A functional molecular marker for detecting blister blight disease resistance in tea (Camellia sinensis L.).

KEY MESSAGE: Identification of an EST-SSR molecular marker associated with Blister blight, a common fungal disease of tea, facilitating marker-assisted selection, marking a milestone in tea molecular breeding. lister blight (BB) leaf disease of tea, caused by the fungus Exobasidium vexans, results in 25-30% crop loss annually. BB is presently controlled by Cu based fungicides, but genetic resistance is the most viable option in disease management. Tea is a naturally out-crossing, woody perennial necessitating a long time for completion of a breeding programme. Marker-assisted selection (MAS) is vital to expedite breeding programmes and also for better accuracy in gene identification. The aim of the current research was to derive marker-trait associations using an F1 population segregating for BB. The population was genotyped at 11 expressed sequence tag simple sequence repeat loci followed by detecting the alleles by fragment analysis. The genotypic and phenotypic data were subjected to single-marker analysis resulting in the identification of EST-SSR073 as a diagnostic marker amplifying three alleles of the sizes, 168, 170 and 190 bp in F1. Of them, alleles 190 and 168 bp were confirmed to concur BB resistance and susceptibility, respectively. The alleles were validated in a panel of 64 tea cultivars, resulting in the amplification of 12 alleles at EST-SSR073. The EST-SSR073 allele sequences matched with Camellia sinensis photosystem-I reaction center subunit-II. The marker EST-SSR073 can be effectively used in breeding tea against BB, recording a milestone in MAS in tea.

De novo transcriptome assembly and population genetic analyses of an important coastal shrub, Apocynum venetum L.

BACKGROUND: Apocynum venetum L. is an important medicinal plant that is mainly distributed in the coastal areas and northwest of China. In addition to its high medical and economic value, its adaptation to saline-alkali and coastal saline lands makes A. venetum an ideal candidate for use in vegetation restoration. To date, the study of A. venetum has been limited in the northwest region of China, little attention has been paid to the genetic diversity and population structure of A. venetum populations in the coastal region. Here, we performed transcriptome sequencing of total RNA from A. venetum leaves and developed efficient expressed sequence tag-simple sequence repeat (EST-SSR) markers for analyzing the genetic diversity and population structure of A. venetum in the coastal region. RESULTS: A total of 86,890 unigenes were generated after de novo assembly, and 68,751 of which were successfully annotated by searching against seven protein databases. Furthermore, 14,072 EST-SSR loci were detected and 10,243 primer pairs were successfully designed from these loci. One hundred primer pairs were randomly selected and synthesized, twelve primer pairs were identified as highly polymorphic and further used for population genetic analysis. Population genetic analyses showed that A. venetum exhibited low level of genetic diversity (mean alleles per locus, NA = 3.3; mean expected heterozygosity, HE = 0.342) and moderate level of genetic differentiation among the populations (genetic differentiation index, FST = 0.032-0.220) in the coastal region. Although the contemporary (mean mc = 0.056) and historical (mean mh = 0.106) migration rates among the six A. venetum populations were moderate, a decreasing trend over the last few generations was detected. Bayesian structure analysis clustered six populations into two major groups, and genetic bottlenecks were found to have occurred in two populations (QG, BH). CONCLUSIONS: Using novel EST-SSR markers, we evaluated the genetic variation of A. venetum in the coastal region and determined conservation priorities based on these findings. The large dataset of unigenes and SSRs identified in our study, combining samples from a broader range, will support further research on the conservation and evolution of this important coastal plant and its related species.

Elymus nutans genes for seed shattering and candidate gene-derived EST-SSR markers for germplasm evaluation.

