甘薯近缘野生种的抗病性鉴定与新型种间杂种的获得

为了发掘甘薯近缘种抗茎线虫病、病毒病基因资源,改良栽培种抗性,拓宽甘薯遗传基础,本文利用田间自然病圃对30份甘薯近缘野生种进行抗茎线虫病鉴定,利用硝化纤维素膜-酶联免疫(NCM-ELISA)对20份野生种进行了抗病毒病筛选.以筛选到的抗性材料为父本、栽培种徐薯18为母本,采用生长调节剂处理方法进行种间杂交,结果得到8份高抗、10份抗茎线虫病野生种,5份高抗、8份抗病毒病材料;获得4个新型种间杂种.经过染色体计数和ISSR分子标记鉴定,证实了所得杂种的真实性.表明甘薯近缘野生材料中存在着栽培种所需要的抗性基因,通过有性杂交手段可以获得含野生种染色体组的种间杂种新类型,为渐渗野生抗性基因起到"桥梁"作用.     

上海地区番茄黄化曲叶病毒病的鉴定及嫁接接种法研究

番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)是一种由烟粉虱(Bemisia tabaci)和嫁接传播的双生病毒,在热带、亚热带地区给番茄生产造成严重威胁.根据番茄黄化曲叶病毒的保守序列设计一对引物,运用PCR技术从上海地区的感病番茄中扩增出一条575bp的特异带,而健康植株无此带.测序表明该序列与番茄黄化曲叶病毒具有极高的同源性(97%～99%).将健康接穗嫁接到感染番茄黄化曲叶病毒的番茄砧木上,间隔15 d和30 d,分别提取接穗的DNA,并用PCR法检测病毒,发现嫁接15 d后在部分接穗中检测到TYLCV病毒,嫁接30 d后在所有的接穗中均检测到病毒,因此,嫁接法可以作为番茄黄化曲叶病毒病的接种鉴定方法.     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F:5′-ACGCATGCCTCTAATCCAGTGTA-3′,下游引物TYLCV-R:5′-CCAATAAGGCGTAAGCGTGTAGAC-3′),依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒,这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种,从而为蔬菜安全可持续生产提供科技支撑。     

中国番茄黄化曲叶病毒——双生病毒的一个新种

用 2 0个单抗对中国番茄黄化曲叶病毒 (TYLCV CHI)和其他双生病毒进行了测定 ,在血清学水平上证实中国番茄黄化曲叶病毒与中国烟草曲叶病毒有较大的亲缘关系 ;同时报道了TYLCV CHI部分共同区、外壳蛋白N端基因和AV1基因的PCR及其克隆和序列分析 ,从分子水平上证实TYLCV CHI与世界各地的其他双生病毒不同 ,是一种新的粉虱传双生病毒     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F：5′-ACGCATGCCTCTAATCCAGTGTA-3′, 下游引物TYLCV-R：5′-CCAATAAGGCGTAAGCGTGTAGAC-3′), 依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒, 这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种, 从而为蔬菜安全可持续生产提供科技支撑。     

单性结实樱桃番茄种质材料研究与抗病新种质创制

以5份单性结实樱桃番茄种质材料为研究对象,调查了果实内的种子数量、鉴定了黄化曲叶病毒病(TYLCVD)和灰叶斑病(GLSD)的抗病性;以材料CP28为研究对象,分析了单性结实性状的遗传规律和子房的发育过程;开展了抗番茄黄化曲叶病毒病和番茄灰叶斑病单性结实新种质的创制。结果表明:单性结实材料单果种子数在不同位置花序间表现差异较大,变异系数高达120%以上,平均单果种子数在6.44～7.41粒之间,明显少于普通材料;5份材料都不抗番茄黄化曲叶病毒病,材料CP24和CR3表现出抗番茄灰叶斑病;通过遗传群体内单性结实性状分离情况分析,得出材料CP28的单性结实性状由隐性单基因控制的结论;通过石蜡切片看出,日温15℃/夜温5℃低温栽培条件下子房内几乎没有胚珠,日温25℃/夜温15℃温度栽培条件下子房内有数量多少不等的胚珠,并且能正常发育;创制了3份兼抗番茄黄化曲叶病毒病和番茄灰叶斑病单性结实新种质,为多抗性单性结实新品种的培育奠定了基础。     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

