Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.     

HXKs基因在香蕉果实后熟过程中功能初探

香蕉是世界主要水果之一,对乙烯非常敏感,属于呼吸跃变型果实。目前,香蕉果实后熟机理还未完全探明,后熟过程中己糖激酶(HXK)与乙烯的关系还不清楚。通过BLAST比对从香蕉基因组数据库中发现HXK基因家族的11个成员,经克隆获得这些基因的序列,并研究HXKs基因在香蕉果实自然后熟过程中的转录表达谱,重点研究乙烯利、甘露糖、NAG和1-MCP对HXKs基因表达、HXK酶活和内源乙烯生物合成的影响。结果表明:在香蕉果实后熟过程中HXKs基因呈现差异性表达,乙烯利上调大多数HXKs基因的表达和HXK酶活,1-MCP的作用正好相反,这表明外源乙烯正作用于HXK。另一方面,甘露糖加快内源乙烯生物合成,NAG却推迟内源乙烯生物合成,这说明HXK正作用于内源乙烯生物合成。所以,在香蕉果实后熟过程中乙烯和HXK之间存在相互促进的关系。这将为进一步阐明香蕉果实后熟机制提供理论依据,也将为香蕉果实保鲜新技术的挖掘提供新思路。     

虾源哈维氏弧菌的致病性与生物学特性比较分析

哈维氏弧菌(Vibrio harveyi)是导致养殖对虾暴发弧菌病的重要病原之一。从我国南方养殖凡纳滨对虾(Penaeus vannamei)分离、鉴定了10株哈维氏弧菌(Vh00947、Vh00949、Vh11011、Vh11014、Vh21217、Vh21218、Vh21220、Vh21229、Vh21231和Vh31487),分别肌注感染健康凡纳滨对虾后发现,菌株Vh21229致病性很弱,Vh00949其次,其它菌株毒力较强;对8种哈维氏弧菌常见毒力基因(Vh1、Vh2、Vh3、Vh4、tox S、hcp、zot和pap6)的检测显示,南方虾源哈维氏弧菌无论强弱毒株都很少携带zot、pap6、toxs和4种Vh基因(≤10%),表明这10个菌株的致病性与这7种常见毒力因子的相关度很低;hcp基因在所有菌株中均有检出,其中强毒株中检出率较高(50%),在较弱毒株(Vh00949)中也存在,表明hcp基因与这些菌株的致病性密切相关,但不是决定性因子。因此,这10株南方虾源哈维氏弧菌菌株的致病性差异应由这8种常见毒力因子以外的未知或未检测到的因子所决定。生化特征分析显示,所有菌株中只有弱毒株Vh21229不能利用D-甘露糖、蔗糖、D-海藻糖,而且只有其赖氨酸脱羧酶反应至阳性,而其它菌株均为阴性,推测导致哈维氏弧菌菌株生化特性改变的某种因子可能对菌株的致病性也产生了影响。药敏实验表明,10株哈维氏弧菌均对环丙沙星、氯霉素、恩诺沙星、美罗培南、头孢曲松、多西环素、头孢吡肟、诺氟沙星敏感,而对氨苄西林耐受性强,表明在虾类养殖过程中应当严格规范和控制抗菌药物尤其是青霉素类药物的使用。     

油棕新品种对干旱胁迫的生理响应及其抗旱性评价

在不同程度的人工干旱胁迫下，测定10个油棕品种的叶绿素、游离脯氨酸、可溶性蛋白、可溶性糖、丙二醛含量、相对电导率、SOD与POD酶活性等，并利用SAS8.1统计主成分分析法，对10个油棕品种的抗旱能力进行综合评价。结果表明：(1)在干旱胁迫初期(0～5 d)，油棕各生理指标向着有利于适应抗旱的方向变化；随着干旱时间的延长(5～30 d)和胁迫程度的加剧(trt1～trt3)，各指标反映出油棕植株受害；当胁迫达50 d时，各生理指标几乎都向着抗旱有利的方向转变，表明油棕对干旱有较强的耐受性；(2)主成分分析表明，叶绿素含量、相对电导率、可溶性糖含量和SOD酶活性是反映油棕干旱胁迫的最重要的生理指标，可作为油棕品种抗旱性评价的依据；(3)10个油棕品种的综合抗旱力排序为：RYL34＞RYL39＞RYL38＞RYL32＞RYL37＞RYL31＞RYL36＞RYL40＞RYL33＞RYL35。     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

