应用全外显子组测序筛查一高原肺水肿家系易感基因

高原肺水肿(high-altitude pulmonary edema, HAPE)是一种高原特发性疾病,其发病与遗传因素有一定关联。本研究对一个HAPE相关的家系展开遗传学调查,然后利用外显子组测序筛查了包括先证者在内的6名HAPE病史成员以及先证者的母亲共7个成员的遗传变异,结果发现18个HAPE相关的潜在遗传变异(9个SNVs和9个Indels)。利用SIFT,PolyPhen-2和PROVEAN等3种软件对这些遗传变异进行蛋白功能危害性分析,结果发现定位于CFHR4基因的SNV(p.L85F)以及定位于OXER1基因的SNV(p.R176C)具有高危害性,且OXER1的功能与HAPE低氧诱导通路存在高度关联,它们可作为该家系中HAPE相关的候选病理性变异。此外,还有部分SNVs(NMB p.S150P、APOB p.I4194T和EIF4ENIF1 p.Q763P)以及Indels(KCNJ12 p.EE333-334E、ANKRD31 p.LMN251-253LN和OR2A14 p.HFFC175-178HFC),其遗传变异同样具有一定危害,可作为潜在的HAPE相关遗传变异。本研究首次通过外显子组测序直接筛选与一中国HAPE家系相关的遗传变异,为后续揭示HAPE发病机制提供了新线索。     

360度群体遗传变异扫描——大豆泛基因组研究

大豆(Glycine max)是重要的油料和蛋白作物, 其丰富的遗传变异为生物学性状挖掘和育种改良提供了重要的资源基础。然而, 单个基因组信息无法全面揭示种质资源的遗传变异, 泛基因组研究为解决这一不足提供了新方案。近日, 中国科学院遗传与发育生物学研究所田志喜和梁承志研究团队从2 898份大豆种质中选取26份代表性材料, 并整合已有的3个基因组, 构建了包含野生和栽培大豆的泛基因组和图基因组(graph-based genome), 鉴定了整个群体的绝大多数结构变异数据集, 确定了大豆种质的核心、非必需和个体特异的基因集。利用这些数据系统地揭示了生育期位点E3的等位基因变异和基因融合事件、种皮颜色基因I的单体型和演化关系以及结构变异对铁离子转运基因表达和地区适应性选择的影响。该研究为作物基因组学研究提供了一个新的模式, 同时将加速推动大豆遗传变异的鉴定、性状解析和种质创新。     

复杂疾病的遗传易感基因区域的精细定位

以单核苷酸多态性(Single-nucleotide polymorphism, SNP)为遗传标记, 采用全基因组关联研究(Genome-wide association studies, GWAS)的策略, 已经在660多种疾病(或性状)中发现了3800多个遗传易感基因区域。但是, 其中最显著关联的遗传变异或致病性的遗传变异位点及其生物学功能并不完全清楚。这些位点的鉴定有助于阐明复杂疾病的生物学机制, 以及发现新的疾病标记物。后GWAS时代的主要任务之一就是通过精细定位研究找到复杂疾病易感基因区域内最显著关联的易感位点或致病性的易感位点并阐明其生物学功能。针对常见变异, 可通过推断或重测序增加SNP密度, 寻找最显著关联的SNP位点, 并通过功能元件分析、表达数量性状位点(Expression quantitative trait locus, eQTL)分析和单体型分析等方法寻找功能性的SNP位点和易感基因。针对罕见变异, 则可采用重测序、罕见单体型分析、家系分析和负荷检验等方法进行精细定位。文章对这些策略和所面临的问题进行了综述。     

创伤弧菌毒力相关因子的全基因组关联分析研究

【目的】创伤弧菌是致死率最高的弧菌物种,但目前尚无在全基因组层面挖掘毒力相关因子的研究。本研究以创伤弧菌分离来源(临床和环境)作为不同表型,通过与260株基因组序列进行关联分析,挖掘毒力相关因子,从而进一步了解创伤弧菌致病因素。【方法】对139株创伤弧菌分离株进行高通量测序,获取其全基因组序列;与公共数据库已公开发表的121株基因组整合,使用pyseer软件进行全基因组关联分析,对与不同分离来源显著相关的基因进行注释和解读。【结果】共发现11个基因与临床分离株显著相关,其中9个是本研究新发现的创伤弧菌潜在毒力相关因子。【结论】本研究使用群体基因组学和统计遗传学方法,在全基因组范围扫描挖掘了创伤弧菌毒力相关因子,为深入揭示该物种致病机制、设计新的疫苗和治疗靶点提供了重要依据。     