BACKGROUND: Elymus nutans and E. sibiricus are two important forage grasses of the genus Elymus. But they are difficult to grow for commercial seed production due to serious seed shattering. We conducted a comparative transcriptome analysis of abscission zone to find possible transcription changes associated with seed shattering, explore candidate genes involved in seed shattering and identify candidate gene-based EST-SSR markers for germplasm evaluation. RESULTS: cDNA libraries from abscission zone (AZ) and non-abscission zone (NAZ) tissues of E. nutans were constructed and sequenced. A total of 111,667 unigenes were annotated and 7644 differentially expressed transcripts (DETs) were predicted, corresponding to 6936 up-regulated in AZ and 708 down-regulated in NAZ. We identified 489 candidate genes related to transcription factor, cell wall hydrolysis or modification, hydrolase activity, phytohormone signaling and response, lignin biosynthesis, and signal transduction or protein turnover. Eleven similar candidate genes involved in polygalacturonase activity, hydrolase activity, and mitogen-activated protein kinase were up-regulated in the abscission zone of the two Elymus species, suggesting these genes may have specific function for abscission zone development and seed shattering. A total of 67 polymorphic EST-SSR markers were developed and characterized based on the sequences of these candidate genes. Fourteen polymorphic EST-SSR primers were finally used to study genetic diversity in 48 E. nutans genotypes with contrasting seed shattering habit. The dendrogram based on molecular data showed that most accessions with similar seed shattering degree tended to group together. CONCLUSIONS: The expression data generated from this study provides an important resource for future molecular biological research. Many DETs were associated with abscission zone development, and EST-SSR loci related to candidate genes may have potential application in identifying trait-associated markers in E. nutans in the future.

Genetic diversity and population structure of castor (Ricinus communis L.) germplasm within the US collection assessed with EST-SSR markers.

Castor is an important oilseed crop and although its oil is inedible, it has multiple industrial and pharmaceutical applications. The entire US castor germplasm collection was previously screened for oil content and fatty acid composition, but its genetic diversity and population structure has not been determined. Based on the screening results of oil content, fatty acid composition, and country origins, 574 accessions were selected and genotyped with 22 polymorphic EST-SSR markers. The results from cluster analysis, population structure, and principal component analysis were consistent, and partitioned accessions into four subpopulations. Although there were certain levels of admixtures among groups, these clusters and subpopulations aligned with geographic origins. Both divergent and redundant accessions were identified in this study. The US castor germplasm collection encompasses a moderately high level of genetic diversity (pairwise dissimilarity coefficient = 0.53). The results obtained here will be useful for choosing accessions as parents to make crosses in breeding programs and prioritizing accessions for regeneration to improve germplasm management. A subset of 230 accessions was selected and will be planted in the field for establishing a core collection of the US castor germplasm. Further evaluation of the US castor germplasm collection is also discussed.

Hybrid identification and genetic variation of Elymus sibiricus hybrid populations using EST-SSR markers.

BACKGROUND: Elymus sibiricus is an important native grass in Qinghai-Tibetan Plateau. Seed shattering is a serious problem for E. sibiricus, especially at harvest time. Cross breeding is an effective way to create new varieties with beneficial characteristic or improved traits, and to broaden genetic base. RESULTS: In this study, we created five hybrid populations by crossing seven E. sibiricus genotypes that have seed shattering variation. Then, nine EST-SSR primers were used for hybrid identification based on DNA fingerprinting, and genetic diversity analysis of hybrid populations and their respective parents. A total of 15 hybrids were identified. An analysis of amplified polymorphic bands among genuine hybrids and their respective parents revealed mainly two types of markers: 1) hybrids shared bands exclusively amplified in both parents; 2)hybrids shared bands exclusively amplified in male parents. For each hybrid population, the total number of amplified bands ranged from 37 to 57, the percentage of polymorphism varied from 65.12% to 75.68%, with an average of 70.51%. Novel bands found in each hybrid population varied from 0 to 9 bands, suggesting an occurrence of rearrangements in the hybrid population. The structure analysis revealed that all hybrid populations and parents were assigned to eight groups. The principal coordinate analysis (PCoA) showed similar results. CONCLUSIONS: In general, this study proved EST-SSR markers are efficient for hybrid identification, and suggested more genetic variation could be captured in hybrid populations by crossing breeding.

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora).

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

Development and characterization of expressed sequence tag-simple sequence repeat markers for the near-threatened halophyte, Limonium tetragonum (Thunb.) A. A. Bullock (Plumbaginaceae).

Here, next-generation RNA sequencing analysis was performed to develop 13 novel expressed sequence tag-simple sequence repeat markers to evaluate the genetic variation in the near-threatened halophyte, Limonium tetragonum (Thunb.) A. A. Bullock. In the four populations, the total number of alleles at each locus ranged from two to seven, with an average of 3.1 and average observed and expected heterozygosities ranged from 0.00 to 0.13 and 0.28 to 0.78, respectively. Three of thirteen loci had the same homo alleles within populations, however, different alleles among different populations. Compared to other halophytes, relatively low genetic diversity was observed in this species. Further studies are necessary to determine the population demography of L. tetragonum and clarify the cause of its low genetic diversity.

