水稻NBS-LRR基因选择性剪接的全基因组检测及分析

选择性剪接是促进基因组复杂性和蛋白质组多样性的一种主要机制,但是对水稻NBS-LRR序列选择性剪接的全基因组分析却未见报道。通过隐马尔柯夫模型搜索,从TIGR数据库里得到了855条编码NBS-LRR基序的序列。利用这些序列在KOME、TIGR基因索引及UniProt三个数据库中进行同源搜索,获得同源的完整cDNA序列、假设一致性序列和蛋白质序列。再利用Spidey和SIM4程序把完整cDNA序列和假设一致性序列联配到相应的BAC序列上来预测选择性剪接。蛋白质序列和基因组序列之间的联配使用tBLASTn。在这875个NBS-LRR基因中,119个基因具有选择性剪接现象,其中包括71内含子保留,20个外显子跳跃,25个选择性起始,16个选择性终止,12个5′端的选择性剪接和16个3′端选择性剪接。大多数选择性剪接都为两个和多个转录本所支持。可以通过访问http://www.bioinfor.org查询这些数据。进而通过生物信息学分析剪接边界发现外显子跳跃和内含子保留的‘GT…AG’的规则不如组成型的保守。这暗示了它们是通过不同的调控机制来指导剪接变构体的形成。通过分析内含子保留对蛋白质的影响,发现选择性剪接的蛋白更倾向于改变其C端氨基酸序列。最后对选择性剪接的组织分布和蛋白质定位进行分析,结果表明选择性剪接的最大类的组织分布是根和愈伤组织。超过1/3剪接变构体的蛋白质定位是质膜和细胞质。这些选择性剪接蛋白可能在抗病信号转导中起到重要作用。     

山西瘦肉型猪(SD-Ⅲ系)基因组DNA的AFLP检测研究

用分子标记AFLP对山西瘦肉型猪(SDⅢ-系)进行纯度检测,旨在为评价该猪种的遗传特性提供相关参数。实验共用8条引物,对25头猪进行了基因组DNA的分析,共获得171个AFLP标记,单引物获得的标记数在3个~15个之间,群体相似系数为0.928(0.892~0.978);遗传距离为0.072(0.022~0.108)。结果表明:AFLP适宜于基因组DNA遗传结构检测;SDⅢ-系猪纯度较高。     

PMWS病猪猪圆环病毒2型全基因组序列分析

根据GenBank中发表的猪圆环病毒2型(PCV2)全基因序列,设计一对特异性引物,用PCR方法直接从5省病料中分别扩增出5个PCV2毒株的全基因组,分别命名为FujianPCV、GuangxiPCV、HainanPCV、HunanPCV和ShandongPCV.将扩增片段克隆于pMD 18-T载体,进行序列测定,结果表明,除FujianPCV株核苷酸长度为1768bp,其余四株均为1767 bp.应用DNAstar序列分析软件分析表明,本实验的5个PCV2分离株与世界上其它地区的PCV2分离株密切相关,核苷酸序列同源性达93%～98.8%,与PCV1毒株的序列同源性只有68.4%～70%.其中ORF1和ORF2所编码的氨基酸序列与国内外PCV2毒株比较,同源性也很高,分别为98.1%～99.4%和88%～99.1%.     

Otterlace软件在猪全基因组序列人工注释分析中的应用

2009年11月,美、英等国科学家宣布首次绘制出家猪的基因组草图。近两年,随着全基因组序列陆续释放,越来越多的测序片段得到正确拼接组装,从全基因组水平上对猪功能基因进行注释分析显得尤为迫切。文章以丝切蛋白1(Cofilin 1,CFL1)基因的注释过程为例,介绍了运用Sanger研究所开发的Otterlace软件对猪全基因组的免疫基因序列进行人工分析与注释。通过详细说明Zmap、Blixem和Dotter 3个注释工具的使用方法,并给出了注释过程的主要步骤,以期对Otterlace的应用起一个抛砖引玉的作用。运用Otterlace软件对243个免疫相关基因进行分析,其中180个基因得到完整或部分注释,这为后续深入开展这些基因的功能研究奠定了基础。     

近交系小型猪线粒体基因组分析和家猪系统进化关系分析

广西巴马小型猪是长期近交培育而成的实验小型猪,遗传稳定.本研究拟通过线粒体基因组(mitochondrial DNA,mtDNA)分析来了解近交系的遗传背景,并从mtDNA角度进一步认识中国本土猪种的系统进化关系.实验设计29对特异性引物,PCR扩增和产物测序,拼接出广西巴马小型猪近交系7个世代猪的mtDNA序列;用4...     