新冠肺炎遗传易感基因的研究进展

新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)感染人体后,个体间存在显著不同的新冠肺炎(corona virus disease 2019,COVID-19)临床症状。机体遗传因素在新冠病毒感染后的临床转归过程中发挥重要的作用。以全基因组关联研究(genome-wide association studies, GWAS)为代表的遗传关联研究方法,已成功鉴定了多个与新冠肺炎相关的易感基因,为新冠肺炎防诊治措施的研发提供了理论基础。本文综述了新冠肺炎遗传易感基因的研究进展,包括多种表型、多个人群、多种遗传变异类型的新冠肺炎全基因组关联研究以及易感基因区域的精细定位研究等,旨在为新冠肺炎遗传易感基因的后续研究提供参考。     

1株梅花鹿源狂犬病街毒株全基因组测序与分析

对1株梅花鹿源性狂犬病街毒株(DRV)进行全基因组克隆,对全长cDNA进行测序分析.RT-PCR扩增克隆覆盖全基因组9个重叠基因片段,基因组3'和5'末端采取3,-RACE和5'-RACE方法,9个重叠基因片段序列拼接得到DRV全基因组cDNA序列,共11 863个核苷酸.DRV毒株全基因组构成与其他狂犬病毒基因组构成相似,由5个编码区组成,基因起始位点和终止位点高度保守,在核蛋白和糖蛋白的重要抗原位点有个别氨基酸发生变异,对已完成全基因组测序的几个基因1型毒株分别进行了N、P、M、G、L基因核苷酸及氨基酸的同源性比较.与其他具有代表性的毒株进行N基因序列比较建立的系统进化树表明,DRV毒株属于基因1型,与中国人用疫苗株3aG同源性最高为94%,与分类位置未确定的北高加索毒株(WCBV)的同源性最低为71%.本研究结果可为狂犬病毒各项分子生物学研究提供理论参考.     

生物信息学分析方法I: 全基因组关联分析概述

全基因组关联分析(GWAS)是动植物复杂性状相关基因定位的常用手段。高通量基因分型技术的应用极大地推动了GWAS的发展。在植物中, 利用GWAS不仅能够以较高的分辨率在全基因组水平鉴定出各种自然群体特定性状相关的基因或区间, 而且可揭示表型变异的遗传架构全景图。目前, 人们利用GWAS分析方法已在拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、小麦(Triticum aestivum)、玉米(Zea mays)和大豆(Glycine max)等模式植物和重要农作物品系中发掘出与各种性状显著相关的数量性状座位(QTL)及其候选基因位点, 阐明了这些性状的遗传基础, 并为揭示这些性状背后的分子机理提供候选基因, 也为作物高产优质品种的选育提供了理论依据。该文对GWAS的方法、影响因素及数据分析流程进行了详细描述, 以期为相关研究提供参考。     

普通菜豆镰孢菌枯萎病抗性种质资源筛选及全基因组关联分析

普通菜豆镰孢菌枯萎病是严重制约菜豆(Phaseolus vulgaris)产量的主要病害之一。采用下胚轴双孔注射法对601份普通菜豆种质资源进行枯萎病抗性鉴定, 共筛选出4份高抗材料。在此基础上, 基于分布在全基因组上的3 765 456个单核苷酸多态性(SNP)标记, 进行全基因组关联分析, 以P<1×10^-5为阈值。结果检测到57个显著关联的SNP位点, 分布于1、2、6、8和11号染色体上; 共获得8个显著关联区域, 其中位于1号染色体上的区域1包含SNP最多(48个), 最显著SNP P值为2.18E-07。在8个显著关联区域中, 共检测到186个基因, 其中157个基因有注释信息, 编码过氧化物酶、抗病蛋白、转录因子和蛋白激酶等。结合KEGG富集分析和序列同源性比对, 鉴定出9个候选基因可能与抗性相关。     

