Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998     

Isolation by distance and Pleistocene expansion of the lowland populations of the white piranha Serrasalmus rhombeus

The genetic variability and distribution of Amazonian fish species have likely been influenced by major disturbance events in recent geological times. Alternatively, the great diversity of aquatic habitat in the Amazon is likely to shape ongoing gene flow and genetic diversity. In this context, complex patterns of genetic structure originating from a joint influence of historical and contemporary gene flow are to be expected. We explored the relative influence of Pleistocene climatic fluctuations and current water chemistry on the genetic structure of a piranha, Serrasalmus rhombeus, in the Upper Amazon by the simultaneous analysis of intron length polymorphism and mitochondrial DNA sequences. The Madeira river is well suited for that purpose as it is characterized by a great diversity of water types, the presence of one of the largest floodplain of the Amazon and the potential occurrence of two Pleistocene refuges. We found evidence of genetic structure even at a small geographical scale (less than 10 km), indicating that the floodplain is not a homogenizing factor promoting interdrainage dispersal in S. rhombeus. Likewise, the hierarchical genetic structure inferred was correlated to geographical distance instead of habitat characteristic. Our results also support the hypothesis that the area underwent population expansion during the last 800 000 years. In addition, a higher level of genetic diversity was found in the samples from the putative Aripuanã refuge. The present findings suggest that Pleistocene refuges contributed significantly to the colonization of the lowlands in the Upper Amazon valley during the Pleistocene.     

Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)

Gliadins are seed storage proteins which are characterized by high intervarietal polymorphism and can be used as genetic markers. As a result of our work, a considerably extended catalogue of allelic variants of gliadin component blocks was compiled for durum wheat; 74 allelic variants for four gliadin-coding loci were identified for the first time. The extended catalogue includes a total of 131 allelic variants: 16 for locus Gli-A1(d), 19 for locus Gli-B1(d), 41 for locus Gli-A2(d), and 55 for locus Gli-B2(d). The electrophoretic pattern of the standard cultivar and a diagram are provided for every block identified. The number of alleles per family is quite small for loci Gli-A1(d) and Gli-B1(d) of durum wheat, as contrasted to loci Gli-A2(d) and Gli-B2(d) that are characterized by large families including many alleles. The presence of large block families determines a higher diversity of durum wheat for loci Gli-A2(d) and Gli-B2(d) as compared to Gli-A1(d) and Gli-B1(d). The catalogue of allelic variants of gliadin component blocks can be used by seed farmers to identify durum wheat cultivars and evaluate their purity; by breeders, to obtain homogenous cultivars and control the initial stages of selection; by gene bank experts, to preserve native varieties and the original biotypic composition of cultivars.     

Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

Allelic variation,fragment length analyses and population genetic models: a case study on Drosophila microsatellites*

The allelic variation of 16 microsatellite loci from selected species of the Drosophila melanogaster and D. obscura group was determined. Intra‐ and interspecific sequence comparisons allowed discrimination of mutations affecting the repetitive microsatellite from those affecting the flanking regions. The hypotheses that slippage needs a minimum number of repeats in order to become efficient with respect to microsatellite variability, and of an increased mutation rate with increased length of the microsatellite are supported by the results of our analyses. There is in particular at the interrupted complex microsatellite locus BICOID in the species of the D. obscura group, extensive variation in the flanking regions in addition to length and sequence variation of the repetitive microsatellite. The allelic variation at this locus can hardly be explained by slippage alone. Estimates of microsatellite variability by fragment length analyses will pick up only a minor fraction of allelic variation at such loci, and conclusions that are based on the stepwise mutation model will not hold.     

Dinucleotide microsatellite markers from Anopheles funestus

A total of 39 dinucleotide microsatellite‐containing clones were sequenced from a principal African malaria vector, Anopheles funestus. Primers designed to amplify 20 loci were used to genotype A. funestus mosquitoes from Burkina Faso and Kenya. Of nine polymorphic loci that amplified reliably and could be scored unambiguously, the overall within‐sample gene diversity was similar between locales, 0.77 and 0.78, with an allelic richness per locus of five to 11. Both the high level of polymorphism and absence of significant heterozygote deficiency at any locus favour these markers for studies of population structure that are vital to controlling this medically important species.     