猪水泡病病毒全基因组核苷酸序列的测定与分析

猪水泡病是由猪水泡病病毒(Swine vesicular disease virus,SVDV)引起的猪的一种急性传染病,在症状上与口蹄疫极其相似。该病流行性强,发病率高,能造成严重的公共卫生问题。国际兽医局将其列为动物A类传染病,我国农业部列为动物一类传染病。SVDV属于小RNA病毒科肠道病毒属,其核酸类型为单股正链RNA分子,无囊膜,病毒基因组含一个大的开放阅读框,编码一条由2185个氨基酸组成的多聚蛋白。     

猪传染性胃肠炎病毒的分离鉴定及全基因组序列分析

采用ST细胞培养,免疫荧光、理化试验、中和试验、电镜观察等方法,从四川疑似猪腹泻病料中分离到1株猪传染性胃肠炎病毒,命名为SC-Y.分离株在ST细胞上盲传至第8代时可出现稳定的细胞病变,病毒滴度TCID50为10-3.664/0.05m1,中和指数为52.应用长链RT-PCR技术成功地扩增出了覆盖SC-Y株全长基因组的5个片段,通过BioEdit软件对测序结果进行拼接,确认SC-Y株基因组全长28 590bp,包括7个开放阅读框,基因组5非编码区长315nt,3'端非编码区长277nt.TGEV基因组系统进化树显示,SC-Y株与美国Purdue株可能来源于共同的祖先.     

猪源脑心肌炎病毒GXLC株全基因组序列测定与分析

对猪源脑心肌炎病毒(EMCV)GXLC株进行全基因组克隆、测序和分析。采用RT-PCR技术分段扩增基因组可读框(ORF),应用3'-RACE和5'-RACE技术扩增3'-UTR和5'-UTR,分别对扩增片段进行克隆和测序,经拼接后获得GXLC株全基因组序列,共7725个核苷酸。与NCBI GenBank登录的国内外参考毒株进行同源性比较及系统进化分析结果表明,GXLC株与GX0601、GX0602、BJC3、HB1等国内分离株以及CBNU、K3、K11、BEL-2887A、EMCV-R、PV21等国外分离株同源性达99%以上;基于全基因组、结构蛋白及非结构蛋白基因序列绘制的系统进化树拓扑结构图相近,所有EMCV分离株可分成两个群:I群和II群,I群可细分为Ia亚群和Ib亚群,其中猪源EMCV属于Ia或Ib亚群、鼠源EMCV属于Ia亚群、野猪源EMCV属于II群,GXLC株与其它中国分离株均属于Ia亚群。     

猪Ⅱ型圆环病毒浙江株的分离及全基因组结构分析

用PCR方法对从浙江省猪场收集的、以淋巴结肿大和生长迟缓为特征的患猪腹股沟淋巴结进行PCV2检测.将检测为阳性的病料研磨、离心、过滤除菌后,接种PCV-free PK-15细胞进行病毒分离,分离获得3株病毒,命名为HZ0201、HZ0202、NB0301.PK-15细胞增殖所得病毒离心纯化后,在电镜下可观察到直径约为17～20nm,呈二十面体对称的病毒粒子.以组织和病毒的细胞DNA为模板进行PCV2的全基因扩增,测序结果显示,3株病毒分离物全基因序列均由1767bp组成,直接来自病料与细胞分离毒株的核苷酸同源性为100%.基因组内含有11个ORF.通过比较发现,分离毒株与PCV2参考株的同源性介于94.2%～99.7%之间;与PCV1参考株的同源性为77.2%～77.9%.     

