与甘蔗抗黑穗病基因连锁的SSR标记筛选

黑穗病是世界各植蔗区最普遍、最主要的一种气传真菌性病害。为筛选与甘蔗抗黑穗病基因连锁的分子标记,利用高抗和感病亲本杂交的后代群体为材料,分别应用浸渍法和针刺法进行人工接种,通过一年新植一年宿根的抗病性鉴定,采用混合分组分析法（Bulked Segregant Analysis,BSA）构建了抗感池,并结合SSR（Simple Sequence Repeat）分子标记技术筛选出一个与甘蔗抗黑穗病性基因连锁的SSR标记,该标记在甘蔗抗黑穗病育种的辅助选择中具有应用潜力。     

高羊茅单株耐热性相关分子标记的筛选及其与越夏性的关系研究

筛选与高羊茅单株耐热性和越夏性相关的分子标记有利于建立高效育种体系。以来自于耐热性不同的12个高羊茅种质资源的216个单株为耐热性分子标记筛选的基础群体,在人工高温(40℃/35℃,昼/夜各12 h)和夏季田间自然条件下分别进行了耐热性和越夏性鉴定。运用简单重复序列区间(inter-simple sequence repeat,ISSR)和随机扩增多态性DNA(random amplified polymorphism DNA,RAPD)分子标记技术,采用改良的集团分离分析法(modified bulked segregant analysis,BSA),从800个RAPD和100个ISSR分子标记中筛选与耐热性相关分子标记,并研究其与单株越夏性的关系。结果表明,单株的耐热性与越夏性的相关程度较低(决定系数为3.4%);引物351扩增的750 bp条带(351-T750)与耐热性显著相关,而引物138扩增的950 bp条带(138-T950)则与耐热性和越夏性均显著相关。可以推论,耐热性强和/或越夏性强单株材料需要分别在人工高温和夏季田间自然条件下加以鉴定筛选;351-T750可应用于耐热性强单株的辅助选择,而138-T950则可以用于耐热性强与越夏性强单株的辅助选择。     

Identification of allele specific AFLP markers linked with bacterial wilt [Ralstonia solanacearum (Smith) Yabuuchi et al.] resistance in hot peppers (Capsicum annuum L.)

Even though the bacterial wilt is identified as the most destructive disease in hot peppers world-wide, robust molecular markers that facilitate marker assisted selection are absent till date. Kerala Agricultural University (India) has released two hot peppers named Ujwala and Anugraha which show high level field resistance to this pathogen. The variety Anugraha was developed through backcross breeding between a high yielding but highly susceptible variety Pusa Jwala with the highly resistant Ujwala, using Pusa Jwala as a recurrent parent. Thus, Pusa Jwala and Anugraha are near isogenic lines (NILs) differing for the resistance to bacterial wilt only and the resistance is governed by a homozygous recessive (rr) gene action. The F₁s of Anugraha × Pusa Jwala were selfed to generate the segregating F₂ population. The F₂ population has been field screened, 10 highly susceptible and 10 most resistant plants were identified and DNA from these plants were bulked separately. Bulked segregant analysis with AFLP primer combination EcoACT + MseCAC was done using the DNA from donor parent Ujwala, susceptible parent Pusa Jwala, resistant parent Anugraha, bulked susceptible F₂ and bulked resistant F₂ plants. On resolution using capillary electrophoresis system in genetic analyzer, the AFLP products have yielded three polymorphic bands (103, 117, and 161 bp) which were linked with the resistant recessive allele and three polymorphic bands (183, 296, 319 bp) linked with the dominant susceptible allele of the bacterial wilt resistance gene. The results were confirmed through co-segregation analysis in most resistant and susceptible plants of F₂ segregating population.     

Construction of a linkage map and identification of DNA markers linked to Fom-1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon

Resistance to Fusarium oxysporum f.sp. melonis race 2 is conferred by a single dominant gene, Fom-1 in melon. Here, we identified DNA markers tightly linked to Fom-1 that could be used for marker assisted selection in breeding programs. First, we developed 125 F₂ plants derived from the cross between melon lines P11 (fom-1fom-1) and MR-1 (Fom-1Fom-1). Using the F₂ population, we constructed a linkage map including 14 SSR markers which had not been mapped previously. Fom-1 was confirmed to be allocated to linkage group 7. Then, we identified four AFLP markers using bulked segregant analysis. The AFLP marker TAG/GCC-470 was completely linked to Fom-1 and other three markers were mapped near Fom-1. TAG/GCC-470 and TCG/GGT-400 were respectively converted to STS and CAPS markers. Usefulness of DNA markers was confirmed in the analysis with several melon cultivars and lines.     