Characterization of microsatellite DNA markers for the alligator snapping turtle,Macrochelys temminckii

Two trinucleotide and seven tetranucleotide microsatellite loci were isolated from an alligator snapping turtle Macrochelys temminckii. To assess the degree of variability in these nine microsatellite loci, we genotyped 174 individuals collected from eight river drainage basins in the southeastern USA. These markers revealed a moderate degree of allelic diversity (six to 16 alleles per locus) and observed heterozygosity (0.166–0.686). These polymorphic microsatellite loci provide powerful tools for population genetic studies for a species that is afforded some level of conservation protection in every state in which it occurs.     

Carotenoids gene markers for sweetpotato (Ipomoea batatas L. Lam): applications in genetic mapping,diversity evaluation and cross-species transference

Carotenoids play essential biological roles in plants, and genes involved in the carotenoid biosynthesis pathway are evolutionarily conserved. Orange sweetpotato is an important source of β-carotene, a precursor of vitamin A. In spite of this, only a few research studies have focussed on the molecular aspects of carotenoid genes regarding their specific sequence and structure. In this study, we used published carotenoid gene sequences from Ipomoea and other species for “exon-primed intron-crossing” approaches. Fifteen pairs of primers representing six carotenoid genes were designed for different introns, eleven of which amplified scorable and reproducible alleles. The sequence of PCR products showed high homology to the original ones. Moreover, the structure and sequence of the introns and exons from five carotenoid structural genes were partially defined. Intron length polymorphism and intron single nucleotide polymorphisms were detected in amplified sequences. Marker dosages and allelic segregations were analysed in a mapping population. The developed markers were evaluated in a set of Ipomoeas batatas accessions so as to analyse genetic diversity and conservation applicability. Using CG strategy combined with EPIC-PCR technique, we developed carotenoid gene markers in sweetpotato. We reported the first set of polymorphic Candidate Gene markers for I. batatas, and demonstrated transferability in seven wild Ipomoea species. We described the sequence and structure of carotenoid genes and introduced new information about genomic constitution and allele dosage.     

Polymorphisms of GA/GT Microsatellite Loci from Anguilla japonica

Six novel microsatellite loci, containing (GA)_15\N17 or (GT)_10\N19 perfect tandem repeats, were isolated and characterized for the catadromous eel Anguilla japonica. The allelic size of the 6 loci ranged from 79 to 226 bp in length. All loci were polymorphic with a mean number of 14.7 alleles per locus and a mean heterozygosity of 0.67, suggesting higher polymorphism than that of freshwater and anadromous fishes, but lower than that of marine fishes. Genotype diversity of the 6 loci ranged from 0.22 to 0.61 with a mean value of approximately 0.5. Cross-species amplification showed that 5 of the 6 microsatellite primers proved to be useful in addressing questions of population genetics for all Anguilla species. Received October 11, 2000; accepted December 29, 2000.     