野猪、民猪、大白猪μ-钙激活酶基因的变异位点分析

为了进一步研究CAPN1基因变异与肉嫩度的关系, 寻找与猪嫩度性状相关的分子标记, 对CAPN1基因组进行了克隆、测序, 并利用PCR-SSCP方法对其编码区序列进行了分子扫描, 寻找多态位点, 分析不同基因型在野猪、民猪、大白猪中的种间分布规律。获得了猪CAPN1基因的15个内含子序列; 根据GenBank上提供的CAPN1 CDS及克隆的内含子序列设计了5对多态性引物进行PCR-SSCP分析; 共找到8个SNPs, 其中7个位于外显子上, 1个位于内含子上, 并且外显子上的突变有3个是错义突变, 分别造成了蛋白质多肽链上第54位氨基酸的S/T、第192位氨基酸的G/E、第363位氨基酸的V/I替代; χ²独立性检验表明不同基因型在大白猪与野猪、民猪之间存在着极显著的差异(P<0.01), 野猪和民猪之间除了S1引物3种基因型的分布存在显著差异外(0.010.05)。这些多态位点具有成为分子标记的潜在可能。     

Genome‐wide scans to detect positive selection in Large White and Tongcheng pigs

Due to the direction, intensity, duration and consistency of genetic selection, especially recent artificial selection, the production performance of domestic pigs has been greatly changed. Therefore, we reasoned that there must be footprints or selection signatures that had been left during domestication. In this study, with porcine 60K BeadChip genotyping data from both commercial Large White and local Chinese Tongcheng pigs, we calculated the extended haplotype homozygosity values of the two breeds using the long‐range haplotype method to detect selection signatures. We found 34 candidate regions, including 61 known genes, from Large White pigs and 25 regions comprising 57 known genes from Tongcheng pigs. Many selection signatures were found on SSC1, SSC4, SSC7 and SSC14 regions in both populations. According to quantitative trait loci and network pathway analyses, most of the regions and genes were linked to growth, reproduction and immune responses. In addition, the average genetic differentiation coefficient F_ST was 0.254, which means that there had already been a significant differentiation between the breeds. The findings from this study can contribute to further research on molecular mechanisms of pig evolution and domestication and also provide valuable references for improvement of their breeding and cultivation.     

Genome-wide association study identifies quantitative trait loci regions involved in muscle acidic profile in Large White heavy pigs

The widespread use of genome-wide association studies resulted in the discovery of genomic regions associated with fatty acid (FA) composition in different porcine tissues, but little information exists about the genes involved in FA composition of meat obtained from heavy pigs selected for the production of Italian dry-cured hams. To this objective, we genotyped with a single nucleotide polymorphism (SNP) panel 795 Italian Large White heavy pigs to identify the markers and genomic regions associated with Semimembranosus muscle FA profile. Heritability estimates for intramuscular fat FA profile were of low-to-moderate magnitude, suggesting that these traits may be improved with genomic selection. On the whole, 45 SNPs were significantly associated with 14 FAs, and 4 of them (ALGA008109, ALGA0081097, CASI0010164 and SIRI0000267) were associated with more than 1 FA. The palmitoleic : palmitic and oleic : stearic ratios displayed the highest number of significant markers and the most significant associations (Bonferroni adjusted P < 5.00E−07). Of particular interest, the palmitoleic : palmitic ratio was strongly associated with markers located at 111 to 114 Mb on chromosome 14, in the same chromosomal region where Stearoyl-CoA desaturase Δ9 (SCD) gene is located. Several significant chromosomal regions were found; some of them harbour key genes playing pivotal roles in FA desaturation and elongation, such as SCD and some members of the Elongation of Very Long-Chain FA (ELOVL) gene family. The results suggest that the identification of causal mutations in these regions may provide a set of markers useful for selection schemes aimed at improving FA composition in pork products.     

