梨抗黑星病研究进展

梨黑星病(Pear scab)也称为疮痂病,是世界范围内普遍发生的梨树重要病害之一。其病原菌为梨黑星菌(Venturia pirina Aderh)和纳雪黑星菌(V.nashicola Tanaka et Yamamoto)。梨黑星病主要危害梨果实和叶片,梨对黑星病的抗性与栽培措施和气候条件、品种抗病性和黑星病菌生理小种等有关,而品种的抗病性与其抗病性遗传、组织形态结构和生理生化特性等有关。就梨抗黑星病的抗病性遗传、抗病机制、抗病基因分子标记与定位等进行概述,以期为相关研究提供参考。     

梨黑星病研究进展

从梨黑星病（Venturia nashicola）病原菌、发病条件、抗病机制、抗性遗传等方面对梨黑星病的研究进展进行了系统综述。梨黑星病的病原菌有2种,侵染中国梨的为纳雪黑星菌（Venturia nashicola Tanak et Yamamota）,侵染西洋梨的为梨黑星菌（Venturia pirina Aderh）;梨对黑星病的抗性呈显性遗传,抗病性对感病性为显性;目前,梨树对梨黑星病的抗性机理尚不清楚;通过分子标记标记出的抗梨黑星病基因Vnk,和Vf一样均在第1连锁群;通过多种育种手段已培育出一些抗梨黑星病品种并在生产中得以推广应用。     

与梨黑星病抗性基因连锁的AFLP标记筛选及SCAR标记转化

以‘鸭梨’（Pyrus bretschneideri）ב雪青’梨（P. bretschneideri × P. pyrifolia）F1代群体（97株）为试材,采用扩增片段长度多态性（amplified fragment length polymorphism,AFLP）技术和集群分类法（bulked segregant analysis,BSA）筛选与梨抗黑星病基因连锁的分子标记。通过64对AFLP标记引物在亲本和分离群体中的筛选和验证,获得与梨抗黑星病基因紧密连锁标记两个,即ACA/CAA-179和AAC/CAG-198。它们与抗黑星病基因的遗传距离分别为5.2和8.3 cM。对AFLP标记片段的克隆和测序结果显示其长度分别为179和198 bp。根据序列信息设计特异引物,在杂交后代群体上的PCR分析表明AFLPACA/CAA-179标记被成功转换成SCAR标记,命名为SCAR-117。本研究结果将有助于对抗黑星病梨品种资源的分子鉴定及杂种后代的早期辅助选择。     

SSR标记和苗期人工接种鉴定黄瓜抗黑星病种质资源

为了获得抗黑星病黄瓜种质,为分子设计育种提供抗源,利用与黄瓜抗黑星病基因紧密连锁的SSR标记CSWCTT02D对102份黄瓜种质资源进行基因型分析。结果表明,黄瓜种质间抗病基因的标记基因型存在遗传变异性,明确了102份种质抗黑星病基因的标记基因型,3份种质存在抗病标记(2.94%),1份种质为杂合型(0.98%),98份种质不存在抗病标记(96.08%);苗期人工接种鉴定有高度抗病种质4份(3.92%),高度感病种质98份(96.08%)。SSR分子检测结果与苗期人工接种基本一致,有2份材料与人工接种鉴定结果不符。‘南9436’(华南型)接种为感病,标记为抗病;‘K92’(华南型)接种为抗病,标记为感病,符合率达98.04%。抗病材料选择应以人工接种结果为依据,因此初步鉴定出4份抗黑星病种质,分别为日本类型‘Q6’和‘NINIA’、华南型‘南9427’和‘K92’,病情指数均为0。该研究为华北型黄瓜抗黑星病品种的遗传改良奠定了技术与种质基础。     

甜瓜PMR5抗白粉病基因的遗传定位

以甜瓜白粉病抗性品种PMR5,感白粉病甜瓜伽师及杂交F1以及BC1、BC2分离群体为试材,接种白粉菌生理小种1,采用抗病遗传分析,并利用BSA(Bulked Segregation Analysis)结合SSR(Simple Sequence Repeat)分子标记方法,研究抗白粉病基因的遗传规律获得与其连锁的分子标记,利用获得的分子标记对抗白粉病基因进行遗传定位。结果表明:BC1中抗病与感病分离比为64∶27,BC2中分离比为37∶27,推测PMR5在BC1中表现2个显性基因,在BC2中表现单显性基因遗传模式。用BC2对抗病基因进行遗传定位发现,一共有11个位于LGII的SSR分子标记与抗白粉病基因连锁,抗病基因位于标记CMGA36和SSR25208之间约104 113bp。     