重伞灵芝线粒体基因组在灵芝种间鉴定中的应用

线粒体是“动力工厂”,能够进行氧化代谢实现能量的转化,参与三羧酸循环形成ATP。随着分子生物学和生物信息学技术的发展,担子菌灵芝属中已有几种灵芝的线粒体基因组被相继报道,但尚未有对重伞灵芝线粒体基因组的报道。本研究通过对重伞灵芝YX线粒体基因组进行组装注释,比较分析了与其他灵芝属物种的差异,根据差异序列构建分子标记对重伞灵芝菌株快速鉴定。结果显示重伞灵芝线粒体基因组大小为67 340bp的闭合环形结构,含有15个普通蛋白编码基因,28个tRNA基因,以及rRNA的大小亚基基因。共5个基因含有12个内含子,主要为IB型,部分为ID型,包含LAGLIDADG_1 superfamily、GIY-YIG_Cterm superfamily保守结构域。根据重伞灵芝线粒体cox2基因设计的特异性引物扩增结果显示：4株重伞灵芝的cox2基因片段均可准确扩增,且扩增不出其他灵芝物种的cox2基因片段。基于各灵芝菌株线粒体cox2基因序列构建系统进化树,可以将4株重伞灵芝归在同一分支上,并且同源性为98%,与ITS鉴定结果一致。可见,基于线粒体基因cox2特异性引物可以快速地对重伞灵芝进行鉴定,且只需通过观察扩增条带的有无,无需测序,省时省力。     

基于全基因组关联的中国常见肿瘤遗传病因学研究

肿瘤是基因-环境交互作用引起的复杂性疾病.在同样的环境暴露下,不同遗传背景的个体发生肿瘤的风险有很大差异.研究肿瘤相关遗传因素对理解肿瘤发生发展乃至诊断治疗都有重要意义.近年来发展的全基因组关联研究(genome-wide association study,GWAS)可在全基因组范围内发现与复杂疾病或表型关联的遗传因素,为复杂疾病遗传学研究提供了强有力的手段.欧美研究者运用全基因组关联研究的方法,对各种常见肿瘤进行了研究,获得了重要成果.2010年以来,中国科学家在国际核心期刊发表了一系列高水平的肿瘤全基因组关联研究成果,在中国常见肿瘤的遗传病因学研究方面取得了重要进展.     

Testosterone and estradiol concentrations in serum, velvet skin, and growing antler bone of male white-tailed deer

The growth and mineralization of antlers correlate with the seasonal variation of serum androgens. Whereas seasonal levels of testosterone (T) in plasma are well established, steroid concentrations have not yet been determined in the tissues of growing antlers. Therefore, RIA was used to determine T and 17beta estradiol (E2) in serum, and three areas (tip, middle, and base) of the antler bone and the antler skin, called velvet. Blood and antler tissues of white-tailed deer (Odocoileus virginianus) were collected from May to August. The difference between levels of T and E2 among the sites was calculated using the square root transformation followed by a mixed model analysis with individual deer and an interaction of individual and year (individual(*)year) as a random factor. Concentrations of T in serum (799+/-82 pg/ml) were higher than T values in the velvet (589+/-58 pg/ml, P<0.01) and in the antler bone (538+/-58 pg/ml, P<0.001). Estradiol concentrations differed among antler tissues and serum (P<0.001) and between years (P<0.01). Estradiol concentrations in serum (25+/-25 pg/ml) were consistently lower than those in antler bone (208+/-11 pg/ml, P<0.001) and velvet (150+/-12 pg/ml, P<0.001). The E2:T ratio in serum was 1:10-60. The same ratio for the antler bone was only 1:2-3 and for the velvet 1:3.5. It is concluded that higher T and lower E2 concentrations found in plasma, as compared to antler bone or antler velvet, may indicate a partial metabolism of systemic androgens into estrogens xin the tissues of growing antlers.     

Gene expression of axon growth promoting factors in the deer antler

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin.     

