图位克隆技术新型遗传学实验教学项目的设计与实践

染色体重组与连锁互换是遗传学教学的重点和难点。为使学生更好地理解此方面知识, 我们课题组前期利用图位克隆(map-based cloning)技术, 克隆了1个调控水稻(Oryza sativa)类病变表型的基因SPL5, 并基于此设计了一个新的综合型遗传学实验, 即利用DNA分子标记对基因进行定位。实验中学生利用水稻spl5突变体与野生型杂交获得的F₂代定位群体和多态性分子标记, 对spl5突变进行染色体连锁分析、初步定位和遗传作图。该教学实验不仅可有效促进学生对遗传学三大定律的理解, 而且对其开阔视野、提高解决问题和团队协作的能力也有促进作用。     

水稻斑点叶突变体spl101和spl102的筛选及候选基因鉴定

斑点叶突变体是指植物在正常生长条件下叶片或叶鞘上自发形成、且与病原菌侵染产生的病斑类似的一类突变体,筛选并研究斑点叶突变体对揭示植物抗病反应机理具有重要意义。为了进一步研究斑点叶的形成机制,本文通过EMS诱变品种宽叶粳(KYJ),筛选得到两个斑点叶突变体spl101和spl102。这两个突变体在生长发育晚期(抽穗期以后)形成严重的类病斑。遗传分析表明,spl101和spl102均受隐性单基因控制。利用Mutmap方法对候选基因进行克隆,结果显示,spl101和spl102的候选基因均为OsEDR1,该基因与类病斑发生有关。在spl101中,OsEDR1基因突变发生在第6外显子和第6内含子的交接处,该突变导致第6内含子的错误识别,最终造成移码突变。在spl102中,OsEDR1基因突变发生在第10外显子上,导致一个苯丙氨酸(F)变成半胖氨酸(C)。因此,本研究鉴定了两个新的OsEDR1等位突变,对OsEDR1抗病反应机理的进一步研究及丰富水稻种质资源具有积极意义。同时验证了利用Mutmap方法克隆水稻突变基因的有效性。     

水稻顶节间长度控制基因(EUI)的精细定位

通过对一水稻顶节间特异伸长突变体Mh-1进行经典遗传学和基因定位分析,认为该表型是一个核基因隐性突变所致。利用Mh-1与正常的T65-sd1杂交的F2群体对该位点定位研究分析,发现其与水稻第5条染色体长臂STS标记E30531和CAPS标记C903连锁,遗传距离分别为6．7cM和2．8cM。经进一步发展新的分子标记,将该基因精确定位在0．3cM的区域,为进一步克隆和研究该基因的分子机制奠定基础。     

水稻抗白叶枯病基因Xa12区间连锁图的构建

利用SSR标记对143株Java14/珍珠矮F₂随机群体进行分析,构建了水稻第4染色体上抗白叶枯病基因Xa12高饱和度的SSR标记区间连锁图。该分子图谱整合了SSR标记RM349、RM348、RM255、MRG4611,为进一步利用SSR标记在Java14/珍珠矮F₂群体中精细定位Xa12以及克隆该基因奠定了基础。     

基于CSSLs群体定位和图位克隆水稻长芒基因GAD1-2

水稻是世界上最早驯化的重要粮食作物之一。水稻芒可以保护水稻种子不被鸟琢食,是水稻重要的驯化性状之一。芒在野生稻中普遍存在,对野生稻的生存和传播至关重要,然而在驯化和人工选择过程中该性状逐渐被淘汰。定位和克隆水稻长芒相关基因是研究水稻芒驯化遗传机制的基础。本研究以籼稻恢复系东南恢810为受体、漳浦野生稻为供体构建的146个染色体片段置换系(chromosome segment substitution lines, CSSLs)为研究材料,调查了146个CSSLs株系和双亲的芒长,结果表明在4个置换系中检测到1个控制水稻芒长主效基因GAD1-2,位于水稻第8号染色体;利用重叠代换作图法,将GAD1-2定位在Ind8-10和RM4936标记之间,遗传距离约为4.75 Mb。选择分离群体中的显性单株,利用开发的标记,最终将GAD1-2 基因定位在两个 Indel 标记之间,两者间的物理距离约为27 kb,该区域内只有两个候选基因Os08g0485500和Os08g0485400。经测序和分析表明,Os08g0485500是GAD1-2的候选基因,GAD1-2在保守的ORF区域存在6个碱基缺失,导致丝氨酸和半胱氨酸这两个氨基酸缺失,从而表现长芒的性状;在Os08g0485500基因位点已克隆了1个控制水稻芒长的GAD1基因,推测GAD1-2与GAD1为等位基因。本研究为进一步理解水稻起源演化和水稻芒长发育基因的遗传机制奠定了基础。     

