小麦微卫星标记的开发与染色体定位英文文献翻译.doc

Theor Appl Genet (2005) 110: 550-560理论与应用遗传学（Theoretical and Applied Genetics） (2005) 110: 550-560Q. J. Song et al. Development and mapping of microsatellite (SSR) markers in wheat小麦微卫星标记的开发与染色体定位Abstract Microsatellite DNA markers are consistently found to be more informative than other classes of markers in hexaploid wheat. The objectives of this research were to develop new primers flanking wheat microsatellites and to position the associated loci on the wheat genome map by genetic linkage mapping in the ITMI W7984 Opata 85 recombinant inbred line (RIL) population and/or by physical mapping with cytogenetic stocks. We observed that the efficiency of marker development could be increased in wheat by creating libraries from sheared rather than enzyme-digested DNA fragments for microsatellite screening, by focusing on microsatellites with the ATT/TAAn motif, and by adding an untemplated G-C clamp to the 5-end of primers. A total of 540 microsatellite-flanking primer pairs were developed, tested, and annotated from random genomic libraries. Primer pairs and associated loci were assigned identifiers prefixed with BARC (the acronym for the USDA-ARS Beltsville Agricultural Research Center) or Xbarc, respectively. A subset of 315 primer sets was used to map 347 loci. One hundred and twenty-five loci were localized by physical mapping alone. Of the 222 loci mapped with the ITMI population, 126 were also physically mapped. Considering all mapped loci, 126, 125, and 96 mapped to the A, B, and D genomes, respectively. Twenty-three of the new loci were positioned in gaps larger than 10 cM in the map based on pre-existing markers, and 14 mapped to the ends of chromosomes. The length of the linkage map was extended by 80.7 cM. Map positions were consistent for 111 of the 126 loci positioned by both genetic and physical mapping. The majority of the 15 discrepancies between genetic and physical mapping involved chromosome group 5.摘要 微卫星DNA标记不断发现比在六倍体小麦标记其他类信息。本研究的目的是开发新的微卫星引物侧翼小麦和在ITMI W7984奥帕塔85重组自交系（RIL）群体和遗传连锁图谱的相关位点对小麦基因组地图/或物理图谱与细胞遗传学绘图。我们观察到的标记开发效率提高可以通过创建小麦，而不是从剪切酶消化的微卫星DNA片段的筛选，通过与ATT/TAAn的主体微型卫星为重点，并通过添加气相色谱库在5-引物结束。阿微540-侧翼引物共开发测试，从随机基因组文库注明。对引物及相关基因位点被分配与Xbarc分别。一个子集的315套引物，绘347个位点，第125个位点定位的物理图谱是单独的。人类与ITMI映射的222位点，在人的染色体上也有映射。考虑到所有映射的位点，126，125和96映射到的A，B和D基因组。21个新位点定位在与人类之前存在的标记相距10厘米，映射到染色体的末端。谱图谱的长度延长了80.7厘米。地图立场为双方的遗传和物理图谱定位在126位点一致。之间的遗传图谱和物理图谱所涉及的5组15条染色体的差异不多。Introduction DNA markers are required for the creation of genetic maps that can be used to discover the position of genes or quantitative trait loci (QTLs) controlling important traits. The same markers can be used to incorporate genes and QTLs into improved cultivars via marker-assisted selection (MAS). Also, markers tightly linked to important genes provide the starting point for map-based cloning of those genes. In bread wheat, the restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) marker systems have detected only low levels of intraspecific polymorphism. In contrast, microsatellite markers are consistently found to be highly polymorphic, easily visualized, stable, and co-dominant. The development of informative microsatellite markers in wheat is difficult and time-consuming due to its large genome size, polyploidy, and the high level of repetitive sequences in its genome. Approximately 570 publicly available wheat microsatellite primer sequences have been reported, which is a small number relative to the genome size of wheat, the latter estimated to be 16000 M bp/1 C. A map based on recombinant inbred lines (RILs) from the cross W7984 Opata 85 contained a total of 1214 markers, including 944 loci detected by 722 RFLP probes, 261 loci amplified by 223 microsatellite primer pairs, and nine other markers before the addition of markers reported herein. There are 121 gaps that are longer than 10 cM. Although there is an average of one marker every 2.8 cM, the relatively low likelihood that a specific marker locus will be polymorphic in any given single cross dictates the need for much greater marker density. Wheat is one few crops suited for cytogenetic mapping. In common wheat, the mapping of markers of the chromosome, arm, and sub-arm has