小麦指纹图谱数据库的建立及SSR分子标记试剂盒的研发

本研究以国际玉米小麦改良中心（CIMMYT）、国际干旱地区农业研究中心（ICARDA）、法国Agropolis研究所和中国农业科学院作物科学研究所提供的数据为基础，建立了包括134个SSR引物、2457个普通小麦基因型的指纹图谱数据库。利用荧光标记法的分析结果，用代表性基因型在某一位点的扩增片段作为银染法的读带依据，开发了SSR分子标记试剂盒，包括46对SSR引物及其PCR反应程序、代表性基因型的DNA样品、592个等位变异的代表性基因型清单及试剂盒使用说明等。该数据库的建立和试剂盒的开发，为利用SSR分子标记技术进行小麦种质遗传多样性研究，实现小麦遗传资源和信息资源全球共享提供了重要工具和技术平台。     

Identification and validation of a core set of microsatellite markers for genetic diversity analysis in watermelon, <Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb. Matsum. &; Nakai

Watermelon, Citrullus lanatus Thunb. Matsum. & Nakai is an important vegetable crop worldwide. Due to its narrow genetic base, detection and utilization of the genetic variations, cultivar identification and increasing genetic diversity are some important tasks for watermelon breeders. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for these purposes. In the present study, a core set of 23 highly informative SSR markers was developed for watermelon genetic diversity analysis. Based on whole genome sequencing of 17 watermelon inbred lines, we identified 3.9 million single nucleotide polymorphisms (SNPs) which were used to construct a SNP-based dendrogram for the 17 lines. Meanwhile, from the sequenced genome, 13,744 SSRs were developed, of which 704 were placed on a high-resolution watermelon linkage map. To develop the core set SSR markers, 78 of the 704 mapped SSRs were selected as the candidate markers. Using the SNP-based dendrogram as calibration, 23 SSR markers evenly distributed across the genome were identified as the core marker set for watermelon genetic diversity analysis. Each marker was able to detect 2–7 alleles with polymorphism information content values ranging from 0.45 to 0.82. The dendrograms of 17 watermelon lines based on SNPs, the base set of 78 SSRs and the core set of 23 SSRs were highly consistent. The utility of this core set SSRs was demonstrated in 100 commercial watermelon cultivars and elite lines, which could be placed into six clusters that were largely consistent with previous classification based on morphology and parentage data. This core set of SSR markers should be very useful for genotyping and genetic variation analysis in watermelon.     

A physical map of chromosome 4Hch from H. chilense containing SSR, STS and EST-SSR molecular markers

The construction of a physical map of chromosome 4H^ch from Hordeum chilense containing molecular markers capable of detecting segments of this chromosome in a wheat background would be very useful for marker-assisted introgression of 4H^ch chromatin into both durum and common wheat. With this aim, the applicability of 106 barley chromosome 4H primers (62 SSRs and 44 STSs) to amplify markers showing polymorphism between H. chilense and both common or bread and durum wheat was investigated. Twenty-five SSR (40.3%) and six STS (13.6%) barley primer pairs consistently amplified H. chilense products. Eight SSR (12.9%) and four STS (9.1%) barley primers were polymorphic between H. chilense and both common and durum wheat, 10 of them (6 SSRs and 4 STSs) were located on chromosome 4H^ch using both the addition line of chromosome 4H^ch in Chinese Spring wheat and a tritordeum line (an amphiploid between H. chilense and T. turgidum) nullisomic for chromosome 4H^ch. Additionally, 18 EST-SSR barley markers previously located on chromosome 4H^ch were screened for polymorphism; 15 were polymorphic between H. chilense and both durum and common wheat. For physical mapping we used a ditelosomic tritordeum line for the short arm of chromosome 4H^ch and a tritordeum line homozygous for a 70% terminal deletion of the long arm of 4H^ch. A total of 25 markers (6 SSRs, 4 STSs and 15 EST-SSRs) were mapped to chromosome 4H^ch. Eight markers were allocated on the 4H^chS, eight were mapped in the 30% proximal region of 4H^chL and nine were on the 70% distal region of 4H^chL, respectively. Arm location on barley chromosome 4H was also carried out using both 4HS and 4HL ditelosomic addition lines in wheat. All markers mapped may have a role in marker-assisted introgression of chromatin segments of chromosome 4H^ch in both durum and common wheat backgrounds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Validation of a set of informative simple sequence repeats markers for variety identification in Pak‐choi (Brassica rapa L. ssp. chinensis var. communis)