Diversity and diversification of HLA-A,B,C alleles

The nucleotide sequences encoding 14 HLA-A,B,C and 5 ChLA-A,B,C molecules have been determined. Combining these sequences with published data has enabled the polymorphism in 40 HLA-A,B,C and 9 ChLA-A,B,C alleles to be analyzed. Diversity is generated through assortment of point mutations by recombinational mechanisms including gene and allelic conversions. The distribution and frequency of silent and replacement substitutions indicate that there has been positive selection for allelic diversity in the 5' part of the gene (exons 1 to 3) and for allelic homogenization and locus specificity in the 3' part of the gene (exons 4 to 8). These differences may correlate with the lengths of converted sequences in the two parts of the gene and frequency of the CpG dinucleotide. Locus-specific divergence of HLA-A,B, and C demonstrates that recombinational events involving alleles of a locus have been more important than conversion between loci. This contrasts with the predominance of gene conversion events in the evolution of mutants of the H-2Kb gene. However, a striking example of gene conversion involving HLA-B and C alleles of an oriental haplotype has been found. Comparison of human and chimpanzee alleles reveals extensive sharing of polymorphisms, confirming that diversification is a slow process, and that much of contemporary polymorphism originated in ancestral primate species before the emergence of Homo sapiens. There is less polymorphism at the HLA-A locus compared to HLA-B, with greater similarity also being seen between HLA-A and ChLA-A alleles than between HLA-B and ChLA-B alleles. Although greater diversity is seen in the 5' "variable" exons of HLA-B compared to HLA-A, there is increased heterogeneity in the 3' "conserved" exons of HLA-A compared to HLA-B.     

Cervical carcinoma progression-associated genetic alterations on chromosome 6

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.     

Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II β1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a “gene walking” technique to obtain intron 2 sequences that flanked MHC class IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the β1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.     

Balancing selection at a frog antimicrobial peptide locus: fluctuating immune effector alleles?

Balancing selection is common on many defense genes, but it has rarely been reported for immune effector proteins such as antimicrobial peptides (AMPs). We describe genetic diversity at a brevinin-1 AMP locus in three species of leopard frogs (Rana pipiens, Rana blairi, and Rana palustris). Several highly divergent allelic lineages are segregating at this locus. That this unusual pattern results from balancing selection is demonstrated by multiple lines of evidence, including a ratio of nonsynonymous/synonymous polymorphism significantly higher than 1, the ZnS test, incongruence between the number of segregating sites and haplotype diversity, and significant Tajima's D values. Our data are more consistent with a model of fluctuating selection in which alleles change frequencies over time than with a model of stable balancing selection such as overdominance. Evidence for fluctuating selection includes skewed allele frequencies, low levels of synonymous variation, nonneutral values of Tajima's D within allelic lineages, an inverse relationship between the frequency of an allelic lineage and its degree of polymorphism, and divergent allele frequencies among populations. AMP loci could be important sites of adaptive genetic diversity, with consequences for host-pathogen coevolution and the ability of species to resist disease epidemics.     

Eleven novel microsatellite markers developed from the living fossil Ginkgo biloba (Ginkgoaceae)

Ginkgo biloba L. (Ginkgoaceae), a famous and widely cultivated plant, is often referred to as a living fossil. Here we isolated and characterized 11 polymorphic microsatellite loci from G. biloba using a modified biotin-capture method. These loci showed high levels of allelic diversity, alleles sizes ranged from 4 to 16 per locus with polymorphism information content ranging from 0.432 to 0.909 (average = 0.752). The observed and expected heterozygosities ranged from 0.208 to 0.708 (average = 0.484) and from 0.501 to 0.934 (average = 0.795), respectively. These microsatellite markers can be used as genetic markers for further studies on genetic diversity, genetic structure and the gene flow among populations of this species.     

Molecular genetic analysis of polymorphism of the HindII and (CA)-repeat of the factor VIII gene loci in people of the Volgo-Ural region

Molecular genetic analysis of polymorphism of the factor VIII gene at loci HindIII (intron 19) and (CA)-repeats (intron 13) was performed in seven ethnic groups of the Volga-Ural region (Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis). The allelic frequency of the HindIII locus was shown to be similar in these ethnic groups. The ethnic groups were more heterogeneous with regard to the genotype and allele frequencies of (CA)-repeats, which indicates that this polymorphic system can be used to characterize the genetic structure of the Volga-Ural human populations.     

