Polymorphic Expressed Sequence Tags Research Articles

De Novo Transcriptome Assembly and EST-SSR Marker Development and Application in Chrysosplenium macrophyllum.

Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional and unique Chinese herbal medicine. However, the lack of adequate molecular markers has hampered the progress regarding population genetics and evolution within this species. In this research, we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal component analysis, and popular structure analysis revealed that all 60 samples clustered into two major groups that were consistent with their geographical origins. This study provided a batch of highly polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These markers will be of great significance for the study of the genetic diversity and evolutionary history of C. macrophyllum and other Chrysosplenium species.

Enhanced correlations of EST-SSR-based genetic distance with hybrid performance, specific hybridizing ability, and heterosis using effect-increasing and effect-decreasing alleles: a case study in Eucalyptus L’Hér.

A factorial mating population between 10 Eucalyptus urophylla S. T. Blake and 10 E. tereticornis Smith parents tested at two sites was used to investigate the relationship of parental genetic variation to hybrid mean value (MV), specific hybridizing ability (SHA), and heterosis. Based on 184 polymorphic expressed sequence tag (EST) derived simple sequence repeats (SSR) markers, genetic distance (GD) between parents (GDAll) ranged from 0.293 to 0.783, averaging at 0.700. For tree height, breast-high diameter and wood density at age of 7.5 years, GDAll, GDs based on markers beyond 0.70 and 0.80 in polymorphism information content and GDs based on alleles within 0.10–0.90, 0.20–0.80, and 0.30–0.70 in allele frequency were not significantly correlated with MV, SHA, and the majority of heterosis types. GD based on effect-significant alleles was significantly correlated only with a few of traits or for certain site, while GDs based on effect-increasing (GDIA) and effect-decreasing alleles (GDDA) had significantly positive and negative correlations, respectively, with all the MV and SHA as well as across-site mid-parent heterosis. These results suggest the potential utility of GDIA and GDDA for molecular prediction of hybrid performance, SHA, and heterosis in Eucalyptus and may have implications for hybrid breeding in other plants.

Genetic diversity and population structure analyses of Plectranthus edulis (Vatke) Agnew collections from diverse agro-ecologies in Ethiopia using newly developed EST-SSRs marker system

BackgroundPlectranthus edulis (Vatke) Agnew (locally known as Ethiopian dinich or Ethiopian potato) is one of the most economically important edible tuber crops indigenous to Ethiopia. Evaluating the extent of genetic diversity within and among populations is one of the first and most important steps in breeding and conservation measures. Hence, this study was aimed at evaluating the genetic diversity and population structure of this crop using collections from diverse agro-ecologies in Ethiopia.ResultsTwenty polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers were developed for P. edulis based on EST sequences of P. barbatus deposited in the GenBank. These markers were used for genetic diversity analyses of 287 individual plants representing 12 populations, and a total of 128 alleles were identified across the entire loci and populations. Different parameters were used to estimate the genetic diversity within populations; and gene diversity index (GD) ranged from 0.31 to 0.39 with overall mean of 0.35. Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with only 3% of the total variation accounted for variation among populations. Likewise, cluster and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and the reproductive biology of the crop.ConclusionsThese newly developed EST-SSR markers are highly polymorphic within P. edulis and hence are valuable genetic tools that can be used to evaluate the extent of genetic diversity and population structure of not only P. edulis but also various other species within the Lamiaceae family. Among the 12 populations studied, populations collected from Wenbera, Awi and Wolaita showed a higher genetic diversity as compared to other populations, and hence these areas can be considered as hot spots for in-situ conservation as well as for identification of genotypes that can be used in breeding programs.

Effects of the Tanaka Line on the genetic structure of Bombax ceiba (Malvaceae) in dry-hot valley areas of southwest China.

