Hapseeker：一个分析单倍型的C＋＋程序

Hapseeker（Haplotypes Seeker）,即单倍型搜索者,具有辨别单倍型、统计单倍型频率、发现变异位点等功能。Hapseeker能够在Windows、UNIX／Linux操作系统下跨平台运行,具有操作非常简单、运行速度快和需求电脑配置低等特点。在Windows系统下只需要双击Hapseeker．exe．即可运行并自动保存运算结果;而UNIX／Linux系统下,在终端输入命令．／Hapseeker则可运行。Hapseeker被设计用于处理DNA序列或RNA序列,甚至也可对氨基酸序列做类似分析,其输入文件的格式为常用的Fasta格式（序列用Clustal或Mega比对之后另存为Fasta格式即可）。读者通过email：pingchen09@gmail．com联系可以免费获得程序以及详细说明文档,如有需要还可提供源代码。笔者用标准C＋＋语言编写了一个程序——Hapseeker对DNA或RNA序列进行分析。经及复验证,Hapseeker能够鉴定单倍型并统计每种单倍型的频率,快速找出变异位点,具有操作简单、运行速度快,结果准确可靠的优点。     

Genetic Differentiation Analyses Based on mtDNA COⅡ Gene Sequences Among Different Geographic Populations of Aphis glycines(Hemiptera: Aphididae) in Northeast China

Aphis glycines(Hemiptera: Aphididae) is considered as a cosmopolitan pest of cultivated soybean, major diffi culties in its control measures may be due to its higher genetic diversity; however, the knowledge about population genetic diversity of this species is limited. This study aimed to represent the genetic differentiation among different geographic populations of soybean aphid in Northeast China. In order to investigate and assess the genetic diversity, genetic differentiation, molecular variance, population structure, ecological importance and evolutionary history of A. glycines, we sequenced a fragment of one protein-coding gene, the cytochrome c oxidaseⅡof mitochondrial DNA gene. The results showed that four haplotypes were defi ned among COⅡ gene of 180 sequences of soybean aphid in Northeast China including H1 shared by all the populations. Lower haplotype diversity(Hd=0.3590±0.0420) and nucleotide diversity(Pi=0.0012±0.0002) were observed and high gene flow was detected in every two populations, while most of the variation(80.81%) arose from variability within A. glycines from individuals. Low genetic differentiation and high gene fl ow(Nm=2.106) indicated a high migration rate between the populations, which might reveal that gene flow in different geographic populations did not affect by geographical distance. The phylogenetic tree and the haplotype network of A. glycines were obtained based on sequences of COⅡ gene, there were no signifi cant genealogical branches or clusters recognized in NJ tree, and no clear distribution, delineation of haplotypes were demonstrated in the haplotype network according to geographical location. This study rejected the vicariance hypothesis: geographic isolation could be a barrier and it restricted A. glycines gene fl ow among 10 populations.     

Hapseeker：一个分析单倍型的C＋＋程序

开发了一个C＋＋程序——Hapseeker对DNA或RNA序列进行分析。Hapseeker能够鉴定单倍型并统计每种单倍型的频率,快速找出变异住点。具有操作简单、运行速度快、结果准确可靠等优点。     

Hitchhiking Effect Mapping： A New Approach for Discovering Agronomic Important Genes

