基于RNA-seq技术的玉米SNP和InDel标记分析

高温已对玉米(Zea mays L.)生产造成了巨大危害。为了给耐高温胁迫玉米品种选育提供可用的分子遗传标记,本研究以耐高温和高温敏感的两个玉米品种为实验材料,通过花粉总RNA提取、mRNA纯化、反转录、转录组文库构建及测序等工作,对获得的转录组数据进行SNP和InDel分子标记分析。结果显示:高质量碱基数据量平均为6.69 Gb,占原始数据比例平均达94.71%;在SNP分子标记中,Homo位点平均为19450个,Hete位点平均为23598个,转换类型共52337个,颠换类型共27868个;在InDel分子标记中,Homo位点平均为1023个,Hete位点平均为1736个;SNP/InDel位点在外显子区分布最多,达到总量的58.02%,其次是基因间隔区、3'非翻译区和5'非翻译区,其余区域位点占总量比例均低于5%;SNP/InDel造成同义单核苷酸突变比例最高,平均值为60.69%,其次为非同义单核苷酸突变,平均值为36.72%,非移码替换突变为平均值1.64%,其余3种均不到1.00%;在SNP和InDel标记位点基因功能注释中,NR数据库注释最多,超过8000个,其次为KEGG和eggNOG;注释到GO和Swissprot数据库的相对较少。本研究结果为进一步标记辅助耐高温玉米品种选育提供了分子基础,也为其它植物的类似研究提供了参考。     

番茄、茄子和辣椒枯萎病原菌分子鉴定及其致病性测定

调查了福建省番茄、茄子和辣椒3种主要茄科枯萎病的发生与症状情况,通过常规分离,依据Booth鉴定和分子鉴定相结合的方法,对从这3种茄科作物上采集的40个菌株进行了鉴定。结果表明,40株镰刀菌中有35株属于尖孢镰刀菌,占总数的87.5％;3株为串珠镰刀菌,另2株为茄形镰刀菌。进一步利用Koch氏法则进行菌株致病性检测,结果表明,尖孢镰刀菌是本研究中所采集的番茄、茄子和辣椒病株的病原菌,而3株串珠镰刀菌和2株茄形镰刀菌均不造成寄主植物发病。     

小麦抗穗发芽研究进展

穗发芽对小麦的生产、加工和消费等方面带来诸多不利影响。小麦穗发芽受自身与环境因素的影响,其中子粒本身的休眠特性和α-淀粉酶活性与穗发芽联系紧密。近年来,利用分子标记和比较基因组学等方法研究小麦穗发芽发展迅速,已鉴定出大量与穗发芽抗性相关的分子标记,并定位到不同的染色体上。本文从穗发芽的危害、抗性机制、抗性遗传等方面阐述了小麦抗穗发芽的研究进展,并对今后重点研究方向进行了展望,以期为小麦抗穗发芽育种提供理论参考。     

新疆冰川冰缘冻土低温淀粉酶部菌株分子生物学鉴定及酶学特性研究

[目的]对新疆冰川冰缘地区冻土中筛选出的具有良好产低温淀粉酶能力的菌株进行分子生物学鉴定,分析其酶学特性.[方法]用种子培养液活化菌株提取基因组DNA进行PCR扩增,将扩增成功菌株的PCR产物测序结果通过NCBI数据库比对,对冻土中产低温淀粉酶菌株的16S rDNA序列用MegAlign Fasta软件进行系统发育分析.利用Yoo改良法测低温淀粉酶酶活力.[结果]菌株A-2,A-3,A-4经16S rDNA序列分析获得的序列与数据库中解淀粉芽孢杆菌(Bacillus amyloliquefaciens strain NBRC 15535 16S ribosomal RNA,partial sequence(NR_04145))比对相似率达到99;.该菌株产生的低温淀粉酶的最适作用温度为30℃,最适pH为6.[结论]菌株A-2,A-3,A-4均属解淀粉芽孢杆菌.     