Southwest China is an important biodiversity hotspot. The interactions among the complex topography, climate change, and ecological factors in the dry‐hot valley areas in southwest China may have profoundly affected the genetic structure of plant species in this region. In this study, we determined the effects of the Tanaka Line on genetic variation in the wild Bombax ceiba tree in southwest China. We sampled 224 individuals from 17 populations throughout the dry‐hot valley regions. Six polymorphic expressed sequence tag–simple sequence repeat primers were employed to sequence the PCR products using the first‐generation Sanger technique. The analysis based on population genetics suggested that B. ceiba exhibited a high level of gene diversity (H E: 0.2377–0.4775; I: 0.3997–0.7848). The 17 populations were divided into two groups by cluster analysis, which corresponded to geographic characters on each side of the Tanaka Line. In addition, a Mantel test indicated that the phylogeographic structure among the populations could be fitted to the isolation‐by‐distance model (r 2 = .2553, p < .001). A barrier test indicated that there were obstacles among populations and between the two groups due to complex terrain isolation and geographic heterogeneity. We inferred that the Tanaka Line might have promoted the intraspecific phylogeographic subdivision and divergence of B. ceiba. These results provide new insights into the effects of the Tanaka Line on genetic isolation and population differentiation of plant species in southwest China.

New microsatellites revealed strong gene flow among populations of a new outbreak pest, Athetis lepigone (Möschler).

Athetis lepigone (Möschler) (Lepidoptera: Noctuidae) is a new outbreak pest in China. Consequently, it is unclear whether the emergence and spread of the outbreak of this pest are triggered by rapid in situ population size increases in each outbreak area, or by immigrants from a potential source area in China. In order to explore the outbreak process of this pest through a population genetics approach, we developed ten novel polymorphic expressed sequence tags (EST)-derived microsatellites. These new microsatellites had moderately high levels of polymorphism in the tested population. The number of alleles per locus ranged from 3 to 19, with an average of 8.6, and the expected heterozygosity ranged from 0.269 to 0.783. A preliminary population genetic analysis using these new microsatellites revealed a lack of population genetic structure in natural populations of A. lepigone. The estimates of recent migration rate revealed strong gene flow among populations. In conclusion, our study developed the first set of EST-microsatellite markers and shed a new light on the population genetic structure of this pest in China.

Development and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 × PB217 population

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.

Development of gene-based markers for use in construction of the chickpea (Cicer arietinum L.) genetic linkage map and identification of QTLs associated with seed weight and plant height.

Seed weight and plant height are important agronomic traits and contribute to seed yield. The objective of this study was to identify QTLs underlying these traits using an intra-specific mapping population of chickpea. A F11 population of 177 recombinant inbred lines derived from a cross between SBD377 (100-seed weight--48 g and plant height--53 cm) and BGD112 (100-seed weight--15 g and plant height--65 cm) was used. A total of 367 novel EST-derived functional markers were developed which included 187 EST-SSRs, 130 potential intron polymorphisms (PIPs) and 50 expressed sequence tag polymorphisms (ESTPs). Along with these, 590 previously published markers including 385 EST-based markers and 205 genomic SSRs were utilized. Of the 957 markers tested for analysis of parental polymorphism between the two parents of the mapping population, 135 (14.64%) were found to be polymorphic. Of these, 131 polymorphic markers could be mapped to the 8 linkage groups. The linkage map had a total length of 1140.54 cM with an average marker density of 8.7 cM. The map was further used for QTL identification using composite interval mapping method (CIM). Two QTLs each for seed weight, qSW-1 and qSW-2 (explaining 11.54 and 19.24% of phenotypic variance, respectively) and plant height, qPH-1 and qPH-2 (explaining 13.98 and 12.17% of phenotypic variance, respectively) were detected. The novel set of genic markers, the intra-specific linkage map and the QTLs identified in the present study will serve as valuable genomic resources in improving the chickpea seed yield using marker-assisted selection (MAS) strategies.

