基于甘蔗热带种(LA-purple)全基因组序列的SSR开发及特征分析

现代甘蔗栽培品种(2n=100~130)是由甘蔗热带种(2n=80)与割手密(2n=40~128)种间杂交而来,形成异源多倍体、非整倍体作物,使得甘蔗栽培品种中80%~90%的染色体来源于热带种。开发热带种基因组SSR分子标记,有助于甘蔗遗传多样性分析、分子标记辅助选择、遗传图谱的构建等。本研究基于热带种LA-purple的全基因组测序数据的255 398个预测基因序列(累计总长为1 029 222 285 bp),利用Perl程序与生物信息学软件结合,发掘SSR位点,获得了153 150个SSR位点,平均每1.67个基因有1个SSR位点,其中二、三核苷酸重复基序分别为39 556个和50 072个,占总SSR位点数的58.5%。在二核苷酸重复基序中,TA/AT所占比例最高,占41.4%,CG/GC所占比例最低,占4.6%;在三核苷酸碱基重复基序中,TGT/ACA所占比例最高,为15.6%。在TA/AT重复类型中选取100个基序重复次数在60~90之间的SSR位点,进行引物设计与合成,在12个甘蔗属材料中进行PCR扩增分析,从中筛选出52对具有多态性SSR引物,其中有27对引物在研究的2个甘蔗栽培品种间表现为多态。这些基因组SSR标记的开发,不仅可以用于甘蔗栽培品种DNA指纹图谱分析,而且为甘蔗属不同种的遗传图谱构建、遗传多样性分析和重要性状的遗传机制解析奠定基础,为甘蔗分子育种研究提供重要支撑。     

Genomewide association analysis of salt tolerance in soybean [Glycine max (L.) Merr.]

Salinity is a common abiotic stress causing soybean [Glycine max (L.) Merr.] yield loss worldwide. The use of tolerant cultivars is an effective and economic approach to coping with this stress. Towards this, research is needed to identify salt‐tolerant germplasm and better understand the genetic and molecular basis of salt tolerance in soybean. The objectives of this study were to identify salt‐tolerant genotypes, to search for single‐nucleotide polymorphisms (SNPs) and QTLs associated with salt tolerance. A total of 192 diverse soybean lines and cultivars were screened for salt tolerance in the glasshouse based on visual leaf scorch scores after 15–18 days of 120 mM NaCl stress. These genotypes were further genotyped using the SoySNP50K iSelect BeadChip. Genomewide association mapping showed that 62 SNP markers representing six genomic regions on chromosomes (Chr.) 2, 3, 5, 6, 8 and 18, respectively, were significantly associated with salt tolerance (p < 0.001). A total of 52 SNP markers on Chr. 3 are mapped at or near the major salt tolerance QTL previously identified in S‐100 (Lee et al., 2014). Three SNPs on Chr. 18 map near the salt tolerance QTL previously identified in Nannong1138‐2 (Chen, Cui, Fu, Gai, & Yu, 2008). The other significant SNPs represent four putative minor QTLs for salt tolerance, newly identified in this study. The results above lay the foundation for fine mapping, cloning and molecular breeding for soybean salt tolerance.     

小麦白粉菌ADP/ATP载体蛋白基因的克隆及表达特征分析

小麦白粉病是小麦上的一种重要病害。研究小麦白粉菌致病相关基因及其在致病过程中的作用,对于丰富小麦白粉菌致病的分子机制和筛选防治新靶标具有重要意义。前期转录组测序结果提示一个小麦白粉菌ADP/ATP载体蛋白(ADP/ATP carrier protein,AACP)在小麦与白粉菌互作时上调表达。本研究利用cDNA末端快速克隆技术(rapid-amplification of cDNA ends,RACE)获得了长945 bp具有完整开放阅读框的小麦白粉菌ADP/ATP carrier基因序列,暂命名为Bgt AACPx(Gen Bank登录号为M F197857)该基因编码314个氨基酸残基,利用相关软件进行生物信息分析,结果表明该蛋白为疏水性,含有6个跨膜区,亚细胞定位在线粒体上。与已有小麦白粉菌AACP蛋白的同源性为97%,是一个新的蛋白,同时构建了与其同源蛋白的遗传进化树。定量结果表明该基因在小麦白粉菌吸器期(48 h)、次生菌丝(72 h)至白粉菌刚产孢子又未形成孢子堆(120 h)这段时期高表达,提示该基因可能参与小麦白粉菌对小麦的致病过程。     

