Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism

The role of MH class II B (Cyca-DAB1-like) genes in resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV) was analysed. The material consisted of 934 fish from six carp crosses. Fish were challenged with CyHV-3 at an age of 7 and 10 months. During challenge experiments the peak of mortality caused by CyHV-3 was observed at days 8–12 p.i. and the overall cumulative mortality reached 79.9%. Among six Cyca-DAB1-like genotypes, revealed by PCR-RF-SSCP analysis, one genotype (E) was found associated with higher resistance to CyHV-3. Three other genotypes (B, H and J) could be linked to higher susceptibility to CyHV-3. Analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. Our data indicate that MH class II B genes could be used as potential genetic markers in breeding of common carp for resistance to this virus.     

Single nucleotide polymorphisms in randomly selected genes among japonica rice (Oryza sativa L.) varieties identified by PCR-RF-SSCP.

DNA polymorphism of randomly selected genes in rice cultivars was analyzed by the polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) technique. Single DNA fragments were amplified from genomic DNA of the Nipponbare cultivar by 671 primer pairs among the 1000 primer pairs tested. PCR-RF-SSCP analysis using the 671 primer pairs detected polymorphism in 108 DNA fragments between 17 japonica paddy-rice cultivars. An average of 36.9 DNA fragments showed polymorphism between any pair of japonica paddy-rice cultivars. The nucleotide sequences of the polymorphic DNA fragments were determined for 50 alleles of 45 genes together with Nipponbare alleles. In these genes, 142 SNPs and 32 insertions/deletions were identified. Among these 174 sequence variations, 71 were in exons, 78 in introns, and 25 in unassigned regions. There were 28 alleles which had sequence variations in the exons. One allele had a 1-bp deletion in the exon causing a frame-shift mutation, 15 alleles had missense mutations, and the other 12 alleles had synonymous changes and/or sequence variations in 3' untranslated regions. The number of genes having sequence variations between the rice cultivars and the functional implications of the identified SNPs are herein discussed.     

Major-histocompatibility-complex DRB genes of a New-World monkey, the cottontop tamarin (Saguinus oedipus).

The DRB region of the human and great-ape major histocompatibility complex displays not only gene but also haplotype polymorphism. The number of genes in the human DRB region can vary from one to four, and even greater variability exists among the DRB haplotypes of chimpanzees, gorillas, and orangutans. Accumulating evidence indicates that, like gene polymorphism, part of the haplotype polymorphism predates speciation. In an effort to determine when the gene haplotype polymorphisms emerged in the primate lineage, we sequenced three cDNA clones of the New-World monkey, the cottontop tamarin (Saguinus oedipus). We could identify two DRB loci in this species, one (Saoe-DRB1) occupied by apparently functional alleles (*0101 and *0102) which differ by only two nucleotide substitutions and the other (Saoe-DRB2) occupied by an apparent pseudogene. The Saoe-DRB2 gene contains an extra sequence derived from the 3' portion of exon 2 and placed 5' to this exon. This sequence contains a stop codon which makes the translation of the bulk of the Saoe-DRB2 gene unlikely. Preliminary Southern blot hybridization analysis with probes derived from these two genes suggests that both the DRB gene polymorphism and the haplotype polymorphism in the cottontop tamarin may be low. In most individuals the DRB region of this species probably consists of three genes. Comparisons of the Saoe-DRB sequences with those of other primates suggest that probably all of the DRB genes found until now in the Catarrhini were derived from a common ancestor after the separation of the Catarrhini and Platyrrhini lineages. The extant DRB gene and haplotype polymorphism may therefore have been founded in the mid-Oligocene some 33 Mya.     