用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法.通过对水稻(Oryza sativa L.)"安农S-1"和"南京11"的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFLP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1 537.4 cM,相邻标记间的平均间距为9.0 cM,这是在国内建立的第一张AFLP标记连锁图.在建立连锁图谱的同时把一个新基因tms5 (水稻温敏核不育基因)定位在第2染色体上.     

水稻优良性状控制基因的定位进展及其在染色体上的分布

利用分子标记可以寻找并且定位目的基因, 分子标记辅助育种技术则能够快速、高效地筛选出携带目的基因的优良品种。文章总结了近年来水稻中已被定位的优良性状控制基因及与其连锁的分子标记, 其中包括抗病虫害、抗寒、抗旱、不育、育性恢复等194个基因, 这些基因中已有14个被克隆测序。在此基础上探讨了这些基因在染色体上的分布趋势。     

用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法。通过对水稻(Oryza sativa L．)“安农S-1”和“南京11”的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFIP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1537．4cM,相邻标记间的平均间距为9．0cM,这是在国内建立的第一张AFLP标记连锁图。在建立连锁图谱的同时把一个新基因tms5(水稻温敏核不育基因)定位在第2染色体上。     

野败型水稻细胞质雄性不育恢复基因Rf-4的分子标记定位

为了用分子标记准确定位野败型水稻细胞质雄性不育恢复基因Rf-4,将日本水稻基因组项目（Rice Genome Program,RGP)构建的水稻遗传连锁图谱第10染色体分子遗传图上的分子标记R1877和G2155之间对应区域YAC物理图上的6个YAC克隆进行了亚克隆,获得119个片段,对这些探针进行多态性探查,获得了2个多态分子标记,用珍汕97A和恢复基因近等基因系的杂种F2分离群体中的117完全不育株进行连锁分析表明,从YAC4892获得的亚克隆Y3－8与Rf－4座位的连锁距离为0．9cM,从YAC4630获得的亚克隆Y1－10与Rf－4座位的连锁距离为3．2cM,根据以上结果把Rf－4座位定位于第10染色体的特定位置,为该基因的分子标记辅助选择和定位克隆打下了基础。     

基于SSLP分子标记验证遗传学三大定律的教学实践探索与体会

文章对“水稻SSLP分子标记的遗传分析”作为遗传学实验教学案例的实施过程及其效果进行了探讨。利用位于水稻两条染色体上的3个SSLP标记,对两亲本及其杂交构建的F₂代群体进行单株SSLP标记基因型检测,利用检测所得到的基因型结果验证分离定律、独立分配定律及连锁交换定律等遗传学三大定律。实践证明这不仅有利于加深学生对遗传学三大定律的认识,而且在提高学生实验操作技能和综合分析能力的基础上,还有助于培养学生的科研兴趣和创新意识。同时,对该实验的适用范围以及尚需完善之处做了讨论。此综合性实验也是科研成果转化为本科实验教学的一个有益探索。     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

Exploring the genetic characteristics of 93-11 and Nipponbare recombination inbred lines based on the GoldenGate SNP assay

