Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of EST-SSRs in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

In recent years, microsatellites have become the most used markers for studying population genetic diversity. The increased availability of the DNA sequences has given the possibility to develop EST-derived SSR markers. A total of 1,927 ESTs of Eleusine coracana available in the NCBI database were mined for SSRs. Di-nucleotides are the most occurring motifs accounting for more than 50% of the repeats, of which GA was the most abundant motif and tetra-nucleotides are the least occurring motifs. Of the 132 markers identified, 30 primer pairs based were synthesized. SSR markers were used for variety discrimination and genetic assessment in 15 finger millet accessions; 20 primers showed polymorphism and 13 primers were identified as having a PIC value above 0.5. On the basis of the distribution of these polymorphic alleles, the 15 accessions were classified into two groups. This study has demonstrated the potential of EST-derived SSR primer pairs in finger millet. These primers will serve as valuable source for further breeding programs.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

Detection by simple sequence repeat markers of introgression from Coffea canephora in Coffea arabica cultivars

Microsatellites or simple sequence repeats (SSR) were used to assess polymorphism among 16 Coffea arabica and four Coffea canephora accessions, and to identify DNA introgression fragments from C. canephora in four C. arabica lines. Thirty‐one primer pairs allowed for the identification of 92 polymorphic alleles distributed over 37 loci. The C. arabica accessions derived from the genetic bases ‘Typica’ and ‘Bourbon’ were grouped separately according to their genetic origin. Two genotypes derived from a spontaneous hybrid (C arabica×C. canephora) were classified with the C. canephora accessions from Central Africa. Coffea canephora from West Africa were separated from the other accessions studied. Four alleles related to introgression (i.e. present in C. canephora and introgressed lines, and absent in C. arabica) were identified. The SSR markers were used successfully for characterization of a particular cultivar (‘Veranero’) from Costa Rica, which is known for its late maturity.     

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.     

芝麻EST-SSR标记的开发和初步研究

为了加速分子标记在芝麻研究中的应用,利用网上现有的芝麻EST(expressed sequence tags)数据信息,开展了芝麻EST-SSR功能性标记的开发和利用研究。 在所有的3 328条芝麻EST序列中共确认得到1 785条非冗余EST序列。其中,在含有微卫星重复的148条序列中共检测有155个EST-SSR。非冗余EST序列总长为774.266 kb,平均每4.99 kb含有一个EST-SSR。EST-SSR的分布频率和特征分析表明,以AG/TC为重复基元(motif)的SSR出现最多,占总SSR的37.42%。利用这些序列,设计开发了50对EST-SSR引物,并分别选用36个芝麻、2个棉花、2个大豆和2个油葵进行多态性和通用性研究。其中44对引物在供试芝麻材料中扩增出条带,共产生108个位点,平均每对引物产生2.45个位点,多态信息含量(polymorphism information content, PIC)平均值为0.390。根据遗传相似性系数进行聚类,有26个芝麻材料聚类在两个大的亚类(III和IV)中,聚类结果表明芝麻的基因型与地理来源之间没有必然的联系。此外,分别有2对、3对和4对引物可以在棉花、大豆和油葵中进行通用性扩增。本研究证实这种全新开发的芝麻EST-SSR标记在芝麻遗传多样性分析、遗传图谱构建以及比较基因组等研究方面有广阔的利用前景。     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

利用SSR标记分析栽培种花生多态性及亲缘关系

利用11对SSR引物对24个花生栽培品种（包括四大类型）进行PCR扩增分析，其中4对检测到明显的多态性，共检测到33个等位基因变异，每一个位点上检测到的等位变异数为5～13个，平均为8.25个。根据扩增结果可以将24个品种中的21个相互区分。供试品种间的遗传相似系数值在0.2～1.0之间，平均为0.4788。根据UPGMA聚类分析结果,供试     

Transferability of rice SSR markers to Miscanthus sinensis, a potential biofuel crop

Miscanthus sinensis (M. sinensis) is a perennial C₄ photosynthesis grass, with high yield, high efficiency of water usage, low fertilizer requirement, tolerance to extreme environments, and is one of the plant species with good biofuel potential. Simple sequence repeat (SSR) markers are highly informative and widely used in plant genetic studies. In this study, 88 SSR primer pairs derived from the rice genome, including 47 EST-SSRs (eSSRs) and 41 genomic SSRs (gSSRs), were evaluated for cross-species transferability to M. sinensis. Forty-one SSR primer pairs in total could successfully amplify DNA fragments in M. sinensis, showing an overall transferability rate of 46.6 % between rice and M. sinensis. The transferability rate of eSSR (51.1 %) was higher than that of gSSR (41.5 %). A total of 140 SSR loci and 340 alleles in the set of rice and M. sinensis germplasm collections were detected. Nei’s gene diversity varied from 0 to 0.72 and averaged 0.35. Shannon’s information index varied from 0 to 1.49 and averaged 0.56. The observed heterozygosity ranged from 0 to 0.95 and averaged 0.08. Thirty-nine loci (27.86 %) were shown heterozygosity out of 140 SSR loci. A dendrogram based on genetic distance showed a significant geographic differentiation. Our results indicated that 46.6% of the rice SSR markers are transferable to M. sinensis, and are useful for germplasm evaluation and genetic analysis in M. sinensis.     