云南野生茄资源黄萎病苗期人工接种抗性鉴定分析

黄萎病是目前茄子生产中的主要病害之一,广泛收集、鉴定、筛选抗性资源,尤其是从野生近缘种中发掘抗性基因并培育抗病品种,是解决茄子黄萎病危害的优选途径。云南省拥有丰富的野生茄子资源但尚未有效利用。本研究针对云南省茄子主产区收集到的3种黄萎病菌株,通过形态学鉴定和真菌18S r DNA/ITS鉴定,均属于大丽轮枝菌,并通过致病力鉴定,筛选出一株强致病力菌株(QZ-S);应用菌株QZ-S,通过苗期人工接种的方法对45份云南野生茄子资源开展黄萎病抗性鉴定,最终筛选出2份高抗材料(蒜芥茄和喀西茄)、2份抗病材料(水茄和多裂水茄)、6份中抗材料(1份刺天茄和5份红茄);此外,还筛选到了1份黄萎病高感材料239-3-2。本研究筛选出的材料可应用于茄子黄萎病抗病育种,为茄子及其他作物黄萎病抗病育种提供抗源。     

番茄中Rpi-blb2同源基因的挖掘与鉴定

番茄晚疫病是番茄生产中的主要病害之一,经常会造成较大的经济损失。晚疫病生理小种的变异和进化常会导致番茄品种原有的遗传抗性丧失,因此不断挖掘新的抗性基因,改良番茄晚疫病抗性是番茄抗病育种的长期任务。该研究采用BLAST同源比对的方法,以马铃薯野生近缘种的晚疫病抗性蛋白序列Rpi-blb2为种子序列,在NCBI蛋白质序列数据库中检索得到11条番茄蛋白质序列,这些序列与种子序列相似性为78%~83%,属于番茄疾病抗性蛋白家族,并对该家族成员进行了基因结构、基因定位、序列保守结构域和进化关系等分析。结果表明:该家族中10条序列分布在第Ⅵ条染色体上,1条分布在第Ⅴ染色体上;6号染色体上的10序列呈现2个抗病基因簇分布,在染色体上分别占据2个和3个基因位点;10条同源蛋白是Rpi-blb2的共同垂直同源蛋白,但不具有平行同源关系,大多数成员定位于细胞质。按照蛋白质保守结构域和基因定位的不同可分为三类,第一类共4条系列,包含有DUF3542和NB-ARC两个保守结构域特征序列;第二类共6条序列,与马铃薯Rpi-blb2蛋白一样,仅包含NB-ARC保守结构域特征序列,在这2类蛋白序列的NB-ARC结构域均位于序列中部;第三类(仅包含XP_004239406.1)虽然也具有与第一类蛋白相似的DUF3542和NB-ARC结构域,但在结构域两端的非保守区序列较短,且位于5号染色体上,因此将其单独归为1类。前两类蛋白成员相应的基因具有1~2个内含子,第3类蛋白不含内含子。该研究结果为利用生物技术选育番茄抗性品种提供了理论基础。     

转HS1prol基因番茄植株的获得

目的为了减少根结线虫对番茄的危害,研究并获得转抗线虫基因HS1prol番茄植株.方法在鉴定表达载体之后,采用CaCl2法制作农杆菌EHA105感受态细胞,然后用冻融法将HS1prol基因转入农杆菌中.通过农杆菌介导法将HS1prol基因导入无菌番茄外植体中,获得抗根结线虫转化再生植株.用卡那霉素筛选到再生植株后,提取抗性芽的基因组,利用设计好的引物进行PCR鉴定.结果与结论目的基因已整合到番茄基因组中,获得了转HS1prol基因番茄植株.     