11种鲈形目鱼类ITS2特征及系统应用

ITS2 (Internal transcribed spacer 2)是位于核糖体5.8S和28S基因之间的非编码序列。为了探讨该片段的多样性特征以及进化模式,本研究选取了鲈形目(Perciformes) 5科11种鱼类为研究对象,共获得了444条ITS2克隆序列,其长度范围为332～515 bp。比较种内不同序列的长度发现,金带细鲹(Selaroides leptolepis)在种内存在24 bp的差异,剑鱼(Xiphias gladius)在种内存在32 bp的差异,这2种鱼类的差异较为明显;其余9种鱼类的长度相对比较保守,长度差异小于14 bp。依据11种鱼类的保守位点数、变异位点数、简约信息位点数、单倍型数、保守位点比例、单倍型多样性指数、核苷酸多样性等特征分析发现,种内存在着不同程度差异,特别是金带细鲹的ITS2序列存在着Type A、Type B和Type C 3种类型,各类型间差异较大。根据序列的多样性特征推断,金带细鲹和剑鱼的进化方式为非协同进化;蓝圆鲹(Decapterus maruadsi)、大甲鲹(Megalaspis cordyla)、吉打副叶鲹(Alepes djedaba)和日本竹筴鱼(Trachurus japonicas)的长度和变异位点均存在着一定程度的差异,但差异并不明显,视为不严格的协同进化;泰拉鰆鲹(Scomberoides tala)、布氏鲳鲹(Trachinotus blochii)、尖吻鲈(Lates calcarifer)、射水鱼(Toxotes chatareus)和军曹鱼(Rachycentron canadum) 5种鱼类为协同进化;另外,协同和非协同进化状态与分类系统没有相关性。序列比对发现,大甲鲹种内存在着由协同进化方式演变为非严格的协同进化方式的过度序列;在金带细鲹的3个不同个体中,序列间存在着从协同进化、非严格的协同进化演变为非协同进化的3种进化方式。基于ITS2序列构建的11种鱼类的邻接系统树显示,每种鱼类的克隆都分别按种单独聚为一支,鲹科7属鱼类各属也是单独聚支,表明ITS2不仅可以用在种类的分子鉴定,同时也可以作为分子标记应用于鲹科和属级水平的系统关系研究。     

Silicon-mediated genotoxic alterations in Brassica juncea under arsenic stress: A comparative study of biochemical and molecular markers

Arsenic (As), one of the most harmful toxicant at the global level, severely affects plant metabolism when taken up. Interestingly, the presence of silicon (Si) as a fertilizer in As-contaminated soil is an effective strategy to decrease As accumulation in plants. Brassica juncea (var. Varuna) were grown hydroponically to investigate the role of Si at biochemical and molecular levels under arsenite (As³⁺) stress. Seedlings of B. juncea were exposed to As³⁺, Si, and a combination of both elements. Our data demonstrated that seedlings exposed to As³⁺ showed an inhibition in shoot length, chlorophyll, carotenoid, and protein, while co-application of Si improved these growth parameters. Silicon supplementation reduced As accumulation in shoot. Increase/decrease was observed in stress-related parameters (cysteine and proline), antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase), and oxidative stress markers (malondialdehyde and H₂O₂), which were improved upon co-application of Si as compared to As³⁺ alone treatment. Random amplified polymorphic DNA (RAPD) is a suitable biomarker assay for plants for assessing the genotoxicity. Seven RAPD primers produced a total of 39 and 48 bands in the leaves of the untreated and treated seedlings, respectively. The RAPD band-profiles and genomic template stability were consistent with other growth and physiological parameters. In conclusion, the genotoxic alterations along with the biochemical parameters indicate that the exposure to Si mitigates As³⁺-induced oxidative stress by improving the stress-related parameters and antioxidant system in B. juncea.     

绵羊Izumo1基因多态性及其与产羔数关联分析

为研究Izumo1基因多态性及与产羔数关系,本研究利用PCR和直接测序法对小尾寒羊和苏尼特羊的Izumo1基因DNA序列进行扩增、测序和BLAST分析,并和本课题组前期绵羊重测序数据进行比对,筛选出Izumo1基因SNP位点。同时,采用Sequenom MassARRAY~?技术进行基因分型,并对其SNP位点的基因型和等位基因频率在各群体中的分布进行研究。结果表明:小尾寒羊Izumo1基因DNA全长序列3 385 bp,苏尼特羊Izumo1基因DNA全长序列3 382 bp;筛选出8个Izumo1基因SNP位点,经过初步筛选,将在小尾寒羊、苏尼特羊、滩羊、萨福克羊、杜泊羊、草原藏羊这6个绵羊品种中位点分布没有差异的SNP位点排除,最终筛选获得的g.54412135AG和g.54412107CA 2个位点。Izumo1基因g.54412135AG位点在多羔和单羔绵羊品种中存在GG、GA和AA 3种基因型,G基因为优势等位基因;g.54412107CA在多羔品种中存在CC和CA 2种基因型,而在单羔品种中存在CC、CA和AA 3种基因型,C基因为优势等位基因,其基因型频率和基因频率在单、多羔绵羊群体间的分布差异均极显著(P0.01);群体遗传学分析得出g.54412135AG多态位点在6个品种中的多态信息含量(PIC)都属于低度多态(PIC0.25);g.54412107CA多态位点在杜泊羊中属于中度多态(0.25PIC0.5),在其他5个品种中都属于低度多态(PIC0.25),然而,关联分析发现Izumo1基因g.54412135AG和g.54412107CA位点的不同基因型与小尾寒羊不同胎次产羔数之间不存在显著关联(P0.05)。     