Cross‐genera amplification of informative microsatellite markers from common bean and scarlet runner bean for assessment of genetic diversity in mungbean (Vigna radiata)

Mungbean (V. radiata) is an important Asiatic legume supplying inexpensive protein to a vast majority of vegetarian masses. To increase markers repertoire in mungbean, a study was conducted to analyse 384 microsatellite markers derived from common bean, scarlet runner bean and adzuki bean for their transferability and polymorphism. The results showed that 87 (24.71%) primer pairs could amplify DNA loci of 20 mungbean genotypes including one accession of V. trilobata, while 52 showed reliable banding and polymorphism. These showed different degrees of variability at each locus producing 250 alleles with the number of alleles varying from 2 to 9. The major allele frequency varied from 0.17 to 0.95, while the polymorphic information content of SSRs ranged between 0.09 and 0.86 with an average of 0.60 ± 0.16. UPGMA revealed three major clusters accommodating ~95% of the accessions while one accession of V. trilobata (‘NSB‐007’) did not group with any other genotype describing the discriminating power of informative microsatellites. This study identified a set of useful microsatellite markers to accelerate the genetic studies and breeding programme of mungbean.     

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

基于菜心(Brassica campestris)基因组Survey测序开发多态性SSR标记

为解决菜心SSR标记数量不足、已开发的位点多态性差等问题,本研究利用高通量测序技术,对‘四九-19’和‘3T6’两份菜心材料进行基因组Survey测序,规模化开发多态性SSR标记。两个菜心材料分别获得55 649 657个和59 300 433个Clean reads,分开拼接组装得到430 483个和499 876个Contig。在两个材料的Contig中搜索到共有的SSR位点为30 696个,其中以二和三核苷酸重复基序最为丰富,占总SSR位点的67%。分析比较发现,3 652个(12%) SSR位点在两份测序材料间具有潜在多态性,随机挑选50个SSR位点进行PCR扩增验证,48对(96%)引物在4份菜心材料中扩增出清晰的条带,其中31对(62%)引物在两份测序样品间具有多态性,19对(38%)引物在另两份菜心材料间具有多态性。结果表明,利用基因组Survey测序能开发SSR标记和开发具有多态性的SSR标记,本研究开发的多态SSR标记将进一步为菜心分子标记的发展和应用提供基础。     

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。     

Development and characterization of tomato SSR markers from genomic sequences of anchored BAC clones on chromosome 6

Simple sequence repeat motifs are abundant in plant genomes and are commonly used molecular markers in plant breeding. In tomato, currently available genetic maps possess a limited number of simple sequence repeat (SSR) markers that are not evenly distributed in the genome. This situation warrants the need for more SSRs in genomic regions lacking adequate markers. The objective of the study was to develop SSR markers pertaining to chromosome 6 from bacterial artificial chromosome (BAC) sequences available at Solanaceae Genomics Network. A total of 54 SSR primer pairs from 17 BAC clones on chromosome 6 were designed and validated. Polymorphism of these loci was evaluated in a panel of 16 genotypes comprising of Solanum lycopersicum and its wild relatives. Genetic diversity analysis based on these markers could distinguish genotypes at species level. Twenty-one SSR markers derived from 13 BAC clones were polymorphic between two closely related tomato accessions, West Virginia 700 and Hawaii 7996 and were mapped using a recombinant inbred line population derived from a cross between these two accessions. The markers were distributed throughout the chromosome spanning a total length of 117.6 cM following the order of the original BAC clones. A major QTL associated with resistance to bacterial wilt was mapped on chromosome 6 at similar location of the reported Bwr-6 locus. These chromosome 6-specific SSR markers developed in this study are useful tools for cultivar identification, genetic diversity analysis and genetic mapping in tomato.     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Simple sequence repeats for genetic analysis in pear

The development of highly informative DNA markers, such as simple sequence repeats (SSRs), is essential for breeding to select agronomically important traits and for genetic studies in pear. We developed SSR markers by using two approaches, RAHM (random amplified hybridization microsatellites) and 5' anchored PCR methods. Segregation analysis of the SSRs revealed that amplified fragments were derived from the same loci, using 3 sets of progenies from crosses between pear varieties. Genetic diversity was characterized using 32 varieties, including 10 from Japanese pear (Pyrus pyrifolia), 9 from Chinese pear (P. bretschneideri, P. ussuriensis), 10 from European pear (P. communis) as well as 3 wild relatives (P. calleryana). Diversity of SSR genotypes was observed among species as well as within species and 65 putative alleles were detected. The use of seven SSR markers was sufficient to differentiate between all of the 32 varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of microsatellite markers from guineagrass (Panicum maximum) for genetic diversity estimate and cross-species amplification

Guineagrass ( Panicum maximum Jacq.) is one of the major forage grasses in tropical and semitropical regions, largely apomictic and predominantly exist as tetraploid. Non-availability of polymorphic molecular markers has been a major limitation in its characterization and improvement. We report isolation and characterization of microsatellites in P. maximum and cross-species results with other five Panicum species. Based on microsatellite-motifs, 15 functional and polymorphic simple sequence repeat (SSR) primer-pairs were designed, validated and employed in estimating genetic relationship among 34 guineagrass accessions. Thirteen primer-pairs amplified single locus and remaining two generated more than two loci with an average of 3.57 bands per locus amounts to 63 bands with 34 guineagrass accessions. Average expected heterozygosity ( H _E) of 0.35 (maximum 0.97) and observed heterozygosity ( H _O) of 0.37 (maximum 0.91) established the efficiency of developed markers for discriminating guineagrass accessions. Dice's similarity coefficients-based unweighted pair group with arithmetic average method-clustering supported with high bootstrap values (≥40) indicated its significance and distinguished all accessions except IG97-93 and IG97-6. Utility of these new SSR loci in genetic diversity study of P. maximum and other cross–amplified species is discussed.     

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。