been possible through the use of nulli-tetrasomic, ditelosomic, and deletion lines. Using aneuploid stocks, any markers can be used without the necessity of identifying polymorphism, a particularly significant factor in wheat with its low level of DNA polymorphism. These aneuploid stocks have been used to develop cytogenetically based physical maps of the wheat homoeologous groups, to determine the chromosome locations of genes and of molecular markers. These data have provided a wealth of information on the physical positions of genes controlling phenotypic traits and many aspects of wheat chromosome structure and function.Microsatellites are commonly assumed to be abundant in pericentromeric DNA and rare in the euchromatin arms. Alternatively, others have suggested that microsatellite sequences are generated by chance and therefore occur randomly throughout the genome reported that microsatellites are more abundant in transcribed regions. The usefulness of genetic markers for mapbased cloning and MAS is dependent on their proximity to the genes. Thus, it is important to examine the distribution of genes or QTLs versus that of microsatellites.This paper presents information pertinent to the efficient development of 540 new, public domain microsatellite-amplifying primer pairs. This set of primer pairs and associated marker loci bear the prefix BARC, reflective of their origin at the Beltsville Agricultural Research Center. We also report the genetic and/or physical location of 347 loci associated with 315 of the BARC primer pairs. In addition, the distribution of microsatellites in reference to known genes and QTLs is assessed.介绍 DNA标记都需要的，可以用来发现的基因或数量性状位点（QTLs）控制重要性状的遗传图谱的位置设立。同样的标记可以用来改良品种纳入通过标记辅助选择（MAS）的基因及基因定位。此外，标记紧密连锁的基因提供了重要的地图为基础的这些基因克隆的起点。在面包小麦，限制性片段长度多态性（RFLP），扩增片段长度多态性（AFLP），随机扩增多态性DNA（RAPD）标记系统已检测到种内多态性很低。相反，微卫星标记不断发现具有高度多态性，容易直观，稳定，合作占主导地位。在翔实的小麦微卫星标记的发展是困难和费时，由于其庞大的基因组大小，多倍体，以及在其基因组重复序列高的水平。大约570微卫星引物公开小麦序列已报道，这是一个小数目相对于小麦基因组的大小，后者估计为16000平方米的BP / 1三，地图的基础上重组自交系（RIL群体）从十字架W7984 奥帕塔85载有1214标记，包括722个RFLP探针，微卫星引物对223位点扩增出261，和其他9个指标检测前944位点的标记除了本报告的总数。有121的差距，比10厘米长。虽然是一个标记，平均每2.8厘米，相对较低的可能性某个特定的标记基因会在任何给定的单交多态性决定了更大的标记密度的需要。小麦细胞遗传学映射为适合少数农作物之一。在普通小麦中，对染色体，手臂和分部门标记映射有可能通过使用缺体，四体，双端体和删除线。使用非整倍体的股票，可以使用任何标记没有确定多态性，在小麦，特别是其显着的DNA多态性水平低的因素的必要性。这些非整倍体股票已被用于开发基于小麦细胞遗传学部分同源群物理图，确定基因的染色体定位及分子标记。这些数据已对表型性状基因控制的物理位置和小麦染色体结构和功能的许多方面丰富的信息。微卫星通常假定在常染色质在近着丝粒DNA的武器和稀有丰富。另外，一些议员建议，所产生的微卫星序列发生的机会，因此在整个基因组随机报道，微有更丰富的转录区域。为mapbased的克隆和MAS是依赖于他们的邻近的基因遗传标记的效用。因此，重要的是，研究基因或QTL定位与微卫星的分布。本文介绍了有关的资料540个，公共领域的微卫星，引物扩增高效发展。这对引物标记位点的设置及相关承担在贝尔茨维尔农业研究中心的前缀巴巴原子，其原产地反思。我们还报告基因和/或与引物对315的巴巴原子研究相关的347个位点的物理位置。此外，在参考已知基因和QTL的微卫星分布进行评估。DiscussionEfficiency of marker developmentRestriction enzyme-versus nebulizer-derived genomic libraryThere was a marked difference in the spectrum of DNA fragments derived from the two types of libraries, and this difference had a large impact on the success of SSR marker development. The results indicate that microsatellite-containing clones derived from sheared DNA are much less likely to contain sets of clones with similar or nearly identical sequences. We observed that while very few sequences were identical in the libraries constructed by the two methods, there were significantly higher frequencies of similar sequences among SSR-containing clones in the library derived from enzyme digestion. Typical examples of such sets of similar sequences from the restriction enzyme-derived library are shown in Fig.1. The likely explanation for this result is that conserved restriction sites in duplicated and homoeologous regions yielded clones with very similar but not identical sequences. In contrast, the physically sheared library yielded a random sampling of genomic fragments. It may be possible to develop locus-specific PCR primers to many of the sequences within the sets such as those shown in Fig.1.The conversion of microsatellite-containing sequences into useful markers is sometimes quite difficult, especially in species with large genomes. Roder et al. reported that only one of six microsatellite-containing clones yielded a functional marker; the remaining primer pairs either gave gave poor amplification or produced a complex