Developing and characterising Ricinus communis SSR markers by data mining of whole-genome sequences

Ricinus communis is a versatile industrial oil crop that is cultivated worldwide. Genetic improvement and marker-assisted breeding of castor bean have been slowed owing to the lack of abundant and efficient molecular markers. As co-dominant markers, simple sequence repeats (SSRs) are useful for genetic evaluation and molecular breeding. The recently released whole-genome sequence of castor bean provides useful genomic resources for developing markers on a genome-wide scale. In the present study, the distribution and frequency of microsatellites in the castor bean genome were characterised and numerous SSR markers were developed using genomic data mining. In total, 18,647 SSR loci at a density of one SSR per 18.89 Kb in the castor bean genome sequence (representing approximately 352.27 Mb) were identified. Dinucleotide repeats were the most frequently observed microsatellites, although the AAT repeat motif was also prevalent. Using six cultivars as screening samples, 670 polymorphic SSR markers from 1,435 primer pairs (46.7 %) were developed. Trinucleotide motif loci contained a higher proportion of polymorphisms (48.5 %) than dinucleotide motif loci (39.2 %). The polymorphism level in the SSR loci was positively correlated with the increasing number of repeat units in the microsatellites. The phylogenetic relationship among 32 varieties was evaluated using the developed SSR markers. Cultivars developed at the same institute clustered together, suggesting that these cultivars have a narrow genetic background. The large number of SSR markers developed in this study will be useful for genetic mapping and for breeding improved castor-oil plants. These markers will also facilitate genetic and genomic studies of Euphorbiaceae.     

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Background

Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results

Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H _E ) and observed (H _o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion

UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.     

Genome-Wide Characterization of Simple Sequence Repeat (SSR) Loci in Chinese Jujube and Jujube SSR Primer Transferability

Chinese jujube (Ziziphus jujuba), an economically important species in the Rhamnaceae family, is a popular fruit tree in Asia. Here, we surveyed and characterized simple sequence repeats (SSRs) in the jujube genome. A total of 436,676 SSR loci were identified, with an average distance of 0.93 Kb between the loci. A large proportion of the SSRs included mononucleotide, dinucleotide and trinucleotide repeat motifs, which accounted for 64.87%, 24.40%, and 8.74% of all repeats, respectively. Among the mononucleotide repeats, A/T was the most common, whereas AT/TA was the most common dinucleotide repeat. A total of 30,565 primer pairs were successfully designed and screened using a series of criteria. Moreover, 725 of 1,000 randomly selected primer pairs were effective among 6 cultivars, and 511 of these primer pairs were polymorphic. Sequencing the amplicons of two SSRs across three jujube cultivars revealed variations in the repeats. The transferability of jujube SSR primers proved that 35/64 SSRs could be transferred across family boundary. Using jujube SSR primers, clustering analysis results from 15 species were highly consistent with the Angiosperm Phylogeny Group (APGIII) System. The genome-wide characterization of SSRs in Chinese jujube is very valuable for whole-genome characterization and marker-assisted selection in jujube breeding. In addition, the transferability of jujube SSR primers could provide a solid foundation for their further utilization.     

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Construction of fingerprinting for tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) accessions using new genomic SSR markers

