compared to the mainland population. In contrast to the two measures of genetic variation, allele size variance and within-locus heterozygosity, the imbalance index (beta) indicates evidence of recent expansion of population sizes in all islands and in the mainland. High mutation rates of the studied loci together with local drift effects are likely explanations for interisland genetic variation and the observed lack of reduced genetic diversity among the island populations.     

Sequence diversity in the amino-terminal region of the malaria-vaccine candidate serine repeat antigen in natural Plasmodium falciparum populations

The amino-terminal region of the serine repeat antigen (SERA) of Plasmodium falciparum is a major malaria-vaccine candidate. Variation in this molecule is essentially dimorphic and alleles may be grouped into the types FCR3, K1 and Honduras1. The Honduras1-type is thought to be the product of homologous recombination between FCR3 and K1 alleles. Here we have examined patterns of sequence diversity in exon II of SERA gene, which encodes most of the amino-terminal region of the antigen, in wild P. falciparum isolates from Indonesia (n=60), Myanmar (n=10) and Thailand (n=14). Among the Indonesian isolates the FCR-3 type predominated (56/60), twenty of which we characterized as novel alleles. A new K1-type allele was also found. In Myanmar, however, all isolates displayed K1-type SERA sequences, which included one new allele. The Honduras1-type was not detected in both countries. In contrast, the 14 isolates from Thailand displayed all three allelic types, with one new Honduras1-type and three new K1-type alleles. On examining the global distribution of SERA alleles by combining previously published sequence data with our results, the FCR3-type alleles predominated in Indonesia, Brazil, and Solomon Islands, but were not found in wild isolates from Myanmar and Africa. Brazil was the only area where K1-type alleles were not found. The distribution of Honduras1-type alleles seems to be mostly restricted to parasite populations from Vietnam, Thailand and Africa. In the allelic families FCR3 and K1, most diversity resulted from variation in sequence and number of octamer repeat units and of allotypes encoding the stretch of serine residues. Sequence analysis indicated that both insertions and deletions of repetitive motifs (creating variation within dimorphic allelic families) and homologous recombination between alleles belonging to different allelic families (creating Honduras1-type alleles) play a role in generating new SERA alleles. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitory antibodies, sequence variation in exon II may represent one of the parasite's immune-evasion strategies.     

Synergistic and threshold effects of GH1 and GHR promoter size variation on body growth and fat accrual in young Nelore (Bos indicus) bulls

A synergistic effect in the somatotropic axis (GH1-GHR-IGF1) was observed in 736 young Nelore (Bos indicus) bulls under ad libitum grass feeding conditions on irrigated pasture in central Brazil. Stepwise substitution of shorter alleles of the promoter region of the growth hormone gene (GH1) and the P1 promoter of the GH1 receptor gene (GHR) with longer alleles was associated with significantly increased body weight gain (W550, weight at age 550 days; ADG, average daily gain) and fat accrual (FAT, rib eye fat thickness). A threshold effect on ADG was associated with allele size variation at the GH1. A best fit model indicated a 3- to 6-fold effect of GH1 variation on ADG, when compared to the variation at the GHR and a known microsatellite at the somatomedin gene (IGF1, insulin-like growth factor 1). A threshold effect on FAT was associated with substitution of the short GHR allele by the longer GHR alleles; the effect of the GHR variation on FAT was 10-fold that of the variation at the GH1 and IGF1 loci. Among the 10 GH1-GHR-IGF1 multi-genotypes identified, the predominant genotype was homozygous for the large GH1 promoter (long/long, G2/G2 or domestic type), short GHR promoter (short/short or wild type), and short IGF1 microsatellite (short/short or wild type). This predominant multi-genotype suggests that selection pressure in the Nelore breed has been directed towards high ADG and W550, and low FAT. Our results mirror previous findings in the oMtla-oGH transgenic mouse model, in which the level of somatotropic gene expression acts through a threshold mechanism, and low expression results in adipogenesis, while high expression increases body growth.     

Variation in Activities of Amylase Allozymes Associated with Chromosome Inversions in DROSOPHILA PSEUDOOBSCURA, D. PERSIMILIS and D. MIRANDA

Different electrophoretic alleles of amylase show associations with particular chromosome 3 inversions in D. pseudoobscura and D. persimilis. Relative adult amylase activities were compared in 37, 37 and 10 strains of D. pseudoobscura, D. persimilis and D. miranda, respectively. Strains carrying the same electrophoretic allele were compared by crossing these lines individually to a reference strain carrying a different electrophoretic mobility allele. This procedure allows comparisons among species, inversions, electromorphs and strains for genetic variation in amylase activity. F2 analysis established that the activity variation co-segregates with the structural amylase locus. This type of variation could be due to either structural gene differences or differences in closely linked, cis-acting regulatory regions. Variation has been detected among and within electrophoretic mobility classes. Moreover, this variation is clearly nonrandom and reveals more of the genetic structure associated with the chromosomal inversion phylogeny of D. pseudoobscura and D. persimilis. ----Some of the findings are: (1) Similar electromorphs in D. pseudoobscura and D. persimilis usually show different activities. These species show nearly complete differentiation of amylase alleles, based on activities. (2) D. persimilis has the broadest range of variation in amylase activity, about four-fold between the highest and lowest alleles. D. pseudoobscura and D. miranda are also polymorphic for activity, but have more constrained ranges of variation. D. miranda alleles show on the average about four times the activity of D. pseudoobscura alleles. (3) Some association of electrophoretic mobility and activity has been found. Alleles 1.09 of D. persimilis, as well as 1.43 and 1.55 of D. miranda, have relatively high activity. It may be that these high activity alleles are part of an adaptation to cooler habitats. (4) Within electrophoretic classes, associations of activities with inversions have been found. These are especially strong in D. persimilis. The 1.00 alleles in the ST, KL, MD and WT inversions, the 0.92 allele in the ST and MD inversions and the 1.09 allele in the WT and KL inversions have levels of activities that depend upon the arrangement in which they are located. These results demonstrate that suppression of recombination in inversion heterokaryotypes can result in extensive genic divergence between inversions.     