Integrating economic parameters into genetic selection for Large White pigs

The objective of the study was to integrate economic parameters into genetic selection for sow productivity, growth performance and carcass characteristics in South African Large White pigs. Simulation models for sow productivity and terminal production systems were performed based on a hypothetical 100-sow herd, to derive economic values for the economically relevant traits. The traits included in the study were number born alive (NBA), 21-day litter size (D21LS), 21-day litter weight (D21LWT), average daily gain (ADG), feed conversion ratio (FCR), age at slaughter (AGES), dressing percentage (DRESS), lean content (LEAN) and backfat thickness (BFAT). Growth of a pig was described by the Gompertz growth function, while feed intake was derived from the nutrient requirements of pigs at the respective ages. Partial budgeting and partial differentiation of the profit function were used to derive economic values, which were defined as the change in profit per unit genetic change in a given trait. The respective economic values (ZAR) were: 61.26, 38.02, 210.15, 33.34, −21.81, −68.18, 5.78, 4.69 and −1.48. These economic values indicated the direction and emphases of selection, and were sensitive to changes in feed prices and marketing prices for carcasses and maiden gilts. Economic values for NBA, D21LS, DRESS and LEAN decreased with increasing feed prices, suggesting a point where genetic improvement would be a loss, if feed prices continued to increase. The economic values for DRESS and LEAN increased as the marketing prices for carcasses increased, while the economic value for BFAT was not sensitive to changes in all prices. Reductions in economic values can be counterbalanced by simultaneous increases in marketing prices of carcasses and maiden gilts. Economic values facilitate genetic improvement by translating it to proportionate profitability. Breeders should, however, continually recalculate economic values to place the most appropriate emphases on the respective traits during genetic selection.     

The classical hitchhiking model with continuous mutational pressure and purifying selection

Detecting selective sweeps driven by strong positive selection and localizing the targets of selection in the genome play a major role in modern population genetics and genomics. Most of these analyses are based on the classical model of genetic hitchhiking proposed by Maynard Smith and Haigh (1974, Genetical Research, 23, 23). Here, we consider extensions of the classical two‐locus model. Introducing mutation at the strongly selected site, we analyze the conditions under which soft sweeps may arise. We identify a new parameter (the ratio of the beneficial mutation rate to the selection coefficient) that characterizes the occurrence of multiple‐origin soft sweeps. Furthermore, we quantify the hitchhiking effect when the polymorphism at the linked locus is not neutral but maintained in a mutation‐selection balance. In this case, we find a smaller relative reduction of heterozygosity at the linked site than for a neutral polymorphism. In our analysis, we use a semi‐deterministic approach; i.e., we analyze the frequency process of the beneficial allele in an infinitely large population when its frequency is above a certain threshold; however, for very small frequencies in the initial phase after the onset of selection we rely on diffusion theory.     

The genomic basis of adaptation to high‐altitude habitats in the eastern honey bee (Apis cerana)

The eastern honey bee (Apis cerana) is of central importance for agriculture in Asia. It has adapted to a wide variety of environmental conditions across its native range in southern and eastern Asia, which includes high‐altitude regions. eastern honey bees inhabiting mountains differ morphologically from neighbouring lowland populations and may also exhibit differences in physiology and behaviour. We compared the genomes of 60 eastern honey bees collected from high and low altitudes in Yunnan and Gansu provinces, China, to infer their evolutionary history and to identify candidate genes that may underlie adaptation to high altitude. Using a combination of F_ST‐based statistics, long‐range haplotype tests and population branch statistics, we identified several regions of the genome that appear to have been under positive selection. These candidate regions were strongly enriched for coding sequences and had high haplotype homozygosity and increased divergence specifically in highland bee populations, suggesting they have been subjected to recent selection in high‐altitude habitats. Candidate loci in these genomic regions included genes related to reproduction and feeding behaviour in honey bees. Functional investigation of these candidate loci is necessary to fully understand the mechanisms of adaptation to high‐altitude habitats in the eastern honey bee.     

Effect of temperature level on thermal acclimation in Large White growing pigs

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.     