Besides the natural selection, the crops cultivated today have experienced two episodes of strong artificial selection, domestic and modern breeding. Domestication led to giant genetic structure differentiation between cultivars and their wild species, while modern breeding made further genetic structure differentiation between the modern varieties and the landraces. In a population, diversity of the loci under strong selection is significantly lower than that of other loci. At the same time, diversity in the genomic regions flanking these selected loci also declines in the process of selection. This phenomenon is called hitchhiking effects or selection sweep in genetics. Genomic regions with selection sweep （haplotype block） could be detected after draft genome scanning （genome typing） with molecular markers in a number of released varieties or natural populations. Marker/trait association analysis in these regions would detect the loci （or QTLs） even the favored alleles （genes） in breeding or natural adaptation. Fine scanning of these genomic regions would help to determine the sizes of haplotype blocks and to discover the key genes, thereby providing very valuable information for isolation of the key genes and molecular design of new varieties. Establishment of high density genetic linkage maps in the major crops and availability of high throughput genotyping platform make it possible to discover agronomic important genes through marker/trait association analysis. On the basis of available publications, we give a brief introduction of the hitchhiking effect mapping approach in this paper using plant height, 1 000-grain weight, and phosphorus-deficiency tolerance as examples in wheat.     

Sequence Polymorphisms and Phylogenetic Relationships of hina Gene in Wild Barley from Tibet, China

The hina gene encodes a HINA protein in seeds of barley （Hordeum vulgare）, which was known to affect the grain hardness. 171 hina gene sequences from Tibetan wild barley accessions and worldwide were characterized. Across 1 452 nucleotides of 171 hina genes, 152 SNPs were detected, giving an average frequency of one SNP per 9.5 bases. There were 93 singleton variable sites （the nucleotide polymorphism only observed in a single accession）, 59 polymorphic sites （the polymorphisms found in two or more accessions） and 8 indels. A total of 18 haplotypes were defined, and most of the barley accessions shared one gene haplotype. H. spontaneum had a wider haplotype distribution. Through the analysis of median-joining network of the 18 haplotypes, 4 haplotype groups were found, which were testified by neighbor-joining tree based on the complete sequence alignment. Extremely low level of hina gene diversity was observed in Tibetan wild barley accessions, indicating that Tibet is unlikely a center of origin for cultivated barley.     

绿头鸭线粒体ND2基因全序列测定及其研究(英文)

[Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. Combined with ND2 gene sequences of the Anas Linnaeus accessed in GenBank, phylogenetic tree was constructed by Neighbor-joining and maximum parsimony methods. [Result] The ND2 gene sequences of 4 Anas platyrhynchos were identical(1 041 bp in length; the nucleotide contents of A, G, T, and C were 28.91%, 13.35%, 20.75% and 36.98% respectively; A+T content approximated to that of C+G). Sequences of ND2 gene of mallard were same as spotbill duck, and had high homology with others. The phylogenetic trees indicated mallard and spotbilled duck were close in genetic relationship, both shared a haplotype; then Philippine duck, green-winged teal and northern pintail fell into branch 'A". [Conclusion] The domestic duck may be domesticated from mallard and spotbilled duck.     

The Mining of Citrus EST-SNP and Its Application in Cultivar Discrimination

Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.     

Association of Meloidogyne incognita and Fusarium chlamydosporum causing with wilt complex in Northern Karnataka on Coleusforskohlii

A pot culture experiment was carried out to study the interaction of Melodogyne incognita and Fusarium chlamydosporurn on Coleus forskohlii. The nematode multiplication was adversely affected when fungus was inoculated prior to nematode. Simultaneous inoculation of nematode and fungus as well as nematode followed by fungus seven days later, caused 85.5 per cent root rot disease and significant reduction in plant growth as well as nematode multiplication when compared to the inoculation fungus alone or fungus inoculation prior to nematode.     

Direct-fed microbial:Animal nutrition and reproduction responses

Manipulating the gastrointestinal microbial ecosystem to enhance animal performance and reproductive responses has been one of the main goals of animal science researchers and veterinarians.Recent restrictions to the use of antimicrobials as growth promoters led researchers to seek alternative practices that can show promise both from the standpoint of efficacy as well as from the practical and economic aspects.     

Determination of Thioglucoside in Cabbage by Using the Ultraviolet—visible Light Spectrophotometer

The ultraviolet-visible light spectrophotometer method was adopted to determine thioglucoside in cab-bage with the seeds.Individual plant of cabbage was used as test materials,palladium chloride as complexing agent and sodium cellulose glycolate as dispersing agent.The results showed that palladiumd thioglucoside metod could be taken as a quick,easy and precise quantitative analysis method to determine thioglucoside in cabbage.     