Evaluation of genetic diversity and population structure of Fragaria nilgerrensis using EST-SSR markers.

Fragaria nilgerrensis is a diploid wild strawberry widely distributed in Southwest China. Its white color and "peach-like" fragrance of fruits are valuable characters for the genetic improvement of cultivated strawberry plants. Its strong biotic and abiotic resistance and tolerance also enable it to survive in different habitats in the field. In this study, we evaluated the level of genetic variation within and between 16 populations with 169 individuals of F. nilgerrensis using 16 newly developed EST-SSR (expressed sequence tag-simple sequence repeats) markers. The results show that the genetic diversity of this species was high, based on Nei's genetic diversity (0.26) and polymorphic loci (0.41), although it is self-compatible and has clonal propagation. Significant genetic differentiation among populations was also detected by AMOVA analysis (Fst = 0.34), which could be indicative of little gene flow (Nm = 0.43) in F. nilgerrensis. The phylogenetic tree indicates that most of individuals from the same population have clustered together. These populations were not grouped based on the geographical distance, consistent with the Mantel test result (R2 = 0.0063, P > 0.05). All the populations were assigned into two ancestral groups, with some individuals admixed, suggesting ancestral gene flow had occurred between these two groups. Our developed EST-SSR markers as well as the genetic diversity and population structure analysis of F. nilgerrensis are important for genetic improvement in the breeding process. Moreover, the populations that contain high genetic diversity would be a priority for collection and conservation.

Evaluation of genetic diversity and construction of DNA fingerprinting in Polygonatum Mill. based on EST-SSR and SRAP molecular markers.

Polygonatum sibiricum is widely consumed as a traditional Chinese herb and edible plant in China. Despite its nutritional and medical values, research on Polygonatum Mill. has been scarce, particularly as far as its genetic diversity is concerned. In this study, fourteen expressed sequence tag-derived simple sequence repeat (EST-SSR) and seven sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity in fifty Polygonatum Mill. accessions. The EST-SSRs and SRAPs produced 173 (90.58%) and 113 (93.39%) polymorphic bands, respectively. Unweighted Pair-Group Method Analysis (UPGMA) based on the combined data matrices of EST-SSRs and SRAPs divided the fifty Polygonatum Mill. accessions into fourteen groups. In addition, accessions of P. cyrtonema Hua obtained from Anhui and Zhejiang provinces were clustered according to their geographic origin. Furthermore, some accessions were gathered together based on species, such as P. kingianum Coll. et Hemsl, P. punctatum Royle ex Kunth, P. odoratum (Mill.) Druce, and P. sibiricum Red., and bootstrap analysis for clustering fully supported the grouping of the accessions. The Analysis of Molecular Variance (AMOVA) results revealed higher variation within populations (95%) rather than among populations (5%), indicating that Polygonatum Mill. has a low genetic differentiation between populations, and Principal Coordinate Analysis (PCoA) greatly supported the results of cluster analysis and AMOVA analysis. Finally, five markers which could produce abundant and stable bands were used to construct DNA fingerprinting database of Polygonatum Mill.. Our results demonstrated the utility of both EST-SSR and SRAP markers to successfully evaluate and identify Polygonatum Mill..

Development and characterization of EST-SSR markers for Camellia reticulata.

PREMISE: Camellia reticulata, which is native to southwestern China, is an economically important plant belonging to the family Theaceae. We developed expressed sequence tag-simple sequence repeat (EST-SSR) markers for C. reticulata, which can be used to investigate its genetic diversity, population structure, and evolutionary history. METHODS AND RESULTS: We detected 4780 SSRs in C. reticulata from Camellia RNA-Seq data deposited in the National Center for Biotechnology Information's expressed sequence tags database (dbEST). Primer pairs for 70 SSR loci were designed and used for PCR amplification using 90 individuals from four populations of C. reticulata. Of these loci, 50 microsatellite markers were successfully identified, including 11 polymorphic markers. The allele number per locus ranged from two to seven (mean = 4.182), and the levels of observed and expected heterozygosity ranged from 0.044 to 0.567 and from 0.166 to 0.642, respectively. Eleven primer pairs amplified PCR products in three other species of Camellia (C. saluenensis, C. pitardii, and C. yunnanensis). CONCLUSIONS: The set of microsatellite markers developed here can be used to study the genetic variation and population structure of C. reticulata and related species and thereby help to develop conservation strategies for this species.