A core set of 21 simple sequence repeats (SSR) markers was developed for Pak‐choi (Brassica rapa ssp. chinensis var. communis) variety identification. We initially selected 74 SSR markers which exhibited high polymorphism and reproducibility in SSR detection from 2129 SSRs. Using the 74 SSR‐based dendrogram for 45 inbred lines as calibration, 21 core SSRs were selected out. The utility of this core set SSRs was firstly tested in 45 inbred lines and finally verified in 102 commercial varieties. We also constructed a molecular ladder for each core SSR as a reference standard. Diversity analysis of this core SSR panel in 102 varieties demonstrated that each marker generates 2–3 alleles (averaged 2.33), with polymorphism information content values ranging from 0.01 to 0.56 (averaged 0.31). The averaged values of Shannon information index, observed heterozygosity, expected heterozygosity and Wright's fixation index were 0.59, 0.43, 0.38 and −0.09, respectively. Furthermore, the 21 SSR‐based classifications for 102 varieties were consistent with traditional classification based on morphology. This core SSR panel represents an effective tool for genetic variation analysis in Pak‐choi.     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.     

用于区别不同棉花品种基因组特征的微卫星位点筛选

保守性强、重复性好、多态性高的微卫星位点可被有效用于构建作物DNA条形码。选取目前生产上主要推广种植、代表不同来源系统的12个棉花品种作为微卫星位点筛选材料,参考我室构建的四倍体栽培棉种种间高密度遗传图谱信息,从376对覆盖全基因组的SSR引物中,筛选出51对引物可扩增出带型清晰且多态性高的微卫星位点。这些引物在12个供试品种中共产生155个等位位点,每对引物揭示的等位基因位点在2~7之间,平均值为3.04。参照微卫星位点的染色体定位和多态信息,在每条染色体上选择一个多态性相对高的SSR位点,其相应的26对SSR引物被推荐为构建棉花品种DNA条形码的一套首选引物,并初步应用于12个品种的DNA条形码编制。其余25对引物作为候选引物。使用该套引物扩增出的微卫星位点可用于大量棉花品种DNA条形码构建,为棉花品种真实性和纯度的分子鉴定奠定基础。     

普通小麦中国春-百萨偃麦草异染色体系的分子标记分析

综合利用HMW-Glu亚基、STS、SSR和RFLP等分子标记对普通小麦中国春、百萨偃麦草、中国春-百萨偃麦草双二倍体和11个中国春-百萨偃麦草异染色体系进行了分析。结果表明，14对SSR、10对STS引物和6个RFLP标记可以特异追踪百萨偃麦草染色质。C7-17及其后代株系C7-17-2等编码百萨偃麦草特异HMW-Glu亚基，添加染色体涉及与小麦第１部分同源群染色体部分同源的1J；1对STS、3对SSR和1个RFLP探针可以特异追踪二体附加系CH05中的百萨偃麦草染色体，并揭示最初根据分带核型确定的J3与小麦第2部分同源群染色体具有较高的部分同源性；2对STS、1个RFLP探针和1对SSR可以追踪CH09的外源染色体，并揭示最初确定的J7与小麦第3部分同源群染色体具有较高的部分同源性；1对STS和1个RFLP探针在CH03、CH04和CH34中具有相同的多态，3个附加系可能添加了相同染色体，最初确定的J₁、J₂和J_?与小麦第7部分同源群染色体具有较高的部分同源性；3对SSR引物可以特异追踪CH12中附加的大片段易位染色体和CH11中的小片段易位染色体，推测易位可能涉及同一条百萨偃麦草染色体。发现13个标记（5个STS、3个RFLP探针和5个SSR）可以追踪未涉及到的4J和5J等染色体。     