Seasonal variations in red deer (Cervus elaphus) hematology related to antler growth and biometrics measurements

The aim of the study was to relate seasonal hematology changes with the rest of physiological variations suffered by red deer, such as antler and biometrics cycle, and to assess the relationship between hematology and the effort performed in antler development. Blood samples were taken from 21 male red deer every 4 weeks during 18 months. Samples were analyzed for the main hematological parameters. Simultaneously, biometrics measurements were taken, such as antler length, body weight, body condition score, testicular diameter (TD), and thoracic and neck girth. All the blood cell types (erythrocytes, leukocytes, and platelets) showed seasonal variations, increasing as antler cleaning approached, as did hematocrit and hemoglobin. The final size of antlers was negatively related to leukocyte count, nonlymphoid leukocyte count, red cell distribution width, mean corpuscular hemoglobin, mean platelet volume, and TD, whereas it was positively related to body condition during antler growth. Huge seasonal variations in some hematological values have been found to be related to changes in antler and biometrics measurements. Since these variations are even greater than the caused by deer handling, they should be taken into account when evaluating hematology in deer populations.     

Effects of selective harvest on antler size in white-tailed deer: A modeling approach

Selective harvesting in wild deer (Odocoileus spp.) populations is a common practice that may influence antler size. However, in free-ranging populations, response due to selection is unknown or difficult to quantify because antlers are influenced by nutrition and population demographics. We used quantitative genetic models to predict how white-tailed deer (O. virginianus) antlers would respond to selection and what variables (i.e., population size, age structure, mating ratio, and heritability) most affected antler size. We validated our quantitative genetics program by comparing model results with a population of deer used for controlled breeding experiments; modeled antler points (AP) and score increased (2.2–4.3 AP and 48.5–97.7 cm, respectively) after 8 years of selection, similar to observed increases in AP (3.2) and score (92.3 cm) from the controlled population. In modeled free-ranging populations, mating ratio, age structure, and heritability were more important in influencing antler size than size of the population. However, response to selection in free-ranging populations was lower (0.1–0.9 AP) than controlled breeding populations even after 20 years of selection. These results show that selective harvesting of free-ranging white-tailed deer may be inefficient to change population-level genetic characteristics related to antler size. Response of antlers in free-ranging deer will be less than controlled populations, and possibly modeled free-ranging simulations, because individual reproductive success of males is lower, breeding is done by a large group of males, and reproductive and survival rates are lower. These factors, and others, reduce the amount of improvement that can be made to antlers due to selection. Therefore, selective harvesting in free-ranging populations should be justified for managing population demographics and dynamics, but not for changing the genetic characteristics of populations. © 2011 The Wildlife Society.     

麋鹿鹿角生长周期及影响因子

2013年8月至2014年8月,使用望远镜(SWAROVSKI 8×42 WB)和照相机(Canon 550D 70×300)观察北京南海子59只雄性麋鹿茸的生长和角脱落周期,发现麋鹿角总体趋势呈随着年龄的减少,脱角日期越迟,且等级序位高或者鹿王较年老个体先脱落。收集2012年12月至2014年2月两年度麋鹿自然脱落的角,研究表明,个体而言,左右角脱角顺序差异明显,同一天脱落两角者占17.0%,左角先脱落者占34.1%,右角先脱落者占48.8%,重量重和重量轻的角先脱落的个体各占41.5%,个体成对的左右角脱落时间间隔短,平均为1.98d;群体而言,角重量与脱角时间呈显著负相关,总体呈现角重的个体角先脱落,但不是角最重的个体角第一个脱落。2013年6月至2014年5月,对北京南海子、湖北石首、江苏大丰、天津七里海、浙江慈溪、浙江临安、海南海口、河北滦河上游、辽阳千山等9个地区麋鹿种群麋鹿角脱落起止时间进行调查,研究表明野生种群脱角时间比圈养种群早;迁入热带地区海南的麋鹿仍有规律的角周期;同一栖息地不同年份角周期存在差异,不同栖息地间麋鹿角周期存在差异;脱角起始时间与海拔、经度、纬度、年平均气温、圈养情况和气候类型等影响因子不存在统计学上显著的相关性,但光周期和激素直接主导和调节着角周期,年龄大小、角重量、营养以及与营养相关的能量、种群密度、物候等环境因子均影响角周期。     

Regrowth of amputated velvet antlers with and without innervation

The influence of removing portions of the growing antler of yearling red deer stags on subsequent regeneration of the antler in the same season was studied. The influence of the innervation of the antler on such regeneration was the subject of a further study. When the top 0.5-1 cm was removed from antlers 9-17 cm long, growth was slightly reduced in that season. When the antler/pedicle length was reduced to 6-10 cm in antlers 16-38 cm long, branched antlers regrew in 11 out of 13 cases provided the amputation was carried out early in the growing season, i.e., before mid-December. Denervated antlers were shorter, lighter, and of different shape compared with controls, but they were of similar density. Denervation was confirmed histologically. Cleaning of velvet and casting of antlers following castration were unaffected by denervation. It would appear that although nerves affect the size and shape of the antler, they are not essential to the actual control of antler growth and regeneration.     