pattern with multiple bands. A similarly low conversion rate from SSR-containing sequences to useful markers has been observed in pine and spruce. The low conversion rates of primer pairs to useful markers in these latter two species was probably due to the high level of repetitive DNA sequences due to the high level of repetitive DNA sequences or to similar sequences in homoeologous genomes. In the present study, primers were designed to the sequences containing dinucleotide repeats CT/GAn or CA/GTn, the tri-nucleotide motif ATT/TAAn, and the tetra-nucleotide TAGA/ATCTn. The rate of polymorphic markers was 29%, 24%, 36%, and 28% for CT/GAn, CA/GTn, ATT/TAAn and TAGA/ATCTn, respectively. A previous investigation indicated that the ATT/TAAn motif was superior to all other trinucleotide repeats for the successful development of polymorphic microsatellite markers. The current data strongly suggest that the ATT/TAAn motif is also superior to the two most commonly used dinucleotide motifs and one tetra-nucleotide motif in terms of the rate with which polymorphic marker loci can be developed. Although dinucleotide repeats are the most commonly used class of microsatellite markers in plants, dinucleotide-based loci often prove difficult to genotype due to a high frequency of strand-slippage artifacts. In contrast, tri- and tetra-nucleotide repeat-based markers generally produce a higher proportion of discrete PCR products as a result of a reduced level of strand-slippage artifacts.讨论效率指标的发展限制性内切酶抗雾化器源性基因组文库有一个明显的DNA差异片段谱图书馆的两种派生的，而这种差异有对SSR标记的发展成就很大的影响。结果表明，微含从剪切DNA的派生克隆是不太可能含有相同或几乎相同的序列克隆套。我们发现，虽然很少序列，由两种方法构建的库相同，苏维埃社会主义共和国之间也存在着含有从消化酶所产生的类似序列的克隆库中显着高于频率。类似这样的序列集的典型例子，限制性内切酶源库列于图1。产生这一结果的可能的解释是，在重复和地区产生了同源的保守与克隆酶切位点很相似但不完全相同的序列。相反，身体产生了剪切库随机抽样的基因片段。它可能集内发展轨迹特异性PCR引物的序列很多，如在图1所示的。该微含序列转换成有用的标记，有时相当困难，尤其是在物种，与大基因组。Roder等人报告说，只有一六微含克隆产生了一个功能标记，其余引物扩增要么给了穷人或生产具有多个乐队复杂的模式。从苏维埃社会主义共和国含有有用的标记序列已被观察到松树和云杉在同样低的转换率。对引物对这些低转换率后两个物种的有用指标是可能是由于重复的DNA序列，由于高层次的重复DNA序列或在部分同源基因组序列高度相似。在本研究中，设计引物，含二核苷酸重复序列CT /GAn或CA/GTn的，在三核苷酸重复ATT/TAAn和四核苷酸TAGA/ATCTn注多态标记率分别为29，24，36和28%。一个以前的调查显示，ATT/TAAn的主题是优于所有的微卫星多态性成功发展的其他三核苷酸重复序列。目前的数据有力地表明，在ATT/TAAn的主题，也优于两个最常用的多态性标记位点与其中可开发方面的和核苷酸主题。虽然二核苷酸重复是在植物微卫星标记最常使用的一类，核苷酸的基因位点的基因型往往难以证明。相比之下，三个和四个核苷酸重复的标记通常产生一种作为一个串，结果减少离散程度较高比例的PCR产物。MappingIn relation to RFLPs, SSR markers are generally more genome specific. This avoids the ambiguity that sometimes results with RFLP probes that hybridize to two or more positions in the wheat genome. Among the 255 new polymorphic SSR markers reported here, only 20 (7.8%) markers were genetically mapped to two or more genome positions, a proportion similar to that found in rice. This is in contrast to the results obtained with RFLP markers by Nelson et al. and Van Deynze et al.: of 722 RFLP markers, 222 (31%) mapped to more than two positions.One hundred primer pairs did not yield hands which could be physically mapped to a chromosome; of these, 88% were markers that were not polymorphic among the very diverse genotypes: Chinese Spring, Opata 85 and W7984. In contrast, of the 255 markers that were polymorphic for which physical mapping was attempted, only 12 could not be unambiguously positioned on a chromosome. It is our assumption that the loci that could not be physically mapped amplified homoeologous sites with identical or very similar PCR fragment sizes or amplified from cytoplasmic organelle DNA.Deletion mapping provides a simple and rapid method to construct cytogenetically based physical maps. Markers can be used without the necessity of identifying polymorphism, a particularly significant factor in a species in which the lack of DNA polymorphism is a major obstacle. Recombination suppression in proximal regions of the chromosomes limits the power of recombination mapping in ordering proximal loci. The amount of DNA per centi-Morgan generally increases from the telomeres to the centromere, with the exception of recombination “hot spots”. For proximal regions, physical mapping is more powerful for resolving the order of loci. Physical mapping with genetic mapping information could help in the analysis of the physical distribution of recombination within each chromosome region and the ac