中国3种不同生物型烟粉虱的种群DNA多态性研究

利用基因组DNA的多态性研究了中国3种不同生物型烟粉虱的种群分化．烟粉虱种群分别采自北京、扬州和广州的不同寄主上，烟粉虱生物型包括B型、本地型（NaC）和一种新的生物型（Cv型）．对3种生物型烟粉虱的RAPD-PCR结果进行遗传距离聚类分析，结果显示烟粉虱中国本地型和Cv型的种群关系比较近缘，二者与B型种群的亲缘关系较远．3个烟粉虱生物型的种群分化属于种内分化，据此推测Cv型烟粉虱可能起源于中国或其临近地区．     

中国荷斯坦牛SLC11A1基因多态性与乳腺炎的相关性研究

 【目的】通过研究中国荷斯坦牛SLC11A1基因多态性与乳腺炎性状间的相关性,为奶牛乳腺炎抗性选育提供参考。【方法】采用巢式PCR、降落PCR和DNA测序等技术,研究分析771头牛SLC11A1基因外显子10、内含子9和11的基因多态性,采用SHEsis和PHASE软件进行配对连锁不平衡和单倍型分析。【结果】发现4个不连锁的SNPs,分别为内含子9的6 067（A/G）、6 358（C/T）,外显子10的7 155（A/G）和内含子11的7 809（A/T）;其中,6 067（A/G）、6 358（C/T）和7 809（A/T）为新SNPs。不同基因型与乳腺炎相关性分析结果表明,SLC11A1基因的6 358（C/T）和7 155（A/G）对SCS和产奶量有显著影响（P＜0.05）,6 358（C/T）的CC基因型和7 155（A/G）的AA基因型是优良基因型;单倍型分析表明,群体中共发现16种单倍型组合,其中CAAA是优良的单倍型组合,其个体的SCS低、产奶量高。【结论】SLC11A1基因CAAA单倍型组合可能是奶牛SCS和产奶量的优良基因型和单倍型组合,可作为分子标记应用于奶牛乳腺炎抗性筛选。     

MBL1基因第一内含子与第二外显子多态性与中国荷斯坦牛乳腺炎和乳品质的关联分析

【目的】研究中国荷斯坦牛MBL1基因第一内含子与第二外显子多态性及分析MBL1基因遗传多态性与乳腺炎和乳品质的关联性,为培育优质抗性奶牛提供参考依据。【方法】采用PCR、DNA测序和PCR-RFLP技术,对596头中国荷斯坦牛的MBL1基因内含子1和外显子2序列进行了研究,通过SHEsis软件进行配对连锁不平衡分析和单倍型分析。【结果】发现3个新的SNPs,包括内含子1上G855A1个SNP位点;外显子2上G2651A突变为错义突变,导致第24位氨基酸由缬氨酸变为异亮氨酸;T2686C突变为同义突变,仍编码丙氨酸。单位点基因型与泌乳性状关联分析表明,G2651A突变位点AA基因型个体体细胞评分(SCS)显著高于GG、GA基因型个体(P0.05);G855A、T2686C位点的突变与体细胞评分、乳脂率、乳蛋白率、305d产奶量相关性都不显著。共构建出8种单倍型,发现19种单倍型组合;其中H2H2单倍型组合个体乳蛋白率、体细胞评分为最高;H3H7单倍型组合个体体细胞评分最低,与H2H2个体差异极显著;H4H4单倍型组合个体305d产奶量最高。【结论】H3H7可作为乳腺炎抗性选择的分子标记。H2H2单倍型组合个体虽然乳蛋白率最高,但体细胞评分也最高,对乳腺炎易感,为不利单倍型组合。H4H4单倍型组合305d产奶量最高,对乳蛋白率、乳脂率和体细胞评分影响不大,所以此种基因型组合可以用来提高产奶量。     