甘蓝型油菜种皮色泽相关基因的cDNA-SRAP差异显示

为探寻甘蓝型油菜黄黑籽之间的种皮色泽表达差异的分子基础,探索cDNA-SRAP差异显示的可行性,选用培育7年的重组自交系群体（RILs）,根据群体分离分析法（bulked segregant analysis,BSA）构建了种子的RNA混合池,利用cDNA-SRAP法进行了差异显示研究。996对SRAP引物组合扩增出2 100条带,2次扩增重复率为65.2%,其中在黄黑籽材料之间重复稳定出现的差异片段有12条,长度在100~300 bp之间。选择其中7条差异片段,进行回收、克隆和测序,发现2个片段分别与拟南芥的NAD+ADP核糖转移酶及小肽转移蛋白3具有很高的同源性。结果表明,利用SRAP方法可以对甘蓝型油菜cDNA混合池进行差异显示研究,2条差异片段可能与甘蓝型油菜的种皮色泽基因表达相关。     

基于RNA-Seq技术的向日葵油酸形成的转录组学分析

本研究以高油酸材料J9和低油酸材料NK244为研究对象,在授粉后20d取材,采用RNA-Seq技术对其进行转录组学研究。经比较分析,2个材料共鉴定到差异表达基因（DETs）5 447个,其中上调表达基因2 709个,下调表达基因2 738个。对DETs进行生物信息学分析,GO信息分析表明,有2 218个DETs富集到57个功能条目,其中上调表达基因主要涉及催化活性、异构酶活性、碳水化合物结合、DNA结合、脂结合、碳水化合物过程、脂代谢过程和脂肪酸生物合成等;下调表达基因主要涉及蛋白激酶活性、ATP结合、蛋白磷酸化、tRNA氨酰化的蛋白质翻译和纤维素生物合成过程。KEGG分析中799个差异表达片段被显著富集在28个通路,主要为碳水化合物代谢通路和脂代谢通路。本研究为向日葵油酸调控机理研究提供了一定理论依据,为向日葵品质育种与种质创新提供理论参考。     

基于转录组测序的大口黑鲈驯食相关SNP开发及其与生长性状的关联分析

大口黑鲈为肉食性鱼类,传统养殖过程中使用大量的冰鲜鱼为饵料,冰鲜鱼的使用不仅增加了养殖成本,而且多余的冰鲜鱼残饵还会给环境带来严重的污染。为了保护环境并降低养殖成本必须减少冰鲜鱼的使用量,本实验以选育适合人工配合饲料饲喂大口黑鲈为研究目标,用人工配合饲料作为饵料驯化大口黑鲈幼鱼,在驯食后14和16 d,测定其体质量、全长、体高等生长指标。同时在大口黑鲈转录组测序分析获得的SNP位点中,选择其所在序列的基因功能与能量代谢有相关性的7个SNP位点进行SNaPshot分型,用SPSS 19.0进行卡方分析标记与生长性状的相关性。结果发现,序列Unigene031044中的C1332G位点、Unigene085384中的A741G位点和Unigene022319中的A284G位点在体质量、全长及体高性状上存在显著差异。这3个SNP位点所在的基因经预测分别与雌二醇17β脱氢酶12B (hsd17b12b)基因、长链酰基辅酶A合成酶家族成员1(acsl1)基因和琥珀酸脱氢酶组装因子2(sdhaf2)基因具有很高的同源性,推测上述3个SNP位点的变异可能影响了基因表达产物对脂肪酸代谢或三羧酸循环的调节功能,与大口黑鲈对人工配合饲料的利用能力存在密切的相关性。本研究筛选得到的与驯食相关的SNP位点为大口黑鲈功能基因多态性的进一步研究,及加快大口黑鲈食性改良与驯化育种的遗传研究提供了科学的参考依据。     

小麦与叶锈菌互作过程中LRRs类基因在转录水平的表达分析

本试验以小麦近等基因系TcLr26和Thatcher分别与叶锈菌生理小种260组成不亲和及亲和组合,基于RNA-Seq高通量测序数据库,通过基因表达差异分析,筛选了6个在接种后较0h表达明显变化的属于LRRs类基因的Unigenes,利用实时定量PCR(RT-qPCR)技术对这6个LRRs类基因在接种叶锈菌后的表达进行了分析,为深入研究这些LRRs类蛋白在小麦抗叶锈菌侵染过程中的作用机制奠定了重要试验基础。