Sequence diversity analysis of dihydroflavonol 4-reductase intron 1 in common bean.

Variation in common bean (Phaseolus vulgaris L.) was investigated by sequencing intron 1 of the dihydroflavonol 4-reductase (DFR) gene for 92 genotypes that represent both landraces and cultivars. We were also interested in determining if introns provide sufficient variation for genetic diversity studies and if the sequence data could be used to develop allele-specific primers that could differentiate genotypes using a standard PCR assay. Sixty-nine polymorphic sites were observed. Nucleotide variation (pi/bp) was 0.0481, a value higher than that reported for introns from other plant species. Tests for significant deviation from the mutation drift model were positive for the population as a whole, the cultivar and landrace subsets, and the Middle American landrace set. Significant linkage disequilibrium extended about 300 nucleotides. Twenty haplotypes were detected among the cultivated genotypes. Seven recombination events were detected for the whole population, and six events for the landraces. Recombination was not observed among the landraces within either the Middle American or Andean gene pools. Evidence for hybridization between the two gene pools was discovered. Five allele-specific primers were developed that could distinguish 56 additional genotypes. The allele-specific primers were used to map duplicate DFR genes on linkage group B8.     

Demographic factors shaped diversity in the two gene pools of wild common bean Phaseolus vulgaris L.

Wild common bean (Phaseolus vulgaris L.) is distributed throughout the Americas from Mexico to northern Argentina. Within this range, the species is divided into two gene pools (Andean and Middle American) along a latitudinal gradient. The diversity of 24 wild common bean genotypes from throughout the geographic range of the species was described by using sequence data from 13 loci. An isolation–migration model was evaluated using a coalescent analysis to estimate multiple demographic parameters. Using a Bayesian approach, Andean and Middle American subpopulations with high percentage of parentages were observed. Over all loci, the Middle American gene pool was more diverse than the Andean gene pool (π_sil=0.0089 vs 0.0068). The two subpopulations were strongly genetically differentiated over all loci (F_st=0.29). It is estimated that the two current wild gene pools diverged from a common ancestor ∼111 000 years ago. Subsequently, each gene pool underwent a bottleneck immediately after divergence and lasted ∼40 000 years. The Middle American bottleneck population size was ∼46% of the ancestral population size, whereas the Andean was 26%. Continuous asymmetric gene flow was detected between the two gene pools with a larger number of migrants entering Middle American gene pool from the Andean gene pool. These results suggest that because of the complex population structure associated with the ancestral divergence, subsequent bottlenecks in each gene pool, gene pool-specific domestication and intense selection within each gene pool by breeders; association mapping would best be practised within each common bean gene pool.     

Strong signature of natural selection within an FHIT intron implicated in prostate cancer risk

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's chi(2) = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's chi(2) = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (pi = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (pi = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer.     

Genetic diversity and ecological distribution ofPhaseolus vulgaris (Fabaceae) in northwestern South America

Our goal was to investigate in more detail wild and cultivated common bean (Phaseolus vulgaris) accessions from northwestern South America (Colombia, Ecuador, and northern Peru) because prior research had shown this region to be the meeting place of the two major gene pools (Middle American and Andean) of common bean. Explorations were conducted in these countries to collect additional materials not represented in germplasm collections. It was possible to identify wild common bean populations in Ecuador and northern Peru, where they had never been described before. In addition, we were able to extend the distribution of wild common bean in Colombia beyond what was known prior to this study. In all areas, the wild common bean habitat had suffered severely from destruction of natural vegetation. In Colombia, wild common beans were found on the Eastern slope of the Andes (in continuation of its distribution in Venezuela), whereas in Ecuador and northern Peru they were found on the western slope of this mountain range. This geographic distribution was correlated with an ecological distribution in relatively dry environments with intermediate temperatures (known as “dry mountain forest”). Isozyme andphaseolin seed protein analyses of the northern Peruvian and Ecuadoran wild populations showed that they were intermediate between the Middle American and Andean gene pools of the species. Phaseolin analyses conducted on landraces of the Upper Magdalena Valley in Colombia showed that Andean domesticates were grown at a higher altitude than Middle American domesticates suggesting that the former are adapted to cooler temperatures. Our observations and results have the following consequences for the understanding and conservation of genetic diversity in common bean and other crops: 1) Our understanding of the distribution of the wild relative of common bean (and other crops) is imperfect and further explorations are needed to more precisely identify and rescue wild ancestral populations; 2) For crops for which the wild ancestor has not yet been identified, it may be worthwhile to conduct additional explorations in conjunction with genetic diversity studies at the molecular level to guide the explorations; 3) Our study shows the benefit for more efficient germplasm conservation which can be derived from the dynamic interplay between field explorations (and other conservation operations) and molecular analyses to determine genetic distances and diversities; 4) The intermediate materials identified in northern Peru and Ecuador may have basic importance to understand the origin of the common bean and an applied role as a bridge between the Middle American and Andean gene pools; and 5) The differential adaptation to temperature of the two major cultivated gene pools may help breeders select genotypes based at least partially on their evolutionary origin.     

