大口黑鲈肌肉生长抑制素基因单核苷酸多态性位点的筛选及其与生长性状关联性分析

采用PCR-SSCP和PCR-RFLP技术对大口黑鲈肌肉生长抑制素(myostatin,MSTN)基因全序列进行了SNPs位点筛选和分型,共筛选到2个单核苷酸多态性(SNPs)位点(C-1453T和T+33C),其中C-1453T位于启动子E8 box和Octamer(＋)调控元件之间的区域,T+33C的突变位于第一外显子区域,属于同义突变,氨基酸没有发生变化;利用一般线性模型分析单标记位点与大口黑鲈生长性状(体重、体长、体高、体宽和眼间距)相关性,均未达到显著水平(P>0.05)。将2个SNPs位点不同基因型组合成6种双倍型(去掉频率小于3%的组合),关联分析表明,双倍型D2在体重、体长、体高、体宽和眼间距的均值均高于其它双倍型,而双倍型D5在体重、体长、体高、体宽和眼间距的均值均低于其它双倍型,双倍型D2与D5之间在5个主要生长性状均存在差异显著(P＜0.05),推测双倍型D2对生长性状起正相关,而双倍型D5与大口黑鲈的生长性状呈负相关,因此推断MSTN基因突变位点双倍型D2与D5可作为大口黑鲈生长性状的两个标记位点,用其分子标记位点来辅助大口黑鲈育种工作以期加快育种进程。     

Characterization of single‐nucleotide polymorphism markers in the Mediterranean mussel,Mytilus galloprovincialis

The Mediterranean mussel, Mytilus galloprovincialis, is one of the most important aquaculture species in Europe. Appropriate molecular markers are required to evaluate genetic resources and to trace genealogies in breeding programmes for improving mussel culture. Microsatellites have been commonly applied to this purpose in other species. However, Mediterranean mussel microsatellites have demonstrated high frequencies of null alleles that hamper accurate estimates of population parameters and confident parentage inferences. As alternative markers, we have characterized in silico 25 potential single‐nucleotide polymorphism (SNP) markers in the Mediterranean mussel from expressed sequence tag (EST) public databases. The genotyping of SNPs was performed using a single‐base extension approach. Their polymorphism was evaluated in 47 individuals from an Atlantic population. Out of the 25 potential SNPs tested, 12 were technically feasible (producing a single amplicon) and polymorphic. All were biallelic and had an unbiased heterozygosity ranging from 0.160 to 0.504. One SNP was from a mitochondrial gene. The combined potential of nuclear SNPs for parentage assignment gave an exclusion probability of a false couple of parents of 0.9471. These markers will be useful for evaluating resources and tracing genealogies in genetic breeding programmes implemented to solve the main problems of mussel culture.     

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。     

刺参(Apostichopus japonicus)重要经济性状相关SNP标记的验证分析

本研究在前期构建的刺参(Apostichopus japonicus)高密度遗传连锁图谱和QTL分析的基础上,筛选出26个与体长、体重、体宽、棘刺总数、抗病力相关的SNP位点,设计出可用于HRM检测的SNP扩增引物13对。在扩大群体中利用HRM小片段法对这13个刺参重要经济性状相关的候选SNP位点进行分型和多态性检测,并结合扩大群体的相关性状数据进行了QTL位点验证。多态性结果显示,13个位点中有3个单态性位点,其余10个多态性位点中有3个位点为低等位多态性,7个位点为中等位多态性。10个多态性位点的最小等位基因频率(MAF)介于0.016(SNP113)~ 0.332(SNP160)之间,平均值为0.173;各位点的观测杂合度(Ho)介于0.031(SNP113)~0.818(SNP9)之间,平均值为0.433;期望杂合度(He)介于0.031(SNP113)~0.834(SNP9)之间,平均值为0.402;多态信息含量(PIC)介于0.030(SNP113)~0.393(SNP160)之间,平均为0.248,有6个位点偏离Hardy- Weinberg平衡。QTL验证结果表明,SNP40和SNP160位点为与生长(体长、体重、体宽)相关的位点,各位点的优势基因型分别为SNP40(CC)和SNP160(AA);SNP88、SNP112和SNP126这3个位点为与抗病力相关的位点,各位点的优势基因型为SNP88(CC)、SNP112(AA)和SNP126(TT)。基于这5个位点构建生长和抗病二倍型,发现二倍型K1(CC AA TT)抗病力最强,S1(CC AA)、S3(CC AC)在生长方面优势显著,相关研究结果可为分子标记辅助育种在生产中应用提供基础数据。     

Extractive components and taste aspects of fermented fish pastes and bean pastes prepared using different koji molds as starters

We have evaluated four commercially available mold starters (SP-01, NY, M1, and “for kome miso”) for fermented fish pastes with the aim of determining the effect of different koji molds on the nutritional and sensory characteristics of the fermented product. Comparative studies among different fermented fish and bean pastes were also conducted. Protein contents were considerably higher in the fermented fish pastes (22.7–24.1%) than the fermented bean pastes (8.2–12.8%). Free amino acids, oligopeptides, organic acids, mineral content, and sensory scores of the fermented fish pastes were superior to those of the bean pastes in terms of nutritional aspects. Sensory evaluation of fermented fish and bean pastes clearly indicated that different koji molds used as starters had little effect on the taste of the fermented fish pastes. However, in terms of nutritional and sensory characteristics, fermented fish pastes from trash fishes were found to be both commercially valuable and a potential gain to food processing firms among the many new generic fermented food products becoming available.     