大白菜黑斑病抗性遗传规律

采用２ 个抗病品系和２ 个感病品系作为试材,并配制杂交组合,采用真叶期人工接种病菌鉴定Ｆ１ 、Ｆ２ 、Ｂ１Ｃ１ 、Ｂ１Ｃ２ 杂种抗性的方法,研究了大白菜对黑斑病的抗性遗传规律。结果表明,大白菜对黑斑病的抗性为显性遗传,并受单个显性基因控制。     

黄瓜ZYMV-CH抗性遗传与连锁分子标记研究

 小西葫芦黄化花叶病毒中国株系( ZYMV-CH) 是危害我国黄瓜的主要病毒之一。本试验以抗病的‘秋棚’和感病的‘欧洲8号’杂交获得的115份重组自交系(R IL) 为材料, 进行了黄瓜抗ZYMV-CH遗传规律和连锁分子研究。结果表明: 病情指数在黄瓜R IL群体呈双峰分布, 表明其对ZYMV的抗性是受主基因控制的性状, 但也存在微效基因的修饰作用。采用集团分离分析法(BSA) 和AFLP技术, 获得与ZYMV-CH抗性基因连锁的两条特异性片段 E-ACG/M-CAG-182和E-ACG/M-CAG-180) , 遗传连锁距离分别为5 cM 和11 cM。将E-ACG/M-CAG-180 特异片段转化成共显性的SCAR 标记SCAR3-109, 与ZYMV-CH的抗性基因遗传连锁距离为11 cM。该标记可以作为黄瓜抗ZYMV辅助选择的分子标记。     

梨对黑星病的抗病性及其机理研究进展

从梨黑星病(Venturia nashicola)病原菌、发病条件、抗病性及抗病机制等方面对梨黑星病的抗病性及其抗性机理研究进行了系统综述。表明,梨黑星病的病原菌有2种,侵染中国梨的为纳雪黑星菌(Venturia nashicola Tanaket Yamamota),侵染西洋梨的为梨黑星菌(Venturia pirina Aderh);梨对黑星病的抗性呈显性遗传,抗病性对感病性为显性;目前,对梨黑星病的抗性机理尚不清楚。     

茄子青枯病抗性基因的遗传分析及其AFLP标记

 以茄子高感青枯病品种‘三月茄’与高抗品种‘S69’的F1、F2为材料, 对青枯病抗性遗传进行了分析, 同时开展了与抗性基因连锁的AFLP标记鉴定。结果表明: ‘S69’对青枯病的抗病性属于不完全隐性遗传, 感病性属于不完全显性; 抗性由1～2对基因控制。对86个F2单株AFLP分析, 鉴定出1个与‘S69’抗性基因连锁的AFLP标记39A980 (该引物组合为E-ACC /M-CTG) , 该标记与抗性基因间的交换值为4.65% , 遗传距离为4.9 cM。     

茉莉酸甲酯对梨黑星菌侵染下叶片叶绿素降解的缓解效应

为探明茉莉酸甲酯(MeJA)在梨感染黑星菌后叶片损伤上的缓解效应,以感病品种"雪花"和抗病品种‘黄冠"梨叶片为试验材料,分析了黑星菌侵染后梨叶片叶绿体结构和叶绿素含量的变化,以及MeJA处理对叶绿素含量和叶绿素合成关键基因表达的影响。结果表明,黑星菌对"雪花"梨叶片叶绿体结构有更明显的破坏现象,在接种9 d时下降了49%,而"黄冠"梨叶绿素含量比对照下降了37%;MeJA处理可迅速诱导"黄冠"梨CAO的表达,且显著增加了侵染后期(接种5～9 d)"雪花"梨CHLH、CAO基因的相对表达量,缓解了黑星菌侵染后梨叶片叶绿素含量的降低。说明适当浓度的MeJA处理在一定程度上诱导梨增强对黑星菌的抗性,有利保持梨叶片叶绿素含量。     