苦荞全基因组SSR位点特征分析与分子标记开发

目前苦荞SSR多态性标记数量较少,根据已发表的苦荞基因组测序数据,利用MISA软件对1~6核苷酸重复的SSR位点进行了查找和序列特征分析,批量设计引物并对引物进行了有效性和多态性检测。结果表明,苦荞基因组中共检测到1 640个SSR位点,其中三核苷酸重复型SSR最多,占比63.29%,五核苷酸重复型最少,仅占0.12%。AT/TA、AAG/CTT、ACC/GGT和ATC/GAT为出现频率较高的重复基序。苦荞基因组SSR序列长度变化范围为12~476bp,平均长度23.14bp,长度12~19bp的占比71.71%,长度≥20bp的占比28.29%。根据不同类型SSR位点设计并合成引物479对,选择200对引物对5份苦荞资源和3份甜荞资源进行多态性检测,有56对扩增出多态性条带,17对在苦荞种质中产生多态性条带,48对在甜荞种质中产生多态性条带,9对同时在两种种质中产生多态性条带。利用苦荞全基因组序列可实现SSR标记的批量开发,可鉴定出适用于苦荞和甜荞遗传多样性分析、遗传图谱构建和品种鉴定等研究的SSR引物。     

Comparisons of RFLP and PCR-based markers to detect polymorphism between wheat cultivars

Previously chromosome 3A of wheat (Triticum aestivum L.) was reported to carry genes influencing yield, yield components, plant height, and anthesis date. The objective of current study was to survey various molecular marker systems for their ability to detect polymorphism between wheat cultivars Cheyenne(CNN) and Wichita (WI), particularly for chromosome3A. Seventy-seven `sequence tagged site' (STS), 10simple sequence repeat (SSR), 40 randomly amplified polymorphic DNA (RAPD) markers, and 52 restriction fragment length polymorphism (RFLP) probes for wheat homoeologous group 3 chromosomes, were investigated. Three (3.9%) STS-PCR primer sets amplified polymorphic fragments for the two cultivars, of which one was polymorphic for chromosome 3A. Sixty percent of SSR markers detected polymorphism between CNN and WI of which 50% were polymorphic for chromosome 3A. Twenty percent of RAPD markers detected polymorphism between CNN and WI in general, but none of these detected polymorphism for chromosome 3A. Of the fifty-two RFLP probes, 78.8% detected polymorphism between CNN and WI for group 3 chromosomes with one or more of seven restriction enzymes and 42% of the polymorphic fragements were for chromosome 3A. These high levels of RFLP and SSR polymorphisms between two related wheat cultivars could be used to map and tag genes influencing important agronomic traits. It may also be important to reconsider RFLP as the most suitable marker system at least for anchor maps of closely related wheat cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microsatellite polymorphism in Pisum sativum

Pisum sativum sequences were retrieved from Genbank/EMBL databases and searched for all possible dinucleotide and trinucleotide tandem repeats. One‐hundred and seventy‐one simple sequence repeats (SSRs) were found among 663 sequences. The different dinucleotide or trinucleotide motifs occurred at varying frequencies. CT/AG was the most frequent dinucleotide, and TCT/AGA the most frequent trinucleotide. Forty‐three microsatellite markers were generated from these sequences and used to assess the genetic variability among 12 pea genotypes. Thirty‐one were polymorphic among the genotypes and the average number of variants per marker was 3.6 when considering only polymorphic markers. Overall, the number of variants for a given SSR marker was correlated with the length of the SSR but some 12‐bp long SSRs showed the same degree of polymorphism as longer ones. The groupings resulting from the SSR genotyping among the 12 genotypes gave an interesting insight into the possible origin of one recent cultivar. Database‐derived SSR markers are highly variable. They can provide useful information on the genetic diversity among P. sativum cultivated types.     

新疆彩色棉23个品种指纹图谱的构建及遗传多样性分析

以新疆截止2012年审定的23份新彩棉品种为材料,利用SSR标记进行DNA指纹图谱的构建和遗传多样性分析。从5000对SSR引物中,挑选出多态性高、稳定性好、均匀分布在棉花26条染色体上的52对引物,在23份新彩棉品种中筛选出核心引物47对,SSR扩增检测到多态性基因型位点数共计162个,每个标记检测到的基因型位点数在2~7之间,平均为3.45个;引物多态信息量(PIC)值介于0.4537~0.8686之间,平均值为0.7096。结果显示:在23份新彩棉品种中,14份品种采用特异或特征引物可以一次性区分开,其余9份品种需要采用引物组合来实现区别该品种与其他品种。最少选用18对特异引物及组合引物就可以完全区分开新彩棉1~23号品种。利用18对SSR标记构建了新彩棉1号至23号品种的指纹图谱。利用NTSYS-pcV2.10软件聚类分析表明:23个新彩棉品种遗传相似系数变化范围是0.3781~0.9298,平均为0.5511,表明新彩棉品种之间存在着丰富的遗传多样性。     