草鱼连续两代的雌核发育群体及湘江流域群体基因组DNA的微卫星比较分析

对湘江流域草鱼群体,一代及连续两代极体型人工诱导雌核发育草鱼群体基因组DNA的微卫星引物统计分析结果表明:湘江流域草鱼群体存在一定程度的遗传多态性;大多数被检测的微卫星位点上存在两个以上的等位基因,但遗传多样性程度较低。一代人工诱导雌核发育草鱼群体中个体的基因位点已基本纯合,但就整个群体而言,个体之间的基因型还不完全一致,表现出一定的多态性。连续两代人工诱导雌核发育草鱼群体中不仅所检测个体的基因位点已完全纯合,并且各个体的基因型也完全相同。这些观察结果说明所检测的两代人工诱导雌核发育草鱼群体是纯合的,经连续两代人工诱导雌核发育可能建立起草鱼纯系。该实验结果还发现不仅草鱼的微卫星位点上存在等位基因的多态性,而且微卫星位点本身也存在多态性;在人工诱导草鱼雌核发育的过程中不仅存在微卫星等位基因快速丢失的现象,而且也存在微卫星位点丢失的现象。因此,加强对自然水体中草鱼种质资源多样性的保护和利用各种现代生物学技术纯化、筛选和组合优良性状基因,是草鱼遗传育种中同样重要和不可或缺的两个方面。     

锦鲤4个人工雌核发育家系的微卫星标记研究

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpio L.)的8个微卫星DNA标记,对从锦鲤（Cyprinus carpio L.)的红白,大正和昭和3个不同品系中所获得的4个不同人工雌核发育家系的20尾个体进行PCR扩增。电泳结果表明,8对引物在20尾个体中均能重复稳定地扩增出相应的同源序列。随引物不同,各等位基因数为1－11个,大小在68－264bp。在MFW4,MFW7,MFW19,MFW20,MFW23和MFW24 6个微卫星的扩增结果中,20尾个体的扩增图谱呈现了高度的遗传多态性,不同雌核发育家系内个体的遗传异质性也较大。其中大正（TaS)和红白1（RW1）的个体不仅花色分化显著,而且个体间的平均遗传距离分别高达0．28。通过对微卫星等位基因和基因型分析发现,由于锦鲤品系中的每一个体是通过不断地杂交选育而获得,基因组来源复杂,基因高度杂合。因此,只进行1代的人工雌核发育,其家系内仅部分个体的部分座位出现纯合。所获得的人工雌核发育锦鲤为后续的色素遗传调控机制研究提供了必要的实验材料;同时,所鉴定的微卫星分子标记为进行锦鲤的分子标记育种的基因组作图提供了理想的工具。     

应用微卫星多态分析四个鲤鱼群体的遗传多样性

选择12个斑马鱼功能基因的微卫星标记和12个鲤鱼微卫星标记,检测黑龙江鲤(Cyprinus carpiohaematopterusTemminck et Schlegel)、散鳞镜鲤(C.carpio)、荷包红鲤抗寒品系(C.carpiovar.wuyuanensis)、松浦鲤(C.carpioSongpu carp)的群体遗传多样性。共检测到3882个扩增片段,长度为126～489bp,在群体间扩增出1～5个等位基因不等,共计59个等位基因,平均等位基因2.46个。数据经PHYLIP V3.6软件估算和MEGA3软件作图,确立4个群体间的亲缘关系。并应用Bootstrap检验估计系统树中节点的自引导值,并进行了系统发生分析。结果表明①4个群体检测的有效等位基因数都在55个以上,平均观测杂合度为0.36～0.43,平均期望杂合度为0.49～0.53,平均多态信息含量为0.21～0.25,说明这几个群体多态性属于中度偏高水平,遗传多样性较高;②群体间相似系数都在0.84以上,遗传距离较近,为0.067～0.170,与前人研究结果一致。聚类分析显示,松浦鲤与散鳞镜鲤亲缘关系最近,荷包红鲤抗寒品系与它们的亲缘关系较黑龙江鲤更近;③在3个斑马鱼功能基因相关的微卫星位点上,黑龙江鲤缺失特异扩增条带,这可能与几种鱼类的育成史有关。     