马铃薯青枯病抗性的分子标记

对两个马铃薯二倍体分离世代群体ＥＤ和ＣＥ共１４０个基因型进行青枯病人工接种鉴定,并以此为作图群体,综合ＲＡＰＤ,ＳＳＲ,ＡＦＬＰ３种标记技术,利用ＢＳＡ分析方法对抗性基因进行分子标记筛选和定位。筛选与抗病感病性状相连锁的３个ＲＡＰＤ标记ＯＰＧ０５９４０、ＯＰＲ１１８０和ＯＰＯ１３７７０;筛选到一个ＳＳＲ标记ＳＴＭ００３２,被定位于染色体ＸⅡ上,检测到７个ＡＦＬＰ多态性带,初步认为其与抗病性相关,并     

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

生物技术在西瓜遗传育种研究中的应用

离体培养、基因工程与分子标记分析等生物技术应用于西瓜性状遗传改良研究取得重要进展。目前已建立了成熟的西瓜高效离体培养再生体系,主要应用于优良种苗快繁与重要性状遗传转化研究;染色体组水平的倍性操作已成功用于西瓜育种实践;在西瓜中已鉴定克隆多个重要性状基因;采用农杆菌介导或直接转化法,已将一些目的基因导入西瓜中,进行抗病、耐盐等重要性状改良;RAPD、RFLP、SSR、ISSR、AFLP等多种分子标记开始广泛应用于西瓜种质资源分析与系统演化、品种鉴定与倍性分析、遗传图谱构建、性状基因连锁标记与标记辅助育种等方面。     

雪花梨及其亲缘品种S基因型的确定

梨是典型的自交不亲和性果树,自然授粉结实率低,品质差。通过确定梨品种S基因型可寻找简便快速克服自交不亲和性,提高产量,改良品质的方法。根据梨S基因的一级结构特点,利用S基因的保守序列设计特异引物,并对雪花梨及其亲缘品种的基因组DNA进行PCR扩增。将基因组特异扩增电泳只显示一条S基因带雪花梨的特异扩增片段回收并与T载体连接,转化至大肠杆菌中。取其中1个阳性克隆进行测序,并通过生物信息学软件分析确定其核苷酸序列,推导出氨基酸序列,经网上Blast比较,确定该雪花梨S基因片段的S基因型,然后就该S基因进行酶切系统分析,有针对性地挑取另一个阳性克隆测序分析。另外,通过品种间的亲缘关系并经过酶切检测以分析雪花梨亲缘品种的S基因型,并确定各品种的S基因型分别为:雪花S4S16、冀蜜S1S16、雪青S3S16、雪峰S4S16、雪英S3S16、雪芳S4S16。     

AFLP标记在大白菜遗传育种中的应用

AFLP标记是一种新的DNA分子标记技术。对AFLP标记的基本原理、技术流程和关键技术做了简要介绍,从亲缘关系与遗传多样性分析、遗传图谱的构建、特定性状的连锁标记、品种纯度鉴定与QTL分析等方面概述了AFLP标记在大白菜遗传育种中的应用进展,并对其应用前景进行了展望。     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.     

Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

应用RAPD分析新疆主要梨品种的遗传关系

利用RAPD分析了新疆18个梨品种间的亲缘关系。从160个随机十聚体引物中筛选出12条RAPD引物。18份材料共扩增133条带,平均每条引物扩增约11条带,其中14条带为所有材料的共有带,有119个多态性位点,占总带数的89.47%,Nei相似系数为0.5～0.923。用PHYLIP构建MP树,认为(1)对选用的形态学经典分类已较明确的新疆梨、白梨、砂梨、西洋梨、秋子梨,RAPD分析数据完全与其相符;(2)几个芽变品种间高度相似;(3)杂种新梨1号与砀山梨、杂种新梨6号与香梨、杂种早酥梨与苹果梨分别并类;(4)苹果梨独立于供试的5个种之外;(5)库尔勒香梨归属于新疆梨。     

番茄抗叶霉病基因Cf12 的分子标记筛选及种质资源鉴定

 以番茄抗叶霉病的品种05HN36为母本,以感病品种051355为父本配置杂交组合,以亲本及其F₂分离群体为研究材料,采用AFLP技术筛选与抗叶霉病基因Cf12连锁的分子标记。通过对545对引物进行筛选,获得了6个连锁的AFLP标记：E86M51-C、E46M50、E95M59-D、E35M81、E78M36-C和E47M50-C,连锁遗传距离分别为5.1、8.9、9.1、9.2、10.2和10.8 cM。将E86M51-C转化为SCAR标记并应用于种质资源筛选,获得了16份含有该标记的番茄材料,为抗叶霉病育种提供材料基础。