Transcriptome analysis of the endangered Notopterygium incisum: Cold-tolerance gene discovery and identification of EST-SSR and SNP markers.

Notopterygium incisum C. C. Ting ex H. T. Chang (Apiaceae) is an endangered perennial herb in China. The lack of transcriptomic and genomic resources for N. incisum greatly hinders studies of its population genetics and conservation. In this study, we employed RNA-seq technology to characterize transcriptomes for the flowers, leaves, and stems of this endangered herb. A total of 56 million clean reads were assembled into 120,716 unigenes with an N50 length of 850 bp. Among these unigenes, 70,245 (58.19%) were successfully annotated and 65,965 (54.64%) were identified as coding sequences based on their similarities with sequences in public databases. We identified 21 unigenes that had significant relationships with cold tolerance in N. incisum according to gene ontology (GO) annotation analysis. In addition, 13,149 simple sequence repeats (SSRs) and 85,681 single nucleotide polymorphisms were detected as potential molecular genetic markers. Ninety-six primer pairs of SSRs were randomly selected to validate their amplification efficiency and polymorphism. Nineteen SSR loci exhibited polymorphism in three natural populations of N. incisum. These results provide valuable resources to facilitate future functional genomics and conservation genetics studies of N. incisum.

Development of novel EST-SSR markers for Ephedra sinica (Ephedraceae) by transcriptome database mining.

PREMISE OF THE STUDY: Ephedra sinica (Ephedraceae) is a gymnosperm shrub with a wide distribution across Central and Eastern Asia. It is widely cultivated as a medicinal plant, but its wild populations are monitored to determine whether protection is needed. METHODS AND RESULTS: Thirty-six microsatellite markers, including 11 polymorphic markers, were developed from E. distachya RNA-Seq data deposited in the National Center for Biotechology Information dbEST database. Among 100 genotyped E. sinica individuals originating from five different population groups, the allele number ranged from three to 22 per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.866 (average 0.176) and 0 to 0.876 (average 0.491), respectively. Allelic polymorphism information content ranged from 0.000 to 0.847 (average 0.333). Cross-species amplifications were successfully conducted with two related Ephedra species for all 11 di- or trinucleotide simple sequence repeats. CONCLUSIONS: This study provides the first set of microsatellite markers for genetic monitoring and surveying of this medicinal plant.

Characterization and development of EST-SSR markers to study the genetic diversity and populations analysis of Jerusalem artichoke (Helianthus tuberosus L.).

In recent years, Jerusalem artichoke has received widespread attention as a novel source of sugar, biofuel, and animal feed. Currently, only few gDNA-SSRs derived from sunflower were verified in the Jerusalem artichoke; therefore, it is particularly important to develop SSR primer markers that belonged to Jerusalem artichoke resources. Using EST data to develop EST-SSR markers is simple and effective. In order to understand the general characteristics of SSR markers in Jerusalem artichoke EST sequences and accelerate the use of SSR markers in Jerusalem artichoke research. This study used 40,370 sequenced unigene fragments and MISA software to identify SSR loci. The 48 pairs of EST-SSR primers assessed for the identification of 45 varieties of Jerusalem artichoke. Cluster, genetic diversity parameters and AMOVA analysis was conducted using the genetic similarity coefficient, revealing genetic differences between 48 genetic material. A total of 1204 SSR loci were identified with 13 different types of repeats, distributed among 1020 EST sequences, of which trinucleotide repeats were the most common, accounting for 38.21% of the total SSR loci. Among the 44 repeat motifs, AG/CT, AAG/CTT, and ATC/ATG motifs had the highest frequencies, accounting for 22.45, 14.71, and 7.84% of all motifs, respectively. From these sequences, 48 pairs of EST-SSR primers were designed, and 22 primer pairs for loci with high polymorphism were selected to analyze the genetic diversity of 45 Jerusalem artichoke germplasm sources. The results indicated that the variation range of the effective number of alleles for 22 primers ranged between 1.7502 and 4.5660. The Shannon's information index ranged between 0.6200 and 1.6423. The variation range of PIC ranged between 0.3121 and 0.6662 with an average of 0.5184. Cluster analysis was conducted using the genetic similarity coefficient, revealing significant genetic differences between Asian and European genetic material. Cluster analysis revealed a relationship between the genotypes and geographic origins of the Jerusalem artichoke. The results of AMOVA as well as the genetic identity and genetic distance in the Jerusalem artichoke population showed that there presented certain genetic heterogeneity in Jerusalem artichoke genetic structure of 45 samples from seven different geographic populations. The Jerusalem artichoke EST-SSR marker system established in this study provides an effective molecular marker system for future research focused on Jerusalem artichoke genetic diversity and the breeding of new varieties.