Distinct seasonal migration patterns of Japanese native and non‐native genotypes of common carp estimated by environmental DNA

Understanding behavioral differences between intraspecific genotypes of aquatic animals is challenging because we cannot directly observe the animals underwater or visually distinguish morphologically similar counterparts. Here, we tested a new monitoring tool that uses environmental DNA (eDNA), an assemblage of DNA in environmental water, to specifically detect Japanese native and introduced non‐native genotypes of common carp (Cyprinus carpio) in Lake Biwa, Japan, and estimated differences between the two genotypes in the use of inland habitats. We monitored the ratios of native and non‐native single nucleotide polymorphism alleles of a mitochondrial locus of common carp in a lagoon connected to Lake Biwa for 3 years using eDNA. We observed seasonal dynamics in the allele frequency showing that the native genotype frequency peaked every spring, suggesting that native individuals migrated to the lagoon for spawning and then returned to the main lake, whereas non‐native individuals tended to stay in the lagoon. The estimated migration patterns corresponded with the estimates of a previous study, which were based on commercial fish catch data. Our findings suggest that eDNA‐based monitoring can be useful tool for addressing intraspecific behavioral differences underwater.     

鲤鱼微卫星标记与体重、体长和体高性状的相关分析

利用47个鲤鱼微卫星标记, 对柏氏鲤和荷包红鲤抗寒品系自交F2代的92个个体基因组DNA进行检测, 得到162个等位基因, 各座位等位基因数2~6个, 片段长度100~444 bp, 有效等位基因数1.3069~4.2288, 杂合度0.2361~0.7677, 位点多态信息含量0.5368。利用统计软件SPSS的GLM程序对47个微卫星标记与鲤鱼体重、体长和体高相关性进行了分析, 结果表明HLJ695、HLJ716、HLJ739、HLJ759、HLJ774、K16与体重、体长和体高相关, HLJ776与体高相关。对同一标记不同基因型间进行多重比较, 找到与3种性状相关的基因型。     

Frequencies of apolipoprotein E polymorphism in a healthy Kurdish population from Kermanshah, Iran

The molecular polymorphism displayed by apolipoprotein E (APOE) has been listed as a risk factor for susceptibility to various disorders, such as those associated with lipid metabolism, arteriosclerosis, coronary artery disease (CAD), and Alzheimer disease. To evaluate the role of APOE genotypes as risk factors for Alzheimer disease, CAD, and atherosclerosis in the Kurdish population of Kermanshah, Iran, we studied the frequencies of APOE alleles *2, *3, and *4 and genotypes in 914 healthy Kurdish subjects (514 men and 400 women). The highest frequency of APOE in the Kurdish population was found for APOE*3 (87.87%). The APOE*2 and APOE*4 allele frequencies were 6.66% and 5.45%, respectively. Distribution of APOE genotypes and alleles was not significantly different between male and female subjects (p > 0.05). Interestingly, the order of the frequency of APOE alleles (*3-->*2-->*4) in the Kurdish population was quite different from that reported for most populations in the world (*3-->*4-->*2). The findings of the present study can be used to identify individuals with high risk of CAD and atherosclerosis and suggest a preventive measure to reduce their susceptibility.     