First Report of Tomato infectious chlorosis virus on Tomato in Bulgaria

Virus‐like chlorotic symptoms were observed on tomato plants, cv. Velocity, grown in a greenhouse, region of Plovdiv. Samples collected from the leaves with interveinal yellowing and with initial interveinal chlorosis were tested for virus presence. Only the samples collected from the upper leaves with slight interveinal chlorosis were positive for Tomato infectious chlorosis virus (TICV) in indirect ELISA. Further, RT‐PCR analysis with specific primers for Tomato chlorosis virus (ToCV) heat shock protein 70, for TICV heat shock protein 70 and for TICV minor capsid protein was positive for TICV in all tested samples. No signals were obtained with primers for ToCV. Phylogenetic analysis showed that the Bulgarian sequence of Hsp70 and a sequence of Greek isolate clustered together having the highest resampling score. Regarding CPm, the Bulgarian isolate was more relevant to the French isolate. The obtained results from phylogenetic analysis supported the idea of a close relationship between the Bulgarian and Greek isolates.     

Cytopathology of Tomato torrado virus Infection in Tomato and Nicotiana benthamiana

Electron microscopy studies were carried out to investigate the cytopathological changes induced in tomato leaves by Tomato torrado virus (ToTV) that infects tomato plants worldwide causing severe necrotic symptoms. Plants infected with one of the Polish isolates of ToTV were used for cytopathological research. The results revealed severe cellular alterations, especially in Solanum lycopersicum. Moreover, it was shown that crystalline aggregates of virions occurred not only within the phloem cells as it has been previously reported.     

Infectivity of Tomato Leaf Curl Hainan Virus in Tomato and Tobacco in China

Tomato leaf curl Hainan virus (ToLCHnV) was previously reported as a distinct begomovirus infecting tomato in Hainan, China. To investigate the infectivity of ToLCHnV, an infectious clone of ToLCHnV‐[CN: HaNHK7] was constructed and agro‐inoculated into Solanum lycopersicum, Nicotiana benthamiana, Nicotiana glutinosa, Petunia hybrida, Cucumis sativus, Solanum melongena and Capsicum annuum plants; it induced severe leaf curling and crinkling symptoms in these plant species except C. sativus, S. melongena and C. annuum. The induced symptoms were compared with those induced by Papaya leaf curl China virus.     

The Respiration Rate of Tomato Stem Tissue Infected by Tomato Aucuba Mosaic Virus

The oxygen uptake of stem tissue slices of Lycopersicum esculentumvar. ‘Stonor's Moneymaker’, in 0·035 M. Sorensonphosphate buffer was measured manometrically; the optimum pHwas found to be 5·2. With tomato-stem tissue systemicallyinfected by virus (tomato aucuba mosaic), the respiration ratewas always below that of healthy tissue when equal numbers ofrespiratory systems were compared. When the respiration ratewas expressed in terms of oxygen uptake per unit fresh or dryweight, the respiration rate of the virus-infected tissue couldbe greater or less than that of the healthy tissue dependingon the growth conditions of the experimental material. Underconditions favouring the complete development of disease symptoms,virus infection decreased the fresh and dry weight of the cell.For this reason fresh weight and dry weight were not consideredsuitable criteria to express the respiration rates as theseunits contained different numbers of respiratory systems inhealthy and virus-infected tissue.     

In Planta Transformation of Tomato

In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%, and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters.     

Male Sterility in Tomato

Male sterility research has been directed toward two goals: identifying genes required for the pollen development pathway and, more practically, identifying genetically stable lines that can be used in hybrid seed-breeding programs. The present resurgence of interest in male sterility remains true to these goals, but in addition seeks a molecular understanding of pollen development in order to genetically engineer controllable male sterility for hybrid seed production. In this review, we discuss the genetic and histochemical studies of tomato male sterile mutants that were conducted prior to 1970 in the context of gene expression and interaction. We also examine the use of molecular biological techniques in recent studies of male sterility and report on the current strategies being used for hybrid seed production.