Development and characterization of 30 microsatellite loci for Plagiorhegma dubium (Berberidaceae).

PREMISE OF THE STUDY: Plagiorhegma dubium (Berberidaceae) has been listed as an endangered species in Korea due to extensive collection and destruction of natural habitats. In this study, 30 microsatellite loci, including 25 polymorphic loci, were developed for P. dubium for use in population-level genetic analyses. METHODS AND RESULTS: We carried out transcriptome sequencing and isolated a total of 30 expressed sequence tag-simple sequence repeat markers from P. dubium using Illumina HiSeq high-throughput sequencing. To test utility of the developed markers, we genotyped 60 individuals from three populations and estimated the number of alleles and levels of observed and expected heterozygosity. Expected heterozygosity levels ranged from 0.000 to 0.594, 0.000 to 1.000, and 0.000 to 0.744 in the three populations, respectively. CONCLUSIONS: These transcriptome-derived simple sequence repeat markers are highly polymorphic and can be widely used in characterization of the endangered P. dubium. Population genetic studies with these markers will provide valuable insights for conservation by unraveling evolutionary patterns of P. dubium.

Development of novel EST-SSR markers for Rhododendron longipedicellatum (Ericaceae) and cross-amplification in two congeners.

PREMISE OF THE STUDY: To investigate the genetic background and population characteristics of Rhododendron longipedicellatum (Ericaceae), a newly discovered and critically endangered species, expressed sequence tag-simple sequence repeat markers were developed, and transferability was tested in two congeners, R. molle and R. simsii. METHODS AND RESULTS: Based on the transcriptome sequences of R. longipedicellatum, 102 primer sets were designed; 48 primer sets were successfully amplified, with 15 showing polymorphisms in 150 individuals from five extant populations of R. longipedicellatum. The number of alleles per locus ranged from four to 18, and the levels of observed and expected heterozygosity for the 15 loci varied from 0.255 to 0.913 and from 0.306 to 0.851, respectively. All 15 loci were found to amplify in R. molle and R. simsii. CONCLUSIONS: These polymorphic SSR markers can be used in conservation genetic and phylogeographic studies to elucidate the rarity and origin of R. longipedicellatum.

Development of novel EST-SSR markers for Phyllanthus emblica (Phyllanthaceae) and cross-amplification in two related species.

PREMISE OF THE STUDY: A novel set of EST-SSR markers was developed for Phyllanthus emblica (Phyllanthaceae) to investigate the genetic structure and gene flow, identify novel genes of interest, and develop markers for assisted breeding. METHODS AND RESULTS: Based on the transcriptome data of P. emblica, 83 EST-SSR primer pairs were designed; 52 primer pairs were successfully amplified, with 20 showing polymorphisms in 90 individuals from three populations of P. emblica. The number of alleles per locus varied from 11 to 44. The observed and expected levels of heterozygosity for the 20 loci ranged from 0.240 to 0.868 and 0.754 to 0.933, respectively. Cross-species amplification was successful for all 20 loci in each of the two related species, P. reticulatus and Leptopus chinensis. CONCLUSIONS: These markers will be valuable for studying the population genetics and for mining genes of P. emblica, and may be useful for studies of related species.