Understanding genetic characteristics in rice populations will facilitate exploring evolutionary mechanisms and gene cloning. Numerous molecular markers have been utilized in linkage map construction and quantitative trait locus (QTL) mappings. However, segregation-distorted markers were rarely considered, which prevented understanding genetic characteristics in many populations. In this study, we designed a 384-marker GoldenGate SNP array to genotype 283 recombination inbred lines (RILs) derived from 93-11 and Nipponbare Oryza sativa crosses. Using 294 markers that were highly polymorphic between parents, a linkage map with a total genetic distance of 1,583.2 cM was constructed, including 231 segregation-distorted markers. This linkage map was consistent with maps generated by other methods in previous studies. In total, 85 significant quantitative trait loci (QTLs) with phenotypic variation explained (PVE) values≥5% were identified. Among them, 34 QTLs were overlapped with reported genes/QTLs relevant to corresponding traits, and 17 QTLs were overlapped with reported sterility-related genes/QTLs. Our study provides evidence that segregation-distorted markers can be used in linkage map construction and QTL mapping. Moreover, genetic information resulting from this study will help us to understand recombination events and segregation distortion. Furthermore, this study will facilitate gene cloning and understanding mechanism of inter-subspecies hybrid sterility and correlations with important agronomic traits in rice.     

案例教学在林学专业遗传学教学中的应用

遗传学是林学专业本科生普遍反映最难学习的必修课程之一,传统的讲授式教学很难满足遗传学教学的需求。根据林学专业的理论特点和实际需求,结合近几年的教学和科研经验,选取了“开棺验亲”、“黑果枸杞茎刺”、“哈利·波特与魔法力”等一系列典型的案例应用于林学专业的遗传学教学。实践证明针对遗传学的案例教学能培养学生的学习能力和创造力、激发学生学习兴趣和学习的主动性,同时也培养了学生良好的个性品质并在一定程度上增强知识学习的系统性。     

水稻叶片水势的QTL定位与候选基因分析

为探究叶片水势(LWP)相关基因在水稻(Oryza sativa)抗旱中的作用及其遗传机制,以热研2号(Nekken2)和华占(HZ)为亲本以及构建的120个重组自交系(RILs)群体为实验材料,对水稻分蘖期叶片水势进行检测,并利用前期基于高通量测序构建的分子遗传连锁图谱进行数量性状基因座(QTL)分析。结果表明,共检...     

Mapping maize genes: a series of research-based laboratory exercises

Open-ended, inquiry-based multiweek laboratory exercises are the key elements to increasing students' understanding and retention of the major biological concepts. Including original research into undergraduate teaching laboratories has also been shown to motivate students and improve their learning. Here, we present a series of original laboratory exercises on fine mapping novel maize mutations producing interesting phenotypes. In this 4-week lab series, students get involved in the whole process of identifying novel genes controlling specific phenotypes, from phenotype characterization and choosing appropriate molecular markers to calculating the genetic distance between the mutation and the marker and finding possible candidate genes using a complete genome sequence. We chose to use maize mutant lines produced by TILLING project. These lines have been partially mapped to a chromosomal bin by a high-throughput bulk segregant analysis; however, the exact map positions for these mutations have never been determined. Mapping these novel maize mutations provides students with the opportunity to conduct original research as a part of their classroom experience and to contribute to the field of maize genetics. The laboratory series was well received by the students, and the assessment results demonstrated an improvement of student learning of gene mapping, molecular marker analysis, and positional cloning concepts.     

ABO血型综合实验基因分型技术简化及其群体遗传分析拓展

ABO血型是生活中最常见、运用最广泛的遗传性状之一。人类ABO血型由I ^A、I ^B和i 3个复等位基因决定,它们负责编码不同的糖基转移酶,进而决定3种血红细胞表面抗原。ABO血型涉及复等位基因、基因互作、单核苷酸多态(SNP)、基因演化等多个关键知识内容,是理想的遗传学教学案例。本文以ABO血型为研究对象,对遗传学实验进行了创新与整合。首先,在分子遗传学模块中建立了新颖的ABO血型基因分型方法：基于SNP位点设计特异性引物,通过实时定量PCR鉴定基因型; 其次,在群体遗传学模块中创新了基因演化的实验教学方法,开发群体遗传学软件,利用计算机模拟不同条件下ABO血型决定基因频率的演化趋势。这些教学改革举措旨在丰富遗传学实验内容,拓展教学手段,提高学习效率。     

Development and characterization of a genetic linkage map of Cryptococcus neoformans var. neoformans using amplified fragment length polymorphisms and other markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.