Genome-wide association studies for iris pigmentation and heterochromia patterns in Large White pigs

Eye colour genetics have been extensively studied in humans since the rediscovery of Mendel’s laws. This trait was first interpreted using simplistic genetic models but soon it was realised that it is more complex. In this study, we analysed eye colour variability in a Large White pig population (n = 897) and report the results of GWASs based on several comparisons including pigs having four main eye colour categories (three with both pigmented eyes of different brown grades: pale, 17.9%; medium, 14.8%; and dark, 54.3%; another one with both eyes completely depigmented, 3.8%) and heterochromia patterns (heterochromia iridis – depigmented iris sectors in pigmented irises, 3.2%; heterochromia iridum – one whole eye iris of depigmented phenotype and the other eye with the iris completely pigmented, 5.9%). Pigs were genotyped with the Illumina PorcineSNP60 BeadChip and GEMMA was used for the association analyses. The results indicated that SLC45A2 (on chromosome 16, SSC16), EDNRB (SSC11) and KITLG (SSC5) affect the different grades of brown pigmentation of the eyes, the bilateral eye depigmentation defect and the heterochromia iridis defect recorded in this white pig population respectively. These genes are involved in several mechanisms affecting pigmentation. Significant associations for the eye depigmented patterns were also identified for SNPs on two SSC4 regions (including two candidate genes: NOTCH2 and PREX2) and on SSC6, SSC8 and SSC14 (including COL17A1 as candidate gene). This study provided useful information to understand eye pigmentation mechanisms, further valuing the pig as animal model to study complex phenotypes in humans.     

Genetic parameters for tissue and fatty acid composition of backfat, perirenal fat and longissimus muscle in Large White and Landrace pigs

Genetic parameters pertaining to the same chemical characteristics of three porcine tissues, that is backfat (BF), perirenal fat (PF) and longissimus muscle (LM), were estimated in centrally tested Large White and Landrace pigs. Animals were fed ad libitum. They were slaughtered at an average BW of 99.6 kg, and samples of BF (both inner and outer layers) and LM were removed at the 13th to 14th rib level of the carcass on the day after slaughter. The data set included 2483 animals recorded for average daily gain (ADG; 35 to 100 kg), estimated carcass lean percentage (LEAN) and lean tissue growth rate (LTGR). Among these animals, around 950 pigs were recorded for lipid content (L%) and water content (W%) of BF and LM and for fatty acid composition (FAC) of BF, whereas FAC of LM was measured on 297 pigs and L%, W%, and FAC of PF on around 210 pigs. Heritabilities (h2) and genetic correlations (ra) were estimated using REML-animal model methodology. Estimates of h2 for L%, W% and FAC of BF, PF and LM were of moderate-to-high magnitude: for example 0.47 ± 0.09 for L% of LM, 0.59 ± 0.11 for W% of BF, 0.45 ± 0.08 for the ratio of polyunsaturated to saturated fatty acids (P/S) of BF, 0.61 ± 0.15 and 0.29 ± 0.10 for the coefficient of unsaturation of lipids (UNSAT, average number of double bonds of unsaturated fatty acids) of PF and LM, respectively. Genetic correlations of L% with P/S or UNSAT were strongly negative (from -0.4 to -0.9) in BF and LM, but not in PF. The 'between-tissue' genetic correlations for homologous compositional traits were far from being unity (e.g. ra = 0.57 ± 0.05 'between' BF and PF for UNSAT). Genetic relationships between ADG and tissue compositional traits were globally weak. By contrast, genetic correlations were moderate-to-high between carcass leanness and tissue compositional traits, especially those of fat depots: for example -0.66 ± 0.14 between LEAN and L% of BF, 0.50 ± 0.07 between LEAN and UNSAT of PF, -0.44 ± 0.08 between LEAN and L% of LM, and 0.27 ± 0.03 between LEAN and UNSAT of LM. On the basis of the parameter estimates found here, breeding for higher LTGR is expected to increase the ratio of water to lipids and the unsaturation degree of lipids in subcutaneous BF and, to a lesser extent, in PF. Tissue composition and FAC of LM would be less affected.