植物染色体原位杂交技术及其在稻属研究中的应用

本文介绍了植物染色体原位杂交技术,以及该技术在稻属特定DNA序列定位、基因组间关系、外源染色体鉴定等研究中的应用.     

荧光原位杂交技术在植物细胞遗传学和绘制基因图谱中的应用现状与展望

荧光原位杂交是在分子水平上检测外源染色质的一种有效方法。其探针主要有染色体重复序列、总基因组DNA、寡单拷贝序列和染色体涂色集中等,该技术在研究植物细胞遗传学、基因扩增、基因作图及植物进化和亲缘关系的鉴定上已广泛应用。简要概述了荧光原位杂交技术在植物细胞遗传学和绘制基因图谱中的应用现状与展望。     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

转基因水稻BPL9K-2事件特异性检测方法的建立

利用hiTAIL-PCR(high efficient thermal asymmetric interlaced PCR)法扩增获得了转基因水稻BPL9K-2的外源基因插入位点的左旁侧序列450bp,与水稻参考基因组数据比对发现其左边界插入在水稻基因组第10号染色体短臂的1 037 765位核苷酸残基之后。根据水稻参考基因组序列和外源基因右边界序列,设计引物扩增得到485bp的特异片段,通过数据库比对发现其右边界插入在水稻基因组第10号染色体短臂的1 037 825位核苷酸残基之前。因为外源基因插入和非正常重组,水稻基因组上缺失了59个核苷酸。基于左右旁侧序列,建立了转基因水稻BPL9K-2的事件特异性定性PCR检测方法,可以分别扩增到片段大小为449bp和485bp的特异条带。该方法特异性好,灵敏度高,能够在BPL9K-2基因组DNA相对含量为0. 1%的模板中检测出转基因成分。依据旁侧序列,建立了快速鉴定转基因后代植株外源基因型的三引物PCR检测方法。这些方法的建立,为转基因水稻BPL9K-2的应用和检测提供了技术支持。     

远缘杂交早代稳定小麦导入了外源DNA片段并发生了DNA重排

在植物界, 多倍体植物非常普遍. 具有A, B, D三个部分同源染色体组的普通小麦是异源多倍体物种的一个典型代表. 近几年, 模拟普通小麦的起源过程进行的研究表明, 从四倍体小麦注入节节麦整个基因组形成异源六倍体小麦的早期阶段, DNA序列和基因表达发生了可能有利于遗传“二倍化”的变化. 利用普通小麦-黑麦远缘杂交自然结实的早代稳定特异小麦99L2研究发现: (ⅰ) 99L2至少导入了两个黑麦染色体上的DNA片段, 表明可能存在不同于传统的小麦-外源染色体配对重组的外源DNA导入机制; (ⅱ) 99L2自身的DNA序列发生了变化, 表明外源DNA部分片段注入小麦染色体组过程中, 也可能导致小麦自身DNA序列发生变化.     

转Xa21基因水稻中T-DNA整合的遗传定位

利用转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR方法扩增T－DNA整合的侧翼序列。从中筛选属于水稻基因组DNA的T－DNA整合的侧翼序列作为探针,将外源基因整合位点定位到窄叶青／京系17DH群体构建的水稻分子连锁图谱上。共获得属于水稻基因组DNA的T－DNA侧翼序列22个,其中的19个序列在定位群体的两个亲本之间显示RFLP多态性,分别定位在水稻基因组的第3,4,5,7,9,10,11和12染色体上。带有转基因Xa21的T－DNA整合的定位为研究外源基因在不同染色体位点的位置效应和稳定遗传打下基础。     

假基因的组成、分布及其分子进化

假基因（pseudogene）是指基因组中与正常基因序列相似,但是缺乏功能的DNA序列.通过序列同源性搜索,可以收集基因组中假基因的群体特性、染色体分布和同源家族等特性.假基因很好地保留了数百万年前基因组中祖先基因的分子记录,被视为＂基因化石＂,因此假基因在进化和比较基因组学中是重要的资源.应用假基因和基因比较体系,可以探究生物基因的进化史和基因组稳定性.如：用Ka/Ks比值确定假基因的自然选择压、物种亲缘关系和进化距离,分析假基因自身的进化趋势,探讨DNA突变的成因等.     

绵羊胚胎性别鉴定试剂盒的研究

根据绵羊Y-染色体的特异序列和常染色序列分别设计了确定公羊Y-染色体特异序列的3对特异性引物和内标基因的4对特异性引物。单重PCR扩增绵羊基因组DNA,筛选出了3对Y-染色体特异引物和3对羊DNA特异内标引物。将不同的绵羊Y-染色体特异引物与内标引物组合,利用多重PCR扩增绵羊基因组DNA,筛选出了1个可用于羊早期胚胎性别鉴定的PCR引物组合:A0/C1。按照最优PCR扩增DNA条件配制了绵羊PCR性别鉴定试剂盒并成功应用于绵羊血液、已知性别的绵羊成纤维细胞和胚胎,表明本研究建立的体系完全可用于绵羊早期的胚胎性别鉴定。     