中国荷斯坦牛STAT5A基因遗传多态性与泌乳性状的相关分析

 【目的】研究STAT5A基因多态性与中国荷斯坦牛泌乳性状的相关性,为进一步加快育种进程提供参考依据。【方法】利用PCR-SSCP技术对279头中国荷斯坦牛的STAT5A基因的P1和P2两个位点进行群体遗传学检测;计算其基因频率和基因型频率,并对多态性与泌乳性状进行相关性分析。【结果】发现在STAT5AP1和STAT5AP2两个位点上分别存在3种多态以及7种单倍型组合。STAT5AP1位点的AA、GG和AG的基因型频率分别为0.2401、0.1470和0.6129,A和G的基因频率分别为0.5466和0.4534,不同基因型对第1胎产奶量、乳蛋白率和第2胎乳蛋白率存在显著影响;STAT5AP2位点的CC、TT和CT的基因型频率分别为0.7527、0.0036和0.2437,C和T的基因频率分别为0.8746和0.1254,不同基因型对第2胎乳蛋白率有显著影响;7种单倍型组合AACC、AGCC、AACT、AGCT、GGCT、GGCC和AATT的频率分别为0.1864、0.4624、0.0502、0.1505、0.0430、0.1039和0.0036,不同单倍型与第1和2胎产奶量以及乳蛋白率存在相关性。【结论】STAT5A基因不同基因型和单倍型组合与中国荷斯坦牛产奶量和乳蛋白率存在相关性,但与乳脂率不相关。     

中国荷斯坦牛HSF1基因microRNA SNPs 与耐热性能的相关性研究

 【目的】分析638头中国荷斯坦牛HSF1基因多态性与耐热性的关系,以期为中国耐热奶牛分子育种提供参考依据。【方法】设计引物扩增HSF1基因,通过DNA测序、PCR-SSCP和PCR-RFLP技术筛查SNPs并分型,利用miRBASE数据库定位microRNA SNPs,利用SHEsis软件进行配对连锁不平衡分析和单倍型构建,利用SAS软件对不同基因型和单倍型组合个体耐热指标包括红细胞钾浓度、产奶量下降率、直肠温度和耐热系数进行相关分析。应用荧光定量RT-PCR技术研究热应激状态下HSF1 mRNA的转录水平。【结果】发现2个microRNA种子序列新 SNPs,T909C和G4693T。其中909位碱基距离microRNA种子序列5'端3 bp,4 693位突变直接使microRNA种子序列破坏。909位点CC、CT基因型奶牛耐热性能显著高于TT基因型（P＜0.05）;4693位点TT基因型奶牛耐热性能显著高于GG基因型（P＜0.05）。共构建出4种单倍型、发现10种单倍型组合。其中H2H4单倍型组合红细胞钾浓度和直肠温度显著低于H1H3单倍型组合（P＜0.05）,耐热系数显著高于H1H3组合（P＜0.05）,H2H4组合个体产奶量下降率显著低于H1H1组合个体（P＜0.05）,H2H4为耐热单倍型组合。热应激状态下HSF1 mRNA的转录水平在不同组织中存在差异,在心脏中最高,是肌肉中的11.24倍（P＜0.05）。【结论】HSF1基因microRNA SNPs可作为奶牛抗热应激的候选分子标记应用于育种实践。     