中国荷斯坦牛POU1F1基因与PRL基因的多态性及其聚合效应对产奶性状的影响

文章采用DNA测序、PCR-RFLP和CRS-PCR技术对979头中国荷斯坦牛POU1F1基因与PRL基因进行研究,发现了3个新SNPs,分别是POU1F1基因第二外显子G1178C、PRL基因5侧翼区A906G和A1134G。采用SAS统计软件GLM程序,利用最小二乘法拟合线性模型,分析基因多态性与产奶性状的关系。结果表明:POU1F1基因1178位点GC基因型在产奶量、乳蛋白量、乳脂量方面均为优良基因型。PRL基因5侧翼区906位点AG基因型在产奶量方面为优良基因型,1134位点不同基因型产奶性状差异不显著。对PRL基因5侧翼区的906位点和POU1F1基因的1178位点进行基因互作分析,结果在乳脂率、乳蛋白率、产奶量、乳蛋白量和乳脂量方面各基因型组合之间均未观察到显著差异,说明基因聚合效应并不是单基因效应的简单相加,基因聚合效应在分子育种中具有更重要的意义。     

The N-terminus of the Begomovirus nuclear shuttle protein (BV1) determines virulence or avirulence in Phaseolus vulgaris

The BV1 gene of the bipartite Begomovirus genome encodes a nuclear shuttle protein (NSP) that is also an avirulence determinant in common bean. The function of the NSP of two common bean-infecting bipartite begomoviruses, Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV), was investigated using a series of hybrid DNA-B components expressing chimeric BDMV and BGYMV NSP, and genotypes of the two major common bean gene pools: Andean (cv. Topcrop) and Middle American (cvs. Alpine and UI 114). BDMV DNA-A coinoculated with HBDBG4 (BDMV DNA-B expressing the BGYMV NSP) and HBDBG9 (BDMV DNA-B expressing a chimeric NSP with the N-terminal 1 to 42 amino acids from BGYMV) overcame the BDMV resistance of UI 114. This established that the BDMV NSP is an avirulence determinant in UI 114, and mapped the domain involved in this response to the N-terminus, which is a variable surface-exposed region. BDMV DNA-A coinoculated with HBDBG10, expressing a chimeric NSP with amino acids 43 to 92 from BGYMV, was not infectious, revealing an essential virus-specific domain. In the BGYMV background, the BDMV NSP was a virulence factor in the Andean cv. Topcrop, whereas it was an avirulence factor in the Middle American cultivars, particularly in the absence of the BGYMV NSP. The capsid protein (CP) also played a gene pool-specific role in viral infectivity; it was dispensable for infectivity in the Andean cv. Topcrop, but was required for infectivity of BDMV, BGYMV, and certain hybrid viruses in the Middle American cultivars. Redundancy of the CP and NSP, which are nuclear proteins involved directly or indirectly in viral movement, provides a masking effect that may allow the virus to avoid host defense responses.     

Promoter and 3'-untranslated-region haplotypes in the vitamin d receptor gene predispose to osteoporotic fracture: the rotterdam study

Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.     

Activity of the 5′ regulatory regions of the rice polyubiquitin <Emphasis Type="Italic">rubi3</Emphasis> gene in transgenic rice plants as analyzed by both <Emphasis Type="Italic">GUS</Emphasis> and <Emphasis Type="Italic">GFP</Emphasis> reporter genes

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.     

Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies

5-Fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5'-untranslated region (5'-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.     

鸡6个功能基因microRNA靶标区域SNP的生物信息学预测

GDF-8、IGF-I、IGF-III、IGF2R、IGFBP2和GHR是鸡的重要经济性状候选基因。利用miRanda和Targetscan软件预测6个基因3′UTR潜在的microRNA靶标, 并发掘靶标区域SNP位点。结果表明: 在6个基因的26个microRNA靶标区域, 共检测到125个SNP位点, 在靶标及其5′和3′邻接等长侧翼区分别检测到47个、44个和35个SNPs位点, 其中12个SNP定位于靶标种子序列互补区。种子序列互补区及其3′侧翼区的SNP位点可能会影响microRNA的调控, 导致家禽的表型变异     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

Tuber magnatum Pico, a species of limited geographical distribution: its genetic diversity inside and outside a truffle ground

The aim of this work was to clarify the genetic structure of the ectomycorrhizal fungus, Tuber magnatum Pico, in a natural truffle ground located in north Italy. Ascomata of this population of T. magnatum were collected over a period of up to 5 years. For comparative analysis, T. magnatum fruit bodies of different geographical origin were also considered. We used single locus markers, such as the variable region of ribosomal genes (ITS), the beta-tubulin gene and sequence-characterized amplified regions (SCAR), as tools to identify single-nucleotide polymorphisms (SNPs). On the basis of the molecular results, which were indirectly supported by a karyological analysis, a self-fertilization mechanism is suggested. A SCAR region was polymorphic within the samples of the truffle ground, leading to the identification of two genotypes. In addition, both the SCAR and the ITS proved to be polymorphic among samples coming from different geographical regions, revealing a genetic differentiation in T. magnatum.     

Identification and characterization of a new allele for the beta subunit of follicle-stimulating hormone in Chinese pig breeds

During evaluation of follicle-stimulating hormone-beta (FSHB) expression in anterior pituitary glands by an RNase protection assay (RPA), the expected fragment of 205 nucleotides at positions 759-963 was not detected in one boar that had moderate plasma and pituitary FSH concentrations. After subcloning and sequencing, mRNA from this boar lacked an 11-bp fragment (5'-CATTTGGAAAC-3') at nucleotide positions 807-817 of the 3'-untranslated region (3'-UTR, D allele). Wild-type FSHB (WT allele) was present in pituitary RNA and genomic DNA in both Meishan (MS) and White Composite (WC) pigs; whereas the D allele was present only in MS pigs (P < 0.01; 5/6 MS vs. 0/6 WC). Also, we found the D allele in five other Chinese breeds but absent in ten American Landrace, 11 Yorkshire and 17 Berkshire pigs. Additionally, the D allele had one silent nucleotide change in the coding region plus six, single nucleotide changes in the 3'-UTR.