As one of the most popular non-alcoholic beverage crops, the tea plant (Camellia sinensis) plays an important role in human health and lifestyle. Genetic fingerprinting based on genomic-derived markers in tea, however, is still in the initial stages, which has limited tea germplasm resource utilization and cultivar protection. In the current study, we identified whole genome-based simple sequence repeat (SSR) loci and successfully developed 36 new genomic SSR markers, which are highly polymorphic with average allele number and polymorphic information content (PIC) of 14.9 and 0.862, respectively. A phylogenetic tree for 80 tea plant accessions was subsequently constructed based on their genotypic scores for these 36 markers. The phylogenetic relationships among the 80 accessions are highly consistent with their genetic backgrounds or original places. Noteworthy, robust fingerprinting power was performed, and the overall probability of finding two random individuals sharing identical genotypes across the 36 loci was estimated to be 1.5 × 10^?56. We subsequently identified five SSR markers as a recommended core marker set for fingerprinting the tea plant cultivars or accessions. The combined PI and PIsibs of the marker set were 1.49 × 10^?9 and 2.57 × 10^?3, respectively, which allowed us to fully discriminate all 80 tea plant accessions from one another. The SSR markers developed here will provide a valuable resource for tea plant genetics and genomic studies, as well as breeding programs. The fingerprinting profiles can serve as a database that is essential for the tea industry and commercial breeding, and for tea plant cultivar identification, utilization, and protection.     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Analysis of simple sequence repeats (SSRs)dynamics in fungus Fusarium graminearum

The abundance and inherent potential for variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. We describe the organization and abundance of SSRs in fungus Fusarium graminearum (causative agent for Fusarium head blight or head scab of wheat). We identified 1705 SSRs of various nucleotide repeat motifs in the sequence database of F. graminearum. It is observed that mononucleotide repeats (62%) were most abundant followed by di- (20%) and trinucleotide repeats (14%). It is noted that tetra-, penta- and hexanucleotide repeats accounted for only 4% of SSRs. The estimated frequency of Class I SSRs (perfect repeats ≥20 nucleotides) was one SSR per 124.5 kb, whereas the frequency of Class II (perfect repeats >10 nucleotides and ≫20 nucleotides) was one SSR per 25.6 kb. The dynamics of SSRs will be a powerful tool for taxonomic, phylogenetic, genome mapping and population genetic studies as SSR based markers show high levels of allelic variation, codominant inheritance and ease of analysis.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species.

Simple sequence repeat (SSR) markers are widely used in many plant and animal genomes due to their abundance, hypervariability, and suitability for high-throughput analysis. Development of SSR markers using molecular methods is time consuming, laborious, and expensive. Use of computational approaches to mine ever-increasing sequences such as expressed sequence tags (ESTs) in public databases permits rapid and economical discovery of SSRs. Most of such efforts to date focused on mining SSRs from monocotyledonous ESTs. In this study, we have computationally mined and examined the abundance of SSRs in more than 1.54 million ESTs belonging to 55 dicotyledonous species. The frequency of ESTs containing SSRs among species ranged from 2.65% to 16.82%. Dinucleotide repeats were found to be the most abundant followed by tri- or mono-nucleotide repeats. The motifs A/T, AG/GA/CT/TC, and AAG/AGA/GAA/CTT/TTC/TCT were the predominant mono-, di-, and tri-nucleotide SSRs, respectively. Most of the mononucleotide SSRs contained 15-25 repeats, whereas the majority of the di- and tri-nucleotide SSRs contained 5-10 repeats. The comprehensive SSR survey data presented here demonstrates the potential of in silico mining of ESTs for rapid development of SSR markers for genetic analysis and applications in dicotyledonous crops.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

Genome-Wide Analysis of Microsatellite Markers Based on Sequenced Database in Chinese Spring Wheat (Triticum aestivum L.)