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

A Rapid Genotyping Assay for Segregating Human Olfactory Receptor Pseudogenes

Variation in odor perception between individuals is initiated by binding of “odorant” molecules to olfactory receptors (ORs) located in the nasal cavity. To determine the mechanism for variation in odor perception, identification of specific ligands for a large number of ORs is required. However, it has been difficult to identify specific ligands, and ligands have been identified for only 2–3% of the hundreds of mammalian ORs. One way to increase the number of identified ligands is to take advantage of >60 human OR genes that are segregating as a result of a single nucleotide polymorphism, between a functional intact allele and a nonfunctional pseudogene allele. Potential ligands for these ORs can be identified by correlating odor perception of an individual with their genotype [intact/intact (I/I) vs. pseudogene/pseudogene (P/P)] for an OR gene. For this type of study, genotypes must be determined for a large number of individuals. We have developed a PCR-based assay to distinguish between the intact and pseudogene alleles of 49 segregating human OR genes and to determine an individual''s genotype for these genes. To facilitate rapid determination of genotypes for a large number of individuals, the assay uses a small number of simple steps and equipment commonly found in most molecular biology and biochemistry laboratories. Although this assay was developed to distinguish between polymorphisms in OR genes, it can easily be adapted for use in distinguishing single nucleotide polymorphisms in any gene or chromosomal locus.     

Isolation and characterization of major histocompatibility class IIβ genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus)

The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck.     

Association analysis of gene polymorphism in alcohol metabolizing enzymes with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5'-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B (*G (+MslI) allele), CYP2E1 (**C2 (+PstI) allele) and CYP2E1 (*C (-Dra I)2 allele) were 8.48 +/- 1.86%; 1.20 +/- 0.69% and 10.00 +/- 1.90%, respectively. The 2ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7*C (-Sty I) allele was 44.58 +/- 3.21%. A significantly higher frequency of CYP2E1 (*C2 (+Pst I)) allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03-20.01). The tendency to significant effect of A1A2 genotype in ADH1B Msl 1 polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0,042) were also revealed. Association of A1A2 genotype in ADHIB Msl I polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

Haptoglobin gene subtypes in three Brazilian population groups of different ethnicities

Haptoglobin is a plasma hemoglobin-binding protein that limits iron loss during normal erythrocyte turnover and hemolysis, thereby preventing oxidative damage mediated by iron excess in the circulation. Haptoglobin polymorphism in humans, characterized by the Hp(*1) and Hp (*2) alleles, results in distinct phenotypes known as Hp1-1, Hp2-1 and Hp2-2, whose frequencies vary according to the ethnic origin of the population. The Hp(*1) allele has two subtypes, Hp (*1F) and Hp (*1S) , that also vary in their frequencies among populations worldwide. In this work, we examined the distribution frequencies of haptoglobin subtypes in three Brazilian population groups of different ethnicities. The haptoglobin genotypes of Kayabi Amerindians (n = 56), Kalunga Afro-descendants (n = 70) and an urban population (n = 132) were determined by allele-specific PCR. The Hp(*1F) allele frequency was highest in Kalunga (29.3%) and lowest in Kayabi (2.6%). The Hp(*1F)/Hp(*1S) allele frequency ratios were 0.6, 1.0 and 0.26 for the Kayabi, Kalunga and urban populations, respectively. This variation was attributable largely to the Hp(*1F) allele. However, despite the large variation in Hp(*1F) frequencies, results of F (ST) (0.0291) indicated slight genetic differentiation among subpopulations of the general Brazilian population studied here. This is the first Brazilian report of variations in the Hp(*1F) and Hp(*1S) frequencies among non-Amerindian Brazilians.     

Canine olfactory receptor gene polymorphism and its relation to odor detection performance by sniffer dogs

The outstanding sensitivity of the canine olfactory system has been acknowledged by using sniffer dogs in military and civilian service for detection of a variety of odors. It is hypothesized that the canine olfactory ability is determined by polymorphisms in olfactory receptor (OR) genes. We investigated 5 OR genes for polymorphic sites which might affect the olfactory ability of service dogs in different fields of specific substance detection. All investigated OR DNA sequences proved to have allelic variants, the majority of which lead to protein sequence alteration. Homozygous individuals at 2 gene loci significantly differed in their detection skills from other genotypes. This suggests a role of specific alleles in odor detection and a linkage between single-nucleotide polymorphism and odor recognition efficiency.     