中国荷斯坦牛κ-酪蛋白基因第4和第5外显子多态性与泌乳性状的关联分析

 【目的】研究中国荷斯坦牛κ-酪蛋白基因第4和第5外显子多态性与泌乳性状的关联性,为培育高乳蛋白及高乳脂率奶牛提供参考依据。【方法】采用DNA测序、PCR-RFLP和CRS-PCR技术,对544头中国荷斯坦牛κ-酪蛋白基因的外显子4和5序列进行研究,通过SHEsis 软件和PHASE软件进行配对连锁不平衡分析和单倍型分析。【结果】发现6个新的SNPs,包括外显子5上A12907G、G12950A、C12989T、A13028G、T12980C共5个SNP位点,证实前4个位点紧密连锁;外显子4上A10996T共1个SNP位点。单位点基因型与泌乳性状关联分析表明,A10996T突变位点的TT和AT基因型个体乳脂率显著高于AA基因型个体(P＜0.05);T12980C突变位点的TC和CC基因型个体乳脂率、乳蛋白率分别极显著(P＜0.01)、显著(P＜0.05)高于TT基因型个体;紧密连锁的4个突变位点的DE基因型个体乳脂率极显著高于DD基因型个体(P＜0.01)。与泌乳性状的关联分析表明,共构建出7种单倍型,发现16种单倍型组合;其中H6H6单倍型组合个体乳脂率最高,H1H4次之;H1H4单倍型组合个体乳蛋白率最高;H5H6单倍型组合个体乳蛋白率及乳脂率最低。【结论】H1H4单倍型组合在乳蛋白率和乳脂率方面为有利单倍型组合,可作为选择高乳蛋白高乳脂率牛群的分子标记。     

牦牛SMAD1基因第1外显子SNPs检测及与生长性状的关联分析

研究牦牛 SMAD1基因第1外显子SNPs与生长性状的关系，寻找与牦牛生长性状相关的分子标记。采用DNA测序和单倍型分型技术，对青海牦牛 SMAD1基因进行SNPs检测及其基因分型、连锁不平衡和单倍型分析，并对多态位点的基因型及组合单倍型与牦牛生长性状的关联性进行分析。结果发现， SMAD1在第1外显子上存在5个突变位点，其中，g.35084C>T、g.35111C>T和g.35333G>A均存在2种基因型，g.35171G>A和g.35312C>T均存在3种基因型。连锁不平衡分析发现5个位点间不存在强的连锁不平衡效应，单倍型分析发现存在6种不同的单倍型，其中单倍型Hap3的发生频率最高。关联性分析表明，g.35084C>T位点与胸围显著相关，g.35111C>T位点与体斜长、胸围显著相关，g.35171G>A和g.35312C>T位点与体质量、体高、体斜长和胸围显著相关，g.35333G>A位点与胸围、体高显著相关。通过基因型组合发现，4种组合单倍型均与牦牛生长性状有着显著或极显著相关，且H3H6可能是影响牦牛生长性状的最优...     

中国荷斯坦牛IRAK2基因遗传多样性与乳腺炎的相关性研究

 【目的】研究中国荷斯坦牛白细胞介素1受体相关激酶2(IRAK2)基因上5个SNP位点的多态性与奶牛体细胞评分（SCS）的相关性,为奶牛抗乳腺炎育种提供理论基础。【方法】采用PCR-RFLP技术、CRS-PCR技术和DNA测序,对1 292头中国荷斯坦牛、129头鲁西黄牛、32头渤海黑牛IRAK2基因g.28879、g.28916、g.29212、g.40035和g.40120共5个SNP位点进行研究,通过SAS软件进行相关性分析并采用SHEsis软件进行单倍型分析。【结果】共构建出30种单倍型,发现71种单倍型组合;对个体数量大于8头的单倍型组合与SCS进行关联分析表明,H21H23(TTAGGCTCCC)单倍型组合SCS最低。各个位点基因型与SCS关联分析表明,28 879突变位点的CC基因型个体SCS显著低于CT基因型个体(P＜0.05),28 916突变位点的GG基因型个体SCS显著低于AG基因型个体(P＜0.05),而其它3个位点不同基因型之间与SCS没有显著差异(P＞0.05)。【结论】H21H23单倍型组合在SCS方面为有利单倍型组合,可作为选择抗乳腺炎奶牛个体的分子遗传标记。