鱼类组蛋白核苷酸的多态性及其在杀鱼爱德华氏菌感染中的作用

组蛋白是染色体核小体的重要组分,在调控染色质结构、基因转录、个体发育等不同生物学过程中起着重要作用。为了研究鱼类组蛋白基因是否存在核苷酸的多态性以及组蛋白核苷酸多态性是否会影响鱼类的抗病力,本实验以斑马鱼和草鱼为研究对象,通过PCR扩增克隆了组蛋白H2A的全长开放阅读框;利用过表达技术、菌落平板计数、感染存活分析以及荧光定量PCR技术,研究了斑马鱼和草鱼组蛋白H2A核苷酸多态性不同变异体在杀鱼爱德华氏菌感染中的作用。在本研究中,实验发现鱼类组蛋白H2A存在着丰富的核苷酸多态性。序列分析结果显示斑马鱼和草鱼组蛋白H2A核苷酸多态性的变异体核苷酸序列相似性为90%～100%;而两两H2A核苷酸多态性变异体氨基酸序列之间最多只有3个位点存在差异。通过体内和体外抗菌实验可知,斑马鱼和草鱼组蛋白H2A核苷酸的多态性显著影响H2A的抗菌活性。此外,筛选出的抗菌组蛋白H2A核苷酸多态性的变异体在斑马鱼体内的过表达,不仅具有免疫增强的作用,还能显著增强斑马鱼对杀鱼爱德华氏菌感染的抗病力。本研究为筛选具有抗病作用的组蛋白H2A免疫保护原奠定了重要基础。     

重组酶聚合酶等温扩增技术在动物源性成分鉴定中的应用

在打击肉制品掺假走私、维护市场秩序、保护野生动物资源等方面,对动物源性成分鉴定技术的特异性、灵敏性、重复性提出了很高的要求。本文阐述了动物源性成分的检测鉴定技术,有多样性和局限性的特点。归纳了重组酶聚合酶恒等温扩增技术原理与应用,解决了PCR技术在仪器上的局限性,优化了早期恒温扩增技术的缺点。指出重组酶聚合酶恒等温扩增技术为动物源性成分的快速准确鉴定提供了坚实的技术基础,并在动物疫病诊断方面也有很好的应用前景。     

Three SNPs within exons of INHA and ACVR2B genes are significantly associated with litter size in Dazu black goats

The aim of this study was to analyse the association between single-nucleotide polymorphisms within INHA and ACVR2B and litter size in Dazu black goats. In total, twenty-two SNPs were genotyped in 190 individuals by SNaPshot and resequencing. The results showed that three SNPs (SNP_1, SNP_12 and SNP_13 in this study) were detected to have significant additive genetic effect on the recorded goat litter size (p < .05). The SNP_1 (NC_030809.1), a non-synonymous substitution of G for T at chr2-g. 28314990 in the exon 2 of INHA gene (NM_001285606.1), resulted in homozygote 2 (HOM2) contributed 0.25 and heterozygote (HET) contributed 0.12 larger litter than homozygote 1 (HOM1). Meanwhile, SNP_12 (Chr22-g. 11721225 A > T) and SNP_13 (Chr22-g. 11721227 A > C) (NC_030829.1) simultaneously mutated at the first and third position of a triplet AAA (lysine, K) in the exon 4 of ACVR2B gene (XM_018066623.1) had estimated genetic effects of HOM1 (0.00) and HOM2 (0.03) larger than HET (−0.12). In conclusion, one SNPs (chr2-g. 28314990 T > G) within the exon 2 of INHA and two SNPs (Chr22-g. 11721225 A > T and Chr22-g. 11721227 A > C) i n the exon 4 of ACVR2B gene were highly recommended as candidate markers of litter size in Dazu black goats. A large-scale association study to assess the impact of these variants on litter size is still necessary.     

5个肉用绵羊品种血液蛋白(酶)遗传多态性及聚类分析

采用垂直板不连续聚丙烯酰胺凝胶电泳技术对170只甘肃肉用绵羊(波德代羊、无角陶赛特羊、蒙古羊、滩羊和小尾寒羊)的4种血液蛋白(Hb、Alb、Tf和Es)进行多态性检测。计算4种蛋白各位点的基因型频率、等位基因频率和遗传杂合度,并通过聚类分析探讨不同群体间的遗传距离和亲缘关系。结果表明,4个蛋白(酶)位点均表现出不同程度的多态性。4个蛋白(酶)位点的平均杂合度分别为:波德代羊0.4341,无角陶赛特羊0.4176,蒙古羊0.339 3,滩羊0.332 5和小尾寒羊0.371 0。根据Nei氏标准遗传距离进行聚类分析,发现无角陶赛特羊和波德代羊亲缘关系最近,和滩羊亲缘关系最远;3个地方品种和2个引入品种间亲缘关系较远。