元江鲤种群遗传多样性

选择12对微卫星标记检测了于2011年采集自元江(红河上游中国江段)5个样点192尾鲤的群体遗传多样性.共检测到201个等位基因,每个位点等位基因2-27个.各群体各位点平均等位基因(NA)12.25-14.67个,平均有效等位基因(NE)8.28-9.73个,平均观察杂合度(Ho)o.7765-0.8037,平均期望杂合度(HE)0.7761-0.8080,平均多态信息含量(PIC)0.7534-0.7843.元江鲤种群192个个体各位点NA、NE、Ho、HE、PIC分别为16.50、11.26、0.7927、0.8049、0.7966,种群遗传多样性水平高.元江鲤群体之间遗传分化小,可作为一个种群管理单元进行管理.增殖放流要防止遗传多样性丧失.     

Gene duplication and divergence produce divergent MHC genotypes without disassortative mating

Genes of the major histocompatibility complex (MHC) exhibit heterozygote advantage in immune defence, which in turn can select for MHC‐disassortative mate choice. However, many species lack this expected pattern of MHC‐disassortative mating. A possible explanation lies in evolutionary processes following gene duplication: if two duplicated MHC genes become functionally diverged from each other, offspring will inherit diverse multilocus genotypes even under random mating. We used locus‐specific primers for high‐throughput sequencing of two expressed MHC Class II B genes in Leach's storm‐petrels, Oceanodroma leucorhoa, and found that exon 2 alleles fall into two gene‐specific monophyletic clades. We tested for disassortative vs. random mating at these two functionally diverged Class II B genes, using multiple metrics and different subsets of exon 2 sequence data. With good statistical power, we consistently found random assortment of mates at MHC. Despite random mating, birds had MHC genotypes with functionally diverged alleles, averaging 13 amino acid differences in pairwise comparisons of exon 2 alleles within individuals. To test whether this high MHC diversity in individuals is driven by evolutionary divergence of the two duplicated genes, we built a phylogenetic permutation model. The model showed that genotypic diversity was strongly impacted by sequence divergence between the most common allele of each gene, with a smaller additional impact of monophyly of the two genes. Divergence of allele sequences between genes may have reduced the benefits of actively seeking MHC‐dissimilar mates, in which case the evolutionary history of duplicated genes is shaping the adaptive landscape of sexual selection.     

Polymorphisms in xenobiotic-metabolizing genes in the pathogenesis of glucocorticoid-induced osteoporosis in patients with bronchial asthma

Search for and study of risk factors for glucocorticoid-induced (GI) osteoporosis, as one of the most frequent and serious complications of long-term systemic glucocorticoid (SGC) therapy for bronchial asthma are an important problem of prevention medicine. In the present work to determine the frequencies of genotypes and alleles of eight candidate genes of GI osteoporosis in 137 patients with bronchial asthma receiving long-term SGC therapy, using allele-specific hybridization on the biochip. In the analysis of gene polymorphism MTHFR 677C>T showed a statistically significant association between genotypes for this gene and proximal femur mineral density (BMD) for the Z-criterion in patients treated with SGC (non-parametric ANOVA Kruskal-Wallis p = 0.0013). In addition, the analysis of insertion-deletion polymorphism of GSTM1 found that carriers of GSTM1 "null" genotype have lower values of BMD Z-criterion, than carriers of at least one functional allele of GSTM1 gene (Mann-Whitney U-test with Bonferroni correction, p = 0.034). Analysis of gene-gene interactions revealed that carriers of MTHFR genotype 677C/C in combination with GSTM1 "null" genotype characterized by a statistically significant lower values of BMD Z-criterion, than carriers of other variants of genotypes (ANOVA Kruskal-Wallis, p = 0.0012). Thus, the alleles of MTHFR and GSTM1 genes may modulate the risk of GI osteoporosis in patients with bronchial asthma, which is very important to identify group of patients with high risk for osteoporosis among individuals receiving SGC as well as inhaled glucocorticoids.