Microsatellites or simple sequence repeats (SSRs) are distributed across both prokaryotic and eukaryotic genomes and have been widely used for genetic studies and molecular marker-assisted breeding in crops. Though an ordered draft sequence of hexaploid bread wheat have been announced, the researches about systemic analysis of SSRs for wheat still have not been reported so far. In the present study, we identified 364,347 SSRs from among 10,603,760 sequences of the Chinese spring wheat (CSW) genome, which were present at a density of 36.68 SSR/Mb. In total, we detected 488 types of motifs ranging from di- to hexanucleotides, among which dinucleotide repeats dominated, accounting for approximately 42.52% of the genome. The density of tri- to hexanucleotide repeats was 24.97%, 4.62%, 3.25% and 24.65%, respectively. AG/CT, AAG/CTT, AGAT/ATCT, AAAAG/CTTTT and AAAATT/AATTTT were the most frequent repeats among di- to hexanucleotide repeats. Among the 21 chromosomes of CSW, the density of repeats was highest on chromosome 2D and lowest on chromosome 3A. The proportions of di-, tri-, tetra-, penta- and hexanucleotide repeats on each chromosome, and even on the whole genome, were almost identical. In addition, 295,267 SSR markers were successfully developed from the 21 chromosomes of CSW, which cover the entire genome at a density of 29.73 per Mb. All of the SSR markers were validated by reverse electronic-Polymerase Chain Reaction (re-PCR); 70,564 (23.9%) were found to be monomorphic and 224,703 (76.1%) were found to be polymorphic. A total of 45 monomorphic markers were selected randomly for validation purposes; 24 (53.3%) amplified one locus, 8 (17.8%) amplified multiple identical loci, and 13 (28.9%) did not amplify any fragments from the genomic DNA of CSW. Then a dendrogram was generated based on the 24 monomorphic SSR markers among 20 wheat cultivars and three species of its diploid ancestors showing that monomorphic SSR markers represented a promising source to increase the number of genetic markers available for the wheat genome. The results of this study will be useful for investigating the genetic diversity and evolution among wheat and related species. At the same time, the results will facilitate comparative genomic studies and marker-assisted breeding (MAS) in plants.     

Genome-wide identification and characterization of simple sequence repeat loci in grape phylloxera, Daktulosphaira vitifoliae

A genome-wide sequence search was conducted to identify simple sequence repeat (SSR) loci in phylloxera, Daktulosphaira vitifoliae, a major grape pest throughout the world. Collectively, 1524 SSR loci containing mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs were identified. Among them, trinucleotide repeats were the most abundant in the phylloxera genome (34.4%), followed by hexanucleotide (20.4%) and dinucleotide (19.6%) repeats. Mono-, tetra- and pentanucleotide repeats were found at a frequency of 1.3, 11.2 and 12.9%, respectively. The abundance and inherent variations in SSRs provide valuable information for developing molecular markers. The high levels of allelic variation and codominant features of SSRs make this marker system a useful tool for genotyping, diversity assessment and population genetic studies of reproductive characteristics of phylloxera in agricultural and natural populations.     

Development,characterization and utilization of GenBank microsatellite markers in <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis> and related species

Public sequence databases provide a rapid, simple and cost-effective source of microsatellite markers. We analyzed 1,532 bamboo (Phyllostachys pubescens) sequences available in public domain DNA databases, and found 3,241 simple sequence repeat (SSR) loci comprising repeats of two or more nucleotides in 920 genomic survey sequences (GSSs) and 68 cDNA sequences. This corresponded to one SSR per 336 bp of GSS DNA and one SSR per 363 bp of cDNA. The SSRs consisted of 76.6 and 74.5% dinucleotide repeats, 20.0 and 22.3% trinucleotide repeats, and 3.4 and 3.2% higher-number repeats in the GSS DNA and cDNA sequences, respectively. The repeat motif AG/CT (or GA/TC) was the most abundant. Nineteen microsatellite markers were developed from Class I and Class II SSRs, showing that the limited polymorphism in Ph. pubescens cultivars and provenances could be attributed to clonal propagation of the bamboo plant. The transferability of the microsatellites reached 75.3%, and the polymorphism of loci successfully transferred was 66.7% for six additional Phyllostachys species. Microsatellite PBM014 transferred successfully to all six species, showed rich polymorphism, and could serve as species-specific alleles for the identification of Phyllostachys interspecies hybrids.     

Genome Wide Characterization of Short Tandem Repeat Markers in Sweet Orange (Citrus sinensis)

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.     

Mining and characterization of EST derived microsatellites in Curcuma longa L

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST-derived SSRs (Simple sequence repeats) are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non-redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1-6-nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.     

Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.