小麦T、 K、 V型胞质不育系和杂种mtDNA的RAPD分析及育性相关片段的克隆

用RAPD技术对细胞质分别来源于粘果山羊草（Ae.kotschyi）、偏凸山羊草（Ae.ventricosa）、提莫菲维小麦（T.timopheevii）的三种普通小麦雄性不育系--K型、V型、T型及相应的保持系、恢复系以及杂种F1代的线粒体DNA（mtDNA）进行了比较分析。结果如下：（1）发现在mtDNA组织结构上K型、V型、T型不育系之间以及与保持系之间均     

Fluorescence in situ hybridization polymorphism using two repetitive DNA clones in different cultivars of wheat

Twenty‐two wheat cultivars and a wheat line were analysed with two‐colour fluorescence in situ hybridization (FISH) using the pSc119.2 and pAs1 repetitive DNA clones to detect if polymorphism could be observed in the hybridization patterns of different wheat cultivars. The FISH hybridization pattern of ‘Chinese Spring’ was compared with wheat cultivars of different origins. Differences were observed in the hybridization patterns of chromosomes 4A, 5A, 1B, 2B, 3B, 5B, 6B, 7B, 1D, 2D, 3D and 4D. Although a low level of polymorphism exists in the FISH pattern of different wheat cultivars, it is possible to identify 17 pairs of chromosomes according to their hybridization patterns with these two probes. This study will help to predict the expected variation in the FISH pattern when analysing wheat genetic stocks of different origin. It is presumed that variation in hybridization patterns are caused by chromosome structural rearrangements and by differences in the amount and location of repetitive sequences in the cultivars analysed.     

Characterization of dinucleotide and trinucleotide EST-derived microsatellites in the wheat genome

Over the past decade microsatellites or simple sequence repeats (SSRs) have attracted a considerable amount of attention from researchers. The aim of the present paper was to analyse expressed sequence tag-derived SSR (EST-SSR) marker variability in wheat and to investigate the relationships between the number and type of repeat units and the level of microsatellite polymorphism. Two hundred and forty-one new EST-SSR markers available in a public database () were characterized in eight durum wheat cultivars (Svevo, Ciccio, Primadur, Duilio, Meridiano, Claudio, Latino, Messapia), two accessions of Triticum turgidum var. dicoccoides (MG4343, MG29896), one accession of T. turgidum var. dicoccum (MG5323) and in the common wheat cv. Chinese Spring. Of these, 201 primer pairs (83.4%) amplified PCR products successfully, while the remaining 40 (16.6%) failed to amplify any product. Of the EST-SSRs analysed, 45.2% of the primer pairs amplified one or two PCR products. Multiple discrete PCR products were observed among both di- and trinucleotide EST-SSR markers (31.2 and 40.5%, respectively). Markers based on dinucleotide microsatellites were more polymorphic than those based on trinucleotide SSRs in the 12 wheat genotypes tested (68.9 and 52.7%, respectively). An average of 2.5 alleles for dinucleotide and 2.0 alleles for trinucleotide SSRs was observed. The data reported in the present work indicate the presence of a significant relationship between motif sequence types and polymorphism. The primer set based on the AG repeat motif showed the lowest percentage of polymorphism (55.0%), while the primer set based on the AC repeat motif showed t he highest percentage (85.0%). Among trinucleotide SSRs, the AGG microsatellite markers showed the highest percentage of polymorphism (70.0%), and the ACG motif the lowest value (25.0%). The characterization of these new EST-SSR markers and the results of our studyon the effect of repeat number and type of motifs could have important applications in the genetic analysis of agronomically important traits, quantitative trait locus discovery and marker-assisted selection.     