NFKB1 promoter variation implicates shear-induced NOS3 gene expression and endothelial function in prehypertensives and stage I hypertensives

In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.     

Two-color multiplexed analysis of eight short tandem repeat loci with an electrophoretic microdevice.

Single-channel microfabricated electrophoretic devices equipped with a dual-wavelength laser-induced fluorescence detection system were used for the fast analysis of an eight-loci, two-color multiplex short tandem repeat (STR) system for human identification. Routine analyses of the eight loci (CSF1PO, TPOX, TH01, vWA and D16S539, D7S820, D13S317, D5S818), requiring four-base resolution, were performed in only 2 min. Specific analyses for a microvariant allele (allele 9.3 of the TH01 locus) demanded single-base resolution and was performed in less than 10 min. The high accuracy of the microdevice for real-world STR sample analyses was demonstrated by comparison with conventional slab-gel electrophoresis. Our results show that a fast multiwavelength multichannel electrophoretic microsystem will be capable of routinely processing thousands of complex STR samples per day.     

Population genetics of dinucleotide (dC-dA)n.(dG-dT)n polymorphisms in world populations.

We have characterized eight dinucleotide (dC-dA)n.(dG-dT)n repeat loci located on human chromosome 13q in eight human populations and in a sample of chimpanzees. Even though there is substantial variation in allele frequencies at each locus, at a given locus the most frequent alleles are shared by all human populations. The level of heterozygosity is reduced in isolated or small populations, such as the Pehuenche Indians of Chile, the Dogrib of Canada, and the New Guinea highlanders. On the other hand, larger average heterozygosities are observed in large and cosmopolitan populations, such as the Sokoto population from Nigeria and German Caucasians. Conformity with Hardy-Weinberg equilibrium is generally observed at these loci, unless (a) a population is isolated or small or (b) the repeat motif of the locus is not perfect (e.g., D13S197). Multilocus genotype probabilities at these microsatellite loci do not show departure from the independence rule, unless the loci are closely linked. The allele size distributions at these (CA)n loci do not follow a strict single-step stepwise-mutation model. However, this features does not compromise the ability to detect population affinities, when these loci are used simultaneously. The microsatellite loci examined here are present and, with the exception of the locus D13S197, are polymorphic in the chimpanzees, showing an overlapping distribution of allele sizes with those observed in human populations.     

Phylogeography of the freshwater red alga Sirodotia (Batrachospermales,Rhodophyta) in Brazil

Considering the lack of knowledge on genetic variation on members of the freshwater red algal of the order Batrachospermales in tropical regions, phylogeographic patterns in Sirodotia populations were investigated using two mitochondrial regions: the cox2‐3 spacer and partial cox1 gene (barcode). Individuals identified as Sirodotia delicatula were analyzed from 14 stream segments across its distribution in Brazil. Phylogenetic analyses based on the ribulose‐1,5‐bisphosphate carboxylase/oxygenase large sub‐unit gene showed three clades, one representing S. delicatula, from all locations in southeastern Brazil and other regions from Brazil. The remaining samples formed two clades, which were highly divergent and distantly positioned from those of S. delicatula: 2.5–2.7% and 3.4–3.7%. This level of variation would warrant the species split of these taxa from mid‐western Brazil. A total of eight cox2‐3 spacer and nine cox1 haplotypes were observed among the 122 individuals studied. One location had two cox2‐3 haplotypes and three locations had two cox1 haplotypes; all others had a single dominant haplotype each. The existence of high intraspecific genetic variation among individuals of distinct locations (several haplotypes), but little variation within a location seems to be a pattern for the Batrachospermales. Haplotype networks showed low variation among the haplotypes from southeastern Brazil (10 locations with divergence of 0.3–1.1% for cox2‐3, 0.1–0.3% for cox1) and high variation among the haplotypes from the mid‐west region (four locations, 4.0–9.3% for cox2‐3, 6.2–8.4% for cox1). Thus, the present data clearly suggest the existence of cryptic species in Sirodotia in Brazil.     

New variations in intron 4 of growth hormone gene in Chinese native chickens

Polymorphism in intron 4 of chicken growth hormone (cGH) gene was studied in 20 Chinese native chicken populations and broiler or layer populations. A total of eight restriction digestion profiles were identified in intron 4 and confirmed by sequencing. Among 20 populations, there were distinctively different allele numbers and frequencies of intron 4 restriction fragment length polymorphisms (RFLPs) between Chinese native chickens and broilers or layers. Two new alleles, allele D and allele E, were identified in Taihe Silkies. Allele D was also identified in other Chinese native breeds and a 50 bp fragment deletion was identified in allele E.