变性高效液相色谱法检测鸡HSP70基因单核苷酸多态性

选取我国20个地方鸡种共591个个体作为实验材料,采用变性高效液相色谱(DHPLC)技术,在鸡HSP70基因的全序列内进行未知SNPs的筛选,对有变异的色谱峰型分别选取2个个体测序,再将同一对引物的所有测序样本的测序结果进行序列的同源性比较,从而识别和确认鸡HSP70基因的未知SNPs。结果分别在4对引物扩增的PCR产物中检出并确认了10个SNPs:A258G、C276G、C507T、C1040A、G1044A、C1431A、T1476C、G1500A、A1529G、C1722T。     

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.     

Genome‐wide association analysis of salmon lice (Lepeophtheirus salmonis) resistance in a North American Atlantic salmon population

Our objective was to detect single nucleotide polymorphisms (SNPs) associated with resistance to the salmon louse in the Saint John River aquacultural population of North American Atlantic salmon using estimated breeding values (EBVs) and 6K genotypes from the parent‐generation and lice count phenotypes from the challenged, but ungenotyped, offspring‐generation. In 2011 and 2012, we challenged recent smolts with approximately 100 copepodids each. Fish were euthanized once the lice reached the chalimus stages and lice count, sex, tank and salt water weight were recorded. We used a multiple trait model to estimate breeding values for the parent‐generation using their own fresh water weights and the salt water weights and lice counts of the offspring‐generation. Salmon lice count heritability for untransformed and transformed data was 0.17 and 0.29 respectively. Two different genome‐wide association study methods were compared: (i) forward multiple linear regression and (ii) a mixed linear model using principal components to correct for population stratification as implemented in the egscore function of GenABEL. The two methods detected different SNPs located on different chromosomes. The multiple regression method incorporated 70 SNPs found on chromosomes 2, 7, 9, 12, 14, 15, 21, 22, 1p/23, 24. Many SNPs entered into the forward multiple regression are likely to be false positives from not correcting for the observed population stratification and cryptic relatedness. In contrast, the mixed linear model identified only two SNPs, one on chromosome 1p/23 (6.9%) and one on chromosome 1q (6.1%) consistent with louse‐resistance being a quantitative trait.     

Dietary nucleotide mixture effects on reproductive and performance,ovary fatty acid profile and biochemical parameters of female Pacific shrimp Litopenaeus vannamei

The effects of different dietary nucleotide mixture (NT) levels (0, 2, 4 and 6 g/kg) were investigated on the reproductive performance, fatty acid profile and biochemical parameters in Litopenaeus vannamei female that were co‐fed with fresh food (two times daily) and experimental diets (two times daily) for 30 days. Sampling was carried out at the first day of the trial, before eyestalk ablation (ESA) (day 21) and after ESA (day 30). Reproductive performance parameters including the hepatopancreatic index, absolute fecundity, egg diameter and latency period were higher in the NT‐supplemented groups than the control group (p < .05). Total n‐3 polyunsaturated fatty acids in the ovaries, especially eicosapentaenoic acid, were higher in the NT‐supplemented groups than in the control (222.6 ± 7.3 versus 204.7 ± 3.0 mg/g extracted lipid) (p < .05). Total hemocyte count, granular cells, plasma glucose (Glu), total protein (TP), calcium (Ca²⁺), total cholesterol (Chol), triglyceride (Tg) and high‐density lipoprotein (HDL) levels were significantly increased in the NT‐supplemented groups in comparison with the control group (p < .05). After ESA, the concentration of plasma Glu, TP, Chol and Tg significantly decreased, but HDL levels increased in all groups (p < .05). These results suggested that the application of dietary NT may present a novel strategy for increasing reproductive performance and health status in L. vannamei.     

Dietary supplements of guanosine improve the growth,non‐specific immunity of sea cucumber,Apostichopus japonicus Selenka,and its resistance against Vibrio splendidus

The growth response, non‐specific immune activities and disease resistance were measured in sea cucumber, Apostichopus japonicus Selenka (initial average weight 6.80 ± 0.10 g), when fed diets supplemented with graded levels of guanosine from the guanosine‐5’‐monophosphate disodium (GMP) at 0 (control), 0.3, 0.6 and 1.2 g/kg for 8 weeks. The results showed that GMP supplemented at 0.6 and 1.2 g/kg significantly enhanced the growth of sea cucumber. Sea cucumber fed a diet with 0.6 g/kg of GMP had significantly higher intracellular superoxide anion production, nitric oxide synthase activity, lysozyme activity and the total superoxide dismutase (T‐SOD) activity than those in control group (p < .05). Increased lysozyme activity and T‐SOD activity were also found in sea cucumber fed GMP at 1.2 g/kg. Moreover, there was significantly lower cumulative mortality after the disease challenge in sea cucumber fed the diets with 0.6 and 1.2 g/kg GMP than that in control and 0.3 g/kg GMP groups (p < .05), and no significant difference was observed between 0.6 and 1.2 g/kg GMP groups. These results suggested that feeding GMP at a dose of 0.6 g/kg could enhance growth, non‐specific immunity of sea cucumber as well as its resistance against Vibrio splendidus.