ESTP 표지를 이용한 국내 소나무 집단의 유전변이

소나무의 유전다양성과 유전구조를 추정하기 위해 9개의 ESTP 표지를 13개 소나무 집단에 적용하였다. 소나무 집단의 유전다양성은 관찰된 대립유전자 수(A)가 2.2개, 유효 대립유전자 수(A_e)가 1.8개, 다형적 유전자좌 비율(P)이 98.8%, 이형접합도 관찰치(H_o)가 0.391, 이형접합도 기대치(H_e)가 0.402로 나타났다. 안강과 강릉 집단을 제외한 11개 집단이 하디-바인베르그 평형을 만족하였다. 집단간 유전분화도(F_ST)는 0.057으로, 동위효소나 nSSR 표지분석 결과보다 강하게 나타났다. 군집분석에서 집단의 유전적 거리와 지리적 분포간에 뚜렷한 연관성은 확인할 수 없었으며, 집단의 유전분화와 지리적 인접성도 상관이 없는 것으로 나타났다(Mantel 검증, r = 0.017, P = 0.344). 유전자좌에 대한 F_ST-outlier 분석을 실시한 결과, 빈도주의 방법에서는 F_ST 값이 신뢰하한 이하인 3개 유전자좌와 신뢰상한 이상인 3개 유전자좌가 특이값으로 추정되었고, 베이즈 방법에서는 3개 유전자좌들만 특이값으로 확인되었다. 두 방법에서 공히 특이값으로 판정된 3개 유전자좌(sams2+Alu&#x2160;, sams2+Rsa&#x2160;, PtNCS_p14A9+Hae&#x2162;)중 sams2 표지에서 유래된 2개 유전자좌는 balancing selection의 영향을 받는 것으로 추정되었다. Genetic diversity and genetic differentiation of thirteen Pinus densiflora populations in South Korea were estimated using nine ESTP (Expressed Sequence Tag Polymorphism) markers. The numbers of allele and the effective allele were 2.2 and 1.8, respectively. The percentage of polymorphic loci (P) was 98.8%. The observed and the expected heterozygosity were 0.391 and 0.402, respectively, and the eleven populations except for Ahngang and Gangneung population were under Hardy-Weinberg equilibrium state. The level of genetic differentiation (Wright&#x2019;s F_ST = 0.057) was higher than those of isozyme or nSSR markers. We could not find out any relationship between the genetic distance and geographic distribution among populations from cluster analysis. Also, the genetic differentiation between populations was not correlated with the geographic distance (r = 0.017 and P = 0.344 from Mantel test). From the result of F_ST-outlier analysis to identify a locus under selection, six loci were detected at confidence interval of 99% by the frequentist&#x2019;s method. However, only three loci (sams2+Alu&#x2160;, sams2+Rsa&#x2160;, PtNCS_p14A9+Hae&#x2162;) were presumed as outliers by Bayesian method. The sams2+Alu&#x2160; and sams2+Rsa&#x2160;locus were originated from the sams2 gene and seemed to be the loci under balancing selection.

Development and characterization of polymorphic microsatellite markers (SSRs) for an endemic plant, Pseudolarix amabilis (Nelson) Rehd. (Pinaceae).

Pseudolarix (Pinaceae) is a vulnerable (sensu IUCN) monotypic genus restricted to southeastern China. To better understand levels of genetic diversity, population structure and gene flow among populations of P. amabilis, we developed five compound SSR markers and ten novel polymorphic expressed sequence tags (EST) derived microsatellites. The results showed that all 15 loci were polymorphic with the number of alleles per locus ranging from two to seven. The expected and observed heterozygosities varied from 0.169 to 0.752, and 0.000 to 1.000, respectively. The inbreeding coefficient ranged from −0.833 to 1.000. These markers will contribute to research on genetic diversity and population genetic structure of P. amabilis, which in turn will contribute to the species conservation.

Characterization and development of EST-SSR markers derived from transcriptome of yellow catfish.

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater fish due to its delicious flesh and high nutritional value. However, lack of sufficient simple sequence repeat (SSR) markers has hampered the progress of genetic selection breeding and molecular research for yellow catfish. To this end, we aimed to develop and characterize polymorphic expressed sequence tag (EST)–SSRs from the 454 pyrosequencing transcriptome of yellow catfish. Totally, 82,794 potential EST-SSR markers were identified and distributed in the coding and non-coding regions. Di-nucleotide (53,933) is the most abundant motif type, and AC/GT, AAT/ATT, AAAT/ATTT are respective the most frequent di-, tri-, tetra-nucleotide repeats. We designed primer pairs for all of the identified EST-SSRs and randomly selected 300 of these pairs for further validation. Finally, 263 primer pairs were successfully amplified and 57 primer pairs were found to be consistently polymorphic when four populations of 48 individuals were tested. The number of alleles for the 57 loci ranged from 2 to 17, with an average of 8.23. The observed heterozygosity (HO), expected heterozygosity (HE), polymorphism information content (PIC) and fixation index (FIS) values ranged from 0.04 to 1.00, 0.12 to 0.92, 0.12 to 0.91 and −0.83 to 0.93, respectively. These EST-SSR markers generated in this study could greatly facilitate future studies of genetic diversity and molecular breeding in yellow catfish.