Climate-dependent allocation of resources to secondary sexual traits in red deer

Fitness in highly polygynous male ungulates is related both to body size, weight and antler size. Males must therefore allocate resources both to growth of the body and growth of the antlers, which may lead to tradeoffs whenever resource levels are in limited supply. Several studies have reported how (absolute) growth of antlers and horns are related to environmental conditions, but few have looked for the relative allocation patterns (i.e. relative to body size and weight). We analyzed how the influence of variation in climate (the North Atlantic oscillations, NAO) and population density affected the allocation of resources to antlers, based on data from 2720 red deer stags two years or older harvested during 1965–2002 along the west coast of Norway. Number of antler tines increased up to six years of age, remained stable until 12 years of age, and then decreased significantly (>12 years, n=45). The NAO was positively related to number of antler tines in prime aged males (6–12 years, n=629), also after controlling for both body size and weight. Our study thus suggests that deer have a higher allocation of resources to antlers relative to body weight during favorable environmental conditions.     

Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis)

Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (+/-S.E.M.) testicular volume (118.8+/-4.6 cm(3)), serum testosterone concentration (1.2+/-0.1 ng/mL), semen volume (4.1+/-0.6 mL), sperm concentration (338.3+/-24.9 x 10(6) mL(-1)), percentage of morphologically normal sperm (79.1+/-2.8%), and percentage of motile sperm (66.5+/-1.5%) were higher (P<0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March-May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India.     

Seasonal antler cycle in a herd of pampas deer (Ozotoceros bezoarticus) in Uruguay

To describe the yearling antler cycle of pampas deer bucks (Ozotoceros bezoarticus) 22 bucks and 72 antler cycles have been studied in a semi-captive population. Date of individual antler cast, first day on which the brow and the trez tine were observed, and day of the velvet shedding were registered. Time intervals (days) between first and second antler casts, between first antler cast and the brow tine observation, between brow and trez tines observation and from first antler cast to velvet shedding were calculated. The brow and the trez tine were first observed 22.8±0.6 and 45.9±0.9 days after the first antler cast. Velvet shedding was observed 103.3±2.1 days after the first antler cast. Both antlers casts were observed earlier in adult cycle bucks than in first antler bucks, although the interval between both casts was not different. The interval from the brow tine observation to the trez tine observation was shorter in first antler cycle bucks than in adults. As in other deer species, antler cycle was seasonal, but persistence of cycle-to-cycle antler growth well into adulthood is different from what occurs in most deer species.     

Induction of deer antlers by transplanted periosteum. I. Graft size and shape

When discs of frontal periosteum from presumptive antler sites of 6-8 month old male fawns of the fallow deer are grafted beneath the foreleg skin, they will differentiate into pedicle bones and induce small antlers in the overlying integument. These antlers shed their velvet in the fall, and in succeeding years are replaced by larger outgrowths not exceeding 7 cm in length. Periosteal transplants 1.5 cm in diameter gave rise to ectopic antlers in 100% of the grafts, while discs measuring 1.05 cm, 0.75 cm and 0.4 cm did so in only 20% of the cases. Conversely, the donor sites produced antlers in 20-23% of the cases following removal of 1.05 cm or 1.5 cm of periosteum, while 80% and 100% grew antlers after deletions of 0.75 cm and 0.4 cm discs of periosteum, respectively. Semicircular grafts of periosteum induced antler development in most cases, especially when derived from the lateral halves of the antlerogenic region on the frontal bone. These findings confirm that the histogenesis of a deer's first pedicle and antler resides in the frontal periosteum over an area about 1.5 cm wide. They also show that leg skin is capable of antlerogenic development under the inductive influence of frontal periosteum, and that integumental wounding may enhance inductive interactions.