细菌基因组重复序列PCR技术及其应用

细菌中散在分布的DNA重复序列近年来不断被报道,基因外重复回文序列和肠细菌基因间共有重复序列是两个典型的原核细胞基因组散在重复序列。重复序列在染色体上的分布和拷贝数具种间特异性,用它们的互补序列作为引物,以细菌基因组DNA为模板进行PCR扩增反应,反应产物的琼脂糖电泳可以提供非常清晰的DNA指纹图谱,使用此图谱既可对各种微生物进行快速分型及鉴定,又可对它们进行DNA水平上的遗传多样性分析。细菌基因组重复序列PCR技术具有简捷、快速、结果稳定等特点,可对细菌进行分子标记,用于菌株分型、分类鉴定和亲缘关系等方面的研究。     

侧翼序列获取技术研究进展

侧翼序列是指染色体中特定位点两侧的DNA序列,包含着候选基因、转录调控、染色体结构、生物安全等信息,在基因组学研究中具有重要的作用。侧翼序列获取技术主要应用于启动子和增强子等调控序列的克隆、鉴定T-DNA或转座子插入位点、染色体步移、全基因组空隙填补等,是结构基因组研究以及功能基因组研究的重要手段,在转基因动植物鉴定及安全管理等方面具有重要应用。随着分子生物学的发展,目前已经建立了许多侧翼序列的获取方法,依据技术原理可以分为质粒拯救法、反向PCR法、外源接头介导PCR法、半随机引物PCR法和基因组重测序法等5大类。本文系统总结了近年来侧翼序列获取技术的研究进展,并对这些技术的原理以及应用情况进行了较为系统的综述,为侧翼序列信息的获取提供参考。     

小麦基因组中外源染色体片段的检测和小麦基因分子标记的建立

小麦基因组中外源染色体片段的检测和小麦基因分子标记的建立@石锐$哈尔滨师范大学生物系!150080小麦;;外源染色体;;分子标记     

Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe--a landmark approach for Thinopyrum genome research

The introduction of alien genetic variation from the genus Thinopyrum through chromosome engineering into wheat is a valuable and proven technique for wheat improvement. A number of economically important traits have been transferred into wheat as single genes, chromosome arms or entire chromosomes. Successful transfers can be greatly assisted by the precise identification of alien chromatin in the recipient progenies. Chromosome identification and characterization are useful for genetic manipulation and transfer in wheat breeding following chromosome engineering. Genomic in situ hybridization (GISH) using an S genomic DNA probe from the diploid species Pseudoroegneria has proven to be a powerful diagnostic cytogenetic tool for monitoring the transfer of many promising agronomic traits from Thinopyrum. This specific S genomic probe not only allows the direct determination of the chromosome composition in wheat-Thinopyrum hybrids, but also can separate the Th. intermedium chromosomes into the J, J(S) and S genomes. The J(S) genome, which consists of a modified J genome chromosome distinguished by S genomic sequences of Pseudoroegneria near the centromere and telomere, carries many disease and mite resistance genes. Utilization of this S genomic probe leads to a better understanding of genomic affinities between Thinopyrum and wheat, and provides a molecular cytogenetic marker for monitoring the transfer of alien Thinopyrum agronomic traits into wheat recipient lines.     

C-banding and fluorescence in situ hybridization studies of the wheat-alien hybrid 'Agrotana'.

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat x Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' x 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat-alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat-alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.     

Selection of 29 highly informative InDel markers for human identification and paternity analysis in Chinese Han population by the SNPlex genotyping system

The interest of forensic researchers in single nucleotide polymorphism (SNP) has been attracted because of its potential advantages, such as low mutation rates, amenable to high-throughput automated platform and the improved application in the analysis of degraded samples. In this paper, 29 highly informative insertion/deletion (InDel, a special kind of SNP) markers were selected from the dbSNP () according to the given criteria. 109 unrelated Chinese Han subjects were genotyped for the 29 InDels with SNPlex genotyping system. The allele frequency data revealed that the combined power of discrimination for the 29 InDel markers was 0.999999999990867 and the combined probability of paternity exclusion (PE) was 0.9930. Sensitivity studies were performed to evaluate the flexibility of the SNPlex genotyping system on the set of 29 InDels. Highly reproducible results could be obtained with 40–100 ng genomic DNA and the proportion of total allele drop-in was significantly increased when the amount of DNA added to PCR was lower than 35 ng. These results suggested that the set of 29 InDels was useful in paternity analysis or human identification in the future.     

Development of genome-wide DNA polymorphism database for map-based cloning of rice genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).     

Targeted introgression of a wheat stem rust resistance gene by DNA marker-assisted chromosome engineering

Chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of chromosome engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between wheat chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87-9.15% of the translocated chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of chromosome engineering in genome manipulation for plant improvement.     

Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation

Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare, Secale cereale and Agropyron cristatum in the wheat background was also reported.     

First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome

A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (pi = 0.72 x 10(-3)) was lower when compared with diploid genotypes (pi = 2.31 x 10(-3)). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet.     

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).