The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

Cultivated alfalfa (Medicago sativa L.) is one of the most important forage legume in the world. Here, we report the development of 204 novel polymorphic expressed sequence tag simple sequence repeat (EST-SSRs) markers from transcript sequences via global Illumina sequencing for cultivated alfalfa. Among the synthesized 750 pairs of primers, 204 EST-SSR primer pairs showed polymorphisms among 10 alfalfa accessions (including five individual plants in each accession) generating a total of 1222 alleles, with the number of alleles per locus, the observed heterozygosity (Ho), the corrected heterozygosity (He) and the Shannon-Wiener diversity index (H'c) averaging at 5.99, 0.73, 0.71 and 1.14, respectively. Of the 204 novel EST-SSRs in alfalfa, 120 can be in silico mapped onto the eight Medicago truncatula chromosomes (Mt3.5.2). Considering the high polymorphism, these EST-SSRs can be applied to assess genetic diversity, population structure, relatedness, evolution, linkage mapping and cultivar protection of cultivated alfalfa to facilitate alfalfa breeding programs.

Development of microsatellite markers for, and a preliminary population genetic analysis of, the white-backed planthopper.

For a better understanding of the population structure and dispersal rates of Sogatella furcifera, we developed 21 novel polymorphic expressed sequence tags (EST) derived microsatellites, which were successfully amplified in four multiplex polymerase chain reaction sets. These new microsatellites were firstly assessed in 20 individuals sampled from Wenshan in China. The results showed that all 21 loci were highly polymorphic; the number of alleles ranged from 3 to 9, with an average of 4.8 alleles per locus. The observed and expected heterozygosity ranged from 0.200 to 0.900 and from 0.184 to 0.799, respectively. Nineteen of the 21 microsatellites without null allele, were subsequently used for population genetic structure analyses of five S. furcifera populations sampled in south region of China (sites up to 1314 kilometers apart). The observed and expected heterozygosity for each population ranged from 0.436 to 0.494 and from 0.454 to 0.482, respectively. The level of population differentiation was very low, with an average pairwise F ST of 0.002. Bayesian cluster analysis result suggested that the five S. furcifera populations formed one genetic cluster. Discriminant analysis of principle components detected three genetic clusters. The spread of the three clusters across the five populations explained the lack of population differentiation and the Bayesian cluster result. All the results indicated that long-distance migration of this pest allowed genetic mixing between populations from remote geographical origins. These new microsatellites will be powerful tools for population genetics studies of S. furcifera.

Large-scale development of EST-SSR markers in sponge gourd via transcriptome sequencing

Sponge gourd is a versatile vegetable that can be used as a household cleaning product, industrial material and medicine, and possesses anti-human immunodeficiency virus activity. In the current study, a large dataset composed of 77,858 unigenes derived from the sponge gourd transcriptome was assembled. Of all unigenes, 52,113 (66.93 %) had significant similarity to known proteins from NCBI non-redundant, Swiss-Prot, Clusters of Orthologous Group and Kyoto Encyclopedia of Genes and Genomes databases. Based on these generated sequences, we identified 12,932 putative simple sequence repeats (SSRs) and successfully designed 8,523 high-quality SSR primer pairs. Six hundred and forty-one primer pairs were randomly selected to be verified among S1174 [Luffa acutangula (L.) Roxb.], 93075 [L. cylindrica (L.) Roem.] and their hybrid F1. The result showed that 494 (77.07 %) exhibited successful amplification, of which 201 (40.69 %) revealed polymorphism between S1174 and 93075. Fifty polymorphic expressed sequence tag (EST)-SSRs were used to genotype 60 sponge gourd accessions. Cluster analysis revealed two major clusters, one comprising 42 accessions of L. acutangula and the other 18 accessions belonging to L. cylindrica. Transferability of the 77 polymorphic EST-SSR markers was investigated in six other cucurbits: pumpkin, cucumber, wax gourd, bitter gourd, bottle gourd and chieh-qua, and the percentage of cross-genus amplification was 85.90, 83.33, 83.33, 79.49, 75.64 and 74.36 %, respectively. This indicated that EST-SSRs in sponge gourd could well be applied to other cucurbits. These transcriptome shotgun assembly sequences and EST-SSR data are of great value for the discovery of novel genes and for marker-assisted selection in sponge gourd.