一个棉花GDSL脂肪酶基因的克隆与功能分析

GDSL脂肪酶与GXSXG脂肪酶是2个重要的脂肪酶亚家族。其中, GDSL家族脂肪酶具有水解酶活性, 能水解多种酯类物质。本试验根据新乡小吉无绒无絮(XinWX)和无绒有絮(XinFLM)近等基因系纤维起始期29K芯片竞争杂交结果, 选择了一个在纤维起始期具有极显著表达差异的EST序列(GenBank登录号为DR458916), 以该序列信息为探针, 利用电子克隆方法并进行cDNA及基因组全长基因PCR扩增、测序验证, 克隆获得一个陆地棉GDSL脂肪酶基因(GhGDSL;GenBank登录号为KC186125)。该基因ORF长1065 bp, 编码354个氨基酸, 含有5个外显子和4个内含子。该基因在二倍体棉种基因组中含一个拷贝, 在四倍体棉种基因组中含2个拷贝。序列比对显示该基因在四倍体棉种的2个亚组中独立进化, 且D亚组比A亚组变异大。染色体定位显示该基因2个拷贝分别位于A4 (Chr. 4)和D4 (Chr. 22)染色体上。定量RT-PCR结果表明, GhGDSL在开花后3~10 d的纤维组织中表达量高, 其中在海7124中表达高峰在8DPA, 在TM-1中表达高峰从5DPA持续到10DPA。利用[(TM-1×Hai7124)×TM-1]的BC₁S₁群体开展GhGDSL功能与纤维、种子品质性状关联分析, 发现该基因A亚组的拷贝与种子脂肪含量存在显著相关(P=0.048);D亚组的拷贝与种子蛋白含量存在极显著相关(P=0.008)。推测GhGDSL基因功能与种子中脂肪、蛋白代谢相关, 同时也参与纤维伸长过程。     

大豆分子育种研究进展

分子育种技术的研究和应用是近十余年来世界大豆生产发展的重要科技动力。2011年,国内外在大豆分子育种相关领域取得了新的进展。在分子标记开发与辅助育种方面,发掘出与产量、发育、品质、抗病和抗逆等性状相关的新的分子标记或QTL;在新基因挖掘方面,克隆了与光周期反应、共生固氮、品质及抗逆性相关的基因,并分析了其功能。在大豆转基因育种方面,遗传转化体系的优化取得了新的进展,转化效率有所提高,同时对一些功能基因(包括来自其它物种的一些基因)进行了功能评价和育种利用价值评估。转基因大豆继续保持良好的发展势头,并且出现一些新的发展方向。该文对这些研究进展进行了综述,并对今后的发展趋势进行了展望。     

玉米功能性Insertｉon/Deｌetｉon(InDeｌ)分子标记的挖掘及其在杂交种纯度鉴定中的应用

InDel作为重要的遗传标记已被广泛用于作物连锁图谱的构建及多样性研究。通过生物信息学方法从玉米全基因组水平进行InDel标记的挖掘并对其在玉米杂交种纯度鉴定中的应用进行分析,根据B73全基因组序列(第二版)及Mo17的二代电子拼接序列发现了40 000多个InDel位点,其中约有11 400个含有InDel位点的序列可用于特异性引物的开发。新发展的143个代表基因(<1 kb)或基因内部的功能性InDel标记在B73及Mo17间得到了验证并用于玉米杂交种纯度的鉴定。经过分析,共有13个共显性InDel标记在6个杂交种亲本间表现出明显的长度多态(>50 bp)。结合玉米子粒DNA快速提取方法,利用这些共显性InDel标记对6个杂交种1 400多个样本纯度进行鉴定,结果显示该方法可以快速、准确、经济地鉴定玉米杂交种纯度。     

十字花科作物根肿病的侵染生理与抗性遗传研究进展

十字花科植物根肿病是由芸薹根肿菌侵染而引起的危害最大的病害之一,近年来,该病在世界范围尤其是在欧洲、北美、东亚等地区日趋严重,我国很多地区的十字花科作物也正面临着根肿病的严重威胁。本文就芸薹根肿菌侵染过程中寄主植物的生理生化变化及其分子基础、根肿病抗性遗传规律和分子标记与遗传图谱等多方面的研究进展做一概述,并对该领域今后的研究进行了展望。     

Genetic variability and population structure of <Emphasis Type="Italic">Grapevine virus A</Emphasis> coat protein gene from naturally infected Italian vines

Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.     

Exploiting generic platform technologies for the detection and identification of plant pathogens

The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.