Isolation and characterization of SSR and EST-SSR loci in Chamaecyparis formosensis (Cupressaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) and expressed sequence tag (EST)-SSR markers were developed as tools for marker-assisted selection of Chamaecyparis formosensis and for the molecular differentiation of cypress species. METHODS AND RESULTS: Based on the SSR-enriched genomic libraries and transcriptome data of C. formosensis, 300 primer pairs were selected for initial confirmation, of which 19 polymorphic SSR and eight polymorphic EST-SSR loci were chosen after testing in 92 individuals. The number of alleles observed for these 27 loci ranged from one to 17. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.903, respectively. Most markers also amplified in C. obtusa var. formosana. CONCLUSIONS: The developed SSR and EST-SSR sequences are the first reported markers specific to C. formosensis. These markers will be useful for individual identification of C. formosensis and to distinguish cypress species such as C. obtusa var. formosana.

Development and characterization of polymorphic EST based SSR markers in barley (Hordeum vulgare).

In barley, breeding using good genetic characteristics can improve the quality or quantity of crop characters from one generation to the next generation. The development of effective molecular markers in barley is crucial for understanding and analyzing the diversity of useful alleles. In this study, we conducted genetic relationship analysis using expressed sequence tag-simple sequence repeat (EST-SSR) markers for barley identification and assessment of barley cultivar similarity. Seeds from 82 cultivars, including 31 each of naked and hulled barley from the Korea Seed and Variety Service and 20 of malting barley from the RDA-Genebank Information Center, were analyzed in this study. A cDNA library of the cultivar Gwanbori was constructed for use in analysis of genetic relationships, and 58 EST-SSR markers were developed and characterized. In total, 47 SSR markers were employed to analyze polymorphisms. A relationship dendrogram based on the polymorphism data was constructed to compare genetic diversity. We found that the polymorphism information content among the examined cultivars was 0.519, which indicates that there is low genetic diversity among Korean barley cultivars. The results obtained in this study may be useful in preventing redundant investment in new cultivars and in resolving disputes over seed patents. Our approach can be used by companies and government groups to develop different cultivars with distinguishable markers. In addition, the developed markers can be used for quantitative trait locus analysis to improve both the quantity and the quality of cultivated barley.

Development of EST-SSR markers for Primula ovalifolia (Primulaceae) by transcriptome sequencing.

PREMISE OF THE STUDY: Microsatellite primers were developed for Primula ovalifolia, a member of Primula section Petiolares (Primulaceae), to study the population genetics and species delimitation in this section. METHODS AND RESULTS: A total of 4753 markers were successfully designated from 5139 putative simple sequence repeat loci. We isolated 38 expressed sequence tag-simple sequence repeat markers from 220 selected marker sites and tested polymorphism in three populations of P. ovalifolia, one of P. tardiflora, and one of P. epilosa. The number of alleles per locus ranged from one to 19, and the observed and expected levels of heterozygosity varied from 0 to 0.938 and 0 to 0.915, respectively. Most of the loci could be successfully cross-amplified in the two congeneric species. CONCLUSIONS: These markers will be useful for further population genetic analysis and gene flow estimation of P. ovalifolia and its relatives.

Characterization and development of EST-SSR markers in sweet potato (Ipomoea batatas (L.) Lam).

In this study, a cDNA library was constructed from the total RNA of sweet potato leaves. A total of 789 copies of the cDNA were cloned in Escherichia coli by employing the pGEM-T Easy vector. Sequencing was carried out by Solgent Co. (Korea). As many as 579 expressed sequence tag-simple sequence repeat (EST-SSR) markers were designed (73.38%) from the known cDNA nucleotide base sequences. The lengths of the developed EST-SSR markers ranged from 100 to 499 bp (average length 238 bp). Their motif sequence types were varied, with most being dinucleotides and pentanucleotides, and the most commonly found motifs were CAGAAT (29.0%) and TCT (2.8%). Based on these SSR-containing sequences, 619 pairs of high-quality SSR primers were designed using WebSat and Primer3web. The total number of primers designed was 144. Polymorphism was evident in 82 EST-SSR markers among 20 Korean sweet potato cultivars tested and in 90 EST-SSR markers in the two parents of a mapping population, Yeseumi and Annobeny. In this study, the hexaploid sweet potato (2n = 6x = 90) EST-SSR markers were developed in the absence of full-sequence data. Moreover, by acting as a molecular tag for particular traits, the EST-SSR marker can also simultaneously identify information about the corresponding gene. These EST-SSR markers will allow the molecular analysis of sweet potato to be done more efficiently. Thus, we can develop high-quality sweet potato while overcoming the challenges from climate change and other unfavorable conditions.