对人工合成小麦的微卫星变异分析

比较分析了同一四倍体小麦Langdon与5个不同粗山羊草在合成六倍体小麦前后A、B、D染色体组不同染色体上的微卫星变异, 旨在通过分析异源多倍化引起的微卫星位点和序列变异以期探讨异源多倍体的进化机制。在所检测的位于A、B染色体组上各125个特异微卫星(G-SSR)标记中,分别有5个(4.0%)和6个(4.8%)位点发生变异;而在76个A/B染色体组上的表达序列标签微卫星(EST-SSR)标记中,只有2个(2.6%)发生了变异,比A、B染色体组G-SSR变异频率小,说明功能基因区的变异小于重复序列非编码区。在D染色体组上的103个G-SSR标记中,3个位点(2.9%)发生了序列变化。对表现差异的微卫星位点序列分析发现,人工合成小麦中多倍化引起的微卫星序列变异主要表现为简单序列重复单元次数的增加或减少;发生消除的微卫星序列比普通的微卫星序列更易发生不同类型的序列改变。微卫星序列在异源多倍化过程中对新物种基因组的形成可能起到重要的调节作用。     

Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST–SSRs derived from sucest was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST–SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST–SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76‐314 was grouped apart from the other clones evaluated. The results here demonstrate that EST–SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.     

Cytogenetic Diversity of Korean Hexaploid Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) with Simple Sequence Repeats (SSRs) by Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization

Chromosomes of Korean hexaploid wheat were investigated to compare the chromosomal karyotype for cytogenetic diversity. Chromosomal karyotyping was done with in situ hybridization using two types of simple sequence repeats (SSR)s, (AAG)₅ and (AAC)₅ labeled with tetramethyl-rhodamine-5-dUTP and fluorescein-12-dUPT as a fluorescence, respectively. The two SSRs as cytogenetic markers revealed that the cytogenetic characteristics of the wheat chromosomes were remarkably a B genome. In this study, the chromosomal karyotype of Keumkang, a Korean hexaploid wheat cultivar, was the A, B, and D genomes used as a cytogenetic reference. The expressed signals from the two SSRs showed a difference in the chromosomal karyotype of chromosome 1B among the Korean hexaploid wheat. The distribution pattern and the degree of condensation for the (AAG)₅ and (AAC)₅ signals on the short arm of chromosome 1B were different in the Korean hexaploid wheat shown in descending order: Keumkang > Joeun > Johan > Olgeuru. Olgeuru had a lower level of distribution and condensation for the two SSRs signals compared to the other Korean hexaploid wheat. In the A genome, chromosome 7A showed an unbalanced expression of the (AAG)₅ signal on the distal region of the short and long arms in several Korean hexaploid wheat while Joeun, a Korean hexaploid wheat, showed a definite (AAG)₅ signal on the distal region of each arm of chromosome 7A. Among the Korean hexaploid wheat, Shinmichal1, a Korean hexaploid waxy wheat, had a chromosome with a unique expression pattern for (AAG)₅ and (AAC)₅ compared the other Korean hexaploid wheat. Those cytogenetic differences identified in this study are useful as an indicator to improve the cytogenetic diversity in the Korean wheat breeding program.     

Spanish spelt: a separate gene pool within the spelt germplasm

The renewed interest in spelt (Triticum spelta L.) for wheat improvement programmes requires the study of the available genetic diversity. The purpose of this study was to assess genetic diversity within a Spanish spelt collection. Sixty‐six Spanish spelt accessions, 19 accessions of T. spelta and T. macha from different origins, three bread wheat cultivars (T. aestivum) and one accession of T. dicoccum were screened using simple sequence repeats (SSRs). The diversity observed within the Spanish group was comparable with that observed in the other wheat varieties, despite their broader geographical diversity. Indeed, the highest polymorphic information content value calculated with SSRs for Spanish material (0.90) is similar to that observed for the other wheat varieties (0.98). Principal component analysis explained 46.5% of the cumulative variation and confirmed the Spanish accessions as a separate group. This study showed the Spanish spelt collection to be a variable and unique genetic resource for wheat and spelt breeding programmes.     

甜瓜SSR引物在西葫芦中的通用性研究

本文分析了25对甜瓜SSR引物在西葫芦基因组中的可转移性,以期为西葫芦分子遗传育种研究提供分析工具。结果表明,约24%的甜瓜SSR引物可在西葫芦基因组中有效扩增。多态性分析发现6对引物中有4对在34份西葫芦种质中可以检测到多态性,等位变异数从3~6不等,平均为4。UPGMA 聚类分析表明,来自市场主栽F1的纯化材料和双单倍体材料之间多样性较差,说明现有栽培品种基因谱较窄;而从国家蔬菜种质资源库引进的材料多样性比较丰富,对这些材料的整理和重新评价有助于发掘新的基因源,为西葫芦杂交育种提供新材料。