CHARACTERIZATION OF GRAPE CULTIVARS THROUGH ESTP

Grapevine cultivars developed by Instituto Agronomico de Campinas known as ?IAC 138-22 Maximo?, ?IAC Rainha? and ?IAC Madalena? were characterized to find a distinct genetic profile in order to facilitate patenting of a new cultivar. The characterization was done using the Expressed Sequence Tag Polymorphism (ESTP) methodology. From thirteen primers derived from EST initially chosen, nine amplified DNA fragments in sufficient quantity for sequencing, and only three gave different alleles corresponding to nine single nucleotide polymorphisms (SNP). Although the SNP frequency of about 1.2 per locus, observed in this study was low, by analyzing the sequences it was possible to detect specific restriction sites for the differentiation of cultivars ?IAC 138-22 Maximo?, ?IAC Rainha? and ?IAC Madalena?.

Development and Genotyping Potentials of EST-SSRs in Finger Millet (E. Coracana (L.) Gaertn.)

Finger millet (E. coracana (L.) Gaertn.) provides food for millions of people in Africa and Asia. In this study, sequence data were mined at the database of National Center for Biotechnology Information (NCBI) with the aim of developing polymorphic expressed sequence tags simple sequence repeat (EST-SSRs) markers. Three selected markers which showed clear polymorphism in pre-testing with 5 accessions were used to characterize some randomly selected 48 accessions from the pool of finger millet core set. The polymorphic information content (PIC) of the developed markers gave a value range of 0.6741 for marker UH-Ec-931 to 0.7658 for marker UH-Ec-958. The mean PIC value of 0.7171 was recorded. Marker UH-Ec-958 showed 13 alleles per locus while marker UH-Ec-956 showed 20 alleles per locus. The mean average allele per locus was 17. Following Nei’s approach, the mean gene diversity value of 0.7638 was captured by the three markers. Cluster analysis for the 48 selected accessions of finger millet showed four major clusters. Accessions from Zimbabwe and Zambia are distributed on the cluster I. Accessions from India are mostly found in cluster IV. Accessions from Nepal were found mostly on cluster III while Ugandan accessions are found in cluster II and III respectively. Our investigation showed that the developed EST-SSRs are quite effective in unraveling the nature of diversity in our studied population.

Combination of multipoint maximum likelihood (MML) and regression mapping algorithms to construct a high-density genetic linkage map for loblolly pine (Pinus taeda L.)

Genetic maps have been successfully applied to assist in the dissection of complex traits, provide insight on genome structure, and estimate recombination in conjunction with physical maps. Despite an extensive list of genetic maps developed for loblolly pine (Pinus taeda L.) over the past two decades, a high-density consensus map has not yet been constructed. In this study, we used two reference three-generation outbred pedigrees, base and qtl, obtained from the North Carolina State University Cooperative Tree Improvement Program, to obtain a high-density genetic consensus map. Both populations were genotyped with ≈ 7,000 different markers (restriction fragment length polymorphisms, expressed sequence tag polymorphisms, simple sequence repeats, SNPs). The grouping, ordering, and spacing of the markers on each linkage group were performed with JoinMap® 4.1, which implements the multipoint maximum likelihood algorithm for outbred populations. The final consensus map contains 2,466 markers, with a total length of 1,476 centimorgans (cM). The average marker density across the 12 linkage groups was 0.62 cM/marker. This high-density map provides an important resource for breeders and geneticists and will enable comparative studies across species, as well as improve the loblolly pine genome sequence assembly.

QTL detection for growth traits in Pinus elliottii var. elliottii and P. caribaea var. hondurensis

Quantitative trait locus (QTL) detection was carried out for growth traits in 122 F1 progenies of Pinus elliottii var. elliottii (PEE) × P. caribaea var. hondurensis (PCH) hybrid. The PCH male parent linkage map contained 108 markers in 16 linkage groups, while the PEE female parent contained 93 markers in 19 linkage groups. Sequence-related amplified polymorphism (SRAP), microsatellite (SSR), expressed sequence tag polymorphism (ESTP) and inter-simple sequence repeat (ISSR) were selected from an existing linkage map