The cotton-top tamarin revisited: Mhc class I polymorphism of wild tamarins,and polymorphism and allelic diversity of the class II DQA1, DQB1, and DRB loci

Cotton-top tamarins (Saguinus oedipus) in captivity are unusual in that they exhibit low levels of polymorphism and allelic diversity at the major histocompatibility complex (Mhc) class I loci. Since the polymorphism has previously only been examined in captive tamarins, we analyzed the Mhc class I alleles of a population of wild tamarins. These wild tamarins, like their captive counterparts, exhibited limited class I polymorphism. We also assessed the levels of polymorphism and allelic diversity at the Mhc class II DQA1, DQB1, DQB2, and the DRB loci in captive populations of cotton-top tamarins. In contrast to the extensive polymorphism in Old World monkeys, only two alleles were detected at each of DQA1 and DQB1. Also, the DQB2 locus was monomorphic and conserved between New and Old World monkeys. Sequences derived from four putative DRB loci were obtained, and extensive polymorphism was found at all four loci. Phylogenetic analysis did not indicate that any of the tamarin DRB loci, with the possible exception of Saoe-DRB3, were orthologous to the human DRB loci. At three of the DRB loci (Saoe-DRB11, Saoe-DRB ^* W12, Saoe-DRB ^* W22), the number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites, indicative of positive selection. We found no support for a restriction on the polymorphism at the cotton-top tamarin class II loci. However, the allelic diversity at some of the Saoe-DRB loci is more limited than for the HLA-DRB1, consistent with a restriction imposed by the bone marrow-chimerical lifestyle.     

Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae)

Due to their high degree of polymorphism, microsatellites are considered useful tools for studying population genetics. Nevertheless, studies of genetic diversity in stingless bees by means of these primers have revealed a low level of polymorphism, possibly the consequence of the heterologous primers used, since in most cases these were not specifically designed for the species under consideration. Herein we compared the number of polymorphic loci and alleles per locus, as well as observed heterozygosity in Melipona rufiventris and M. mondury populations, using specific and heterologous primers. The use of specific primers placed in evidence the greater frequency of polymorphic loci and alleles per locus, besides an expressive increase in observed heterozygosity in M. rufiventris and M. mondury, thereby reinforcing the idea that populational studies should be undertaken by preferably using species-specific microsatellite primers.     

Multiple nuclear and mitochondrial genotyping identifies emperors and large-eye breams (Teleostei: Lethrinidae) from New Caledonia and reveals new large-eye bream species

Species identification is fundamental to address questions about community ecology, biodiversity, conservation and resource management, at any life history stage. Current studies on fish larval ecology of tropical species are hampered by the lack of reliable and effective tools for identifying larvae at the species level. Emperors and large-eye breams comprise fish species from the perciform fish family Lethrinidae. They inhabit coastal and coral-reef habitats of the tropical Indo-Pacific, and they are important fishery resources. Their taxonomy is considered difficult and identification to species is often problematic. Lethrinidae larvae and juveniles can be identified on the basis of meristic counts at the sub-family level, but no further. In this study, we developed a set of polymorphic PCR markers (size polymorphisms at the intron regions from 4/5 nuclear protein-coding genes and single-strand conformation polymorphism of a 205-bp fragment at the mitochondrial 16S rRNA locus), to characterize 341 specimens from 21 Lethrinidae species from New Caledonia (southwestern tropical Pacific). A genetic data-bank was constructed using the genotypes screened from the multiple gene loci of adult or sub-adult specimens used as references for these species. The 16S rRNA gene fragment was able to differentiate species for the genus Lethrinus, but it provided little diagnostic resolution among different species within the genus Gymnocranius. A combination of the 16S rRNA marker and 4 nuclear markers developed herein allowed to sort out species within Gymnocranius spp. from New Caledonia. Using genotype distributions at nuclear loci to test for reproductive isolation, we found that three apparently undescribed large-eye bream species may exist, provisionally referred to as Gymnocranius sp. A, sp. B and sp. C. Subsequent genotyping of 137 Lethrinidae larvae collected from the bays of the Noumea peninsula, New Caledonia, found a total of three species (Lethrinus genivittatus, Lethrinus olivaceus and Gymnocranius sp. A).