抗旱性复混肥对土壤物理性状、持水量和马铃薯经济效益的影响

采用田间试验方法,研究抗旱性复混肥配方及其对土壤物理性质、持水量和马铃薯经济效益的影响,以期筛选出抗旱性复合肥最佳配方和施用量。结果表明:影响马铃薯产量的因素由大到小依次为:马铃薯专用肥废弃物组合肥保水剂,抗旱性复合肥最佳配方组合为废弃物组合肥3 000 kg/hm~2、保水剂37.50kg/hm~2、马铃薯专用肥2 230 kg/hm~2。随着抗旱性复混肥施用量的增加,土壤总孔隙度、持水量、马铃薯块茎质量和产量在增加,土壤容重在降低。经回归统计分析,抗旱性复混肥与马铃薯产量间的回归方程为:y=28.87+1.771 3x-0.035 4x2,最佳施肥量为5.28 t/hm~2,计算结果与田间施用抗旱性复混肥5.28 t/hm~2相吻合。     

SSCP Marker Development and Analysis of Candidate Restorer Gene Rf1 for Cotton Cytoplasmic Male Sterility

[Objective] Locating the cotton cytoplasmic male sterility (CMS) restorer gene Rf₁ is important for investigating restorer gene mechanisms and improving restorer lines. In our previous study, a gene cluster, with nine Pentatricopeptide repeat(PPR) genes and nine other genes, was found within the 160-kb Rf₁ target region in Scaffold 333. The objective here was to improve the density of Rf1-linked markers in the target region and determine the expression profiles of candidate genes. [Method] Using the sequences of the 18 genes, we designed 155 single-strand conformation polymorphism (SSCP) primers covering all of the gene sequences to identify the polymorphic SSCP markers between the fertile and sterile pools. Additionally, real-time polymerase chain reaction(PCR) was performed to analyze the expression profiles of eight candidate genes in the four developmental stages of buds of sterile, maintainer and restoring lines, respectively. [Result] In total, 15 polymorphic primers were identified. A genotype analysis of the F₂ population was conducted using the 15 primers and 3 other polymorphic simple sequence repeats (SSR) markers. The markers were distributed in a 4.8 cM range. In addition, owing to the influence of sterile cytoplasm or restorer genes, most of the genes showed different expression patterns in the four developmental stages of the three lines' buds. [Conclusion] SSCP markers tightly linked to Rf₁ were identified and the expression profiles of candidate genes were determined. This study provides a basis for the further fine mapping of restorer genes and for candidate gene screening.     

新疆与美国早实核桃(Juglans regia L.)品种生长结实与成花特性比较

以引进的美国核桃品种‘维纳’、‘艾米格’和新疆本地品种‘温185’和‘新新2号’为试材,研究分析不同品种的植物学和生物学特性、花芽分化过程以及相关成花基因的表达。结果显示:4个品种在树体生长、枝芽发育特性、物侯期以及成花率和坐果率方面没有明显差异。在结果枝同一坐果部位形成双果、三果或以上的能力‘新新2号’明显强于‘维纳’,但‘新新2号’的果实大小和单果重都低于‘维纳’,而‘维纳’平均结果母枝上形成侧花芽的数量和坐果率高于‘新新2号’。从萌芽到开花结束直至当年侧芽发育过程中‘维纳’混合芽中的JrFT和JrLFY基因表达也高于‘新新2号’,而同时期的JrFLC基因表达则相对较低,说明‘维纳’在花芽分化和花器官形成过程中通过高表达JrFT和JrLFY基因,低表达JrFLC基因促进结果母枝上形成大量侧花芽,并利用大量侧花芽抽生结果枝坐果,形成较大的果实,使得最终产量并不低于同一个坐果部位大量着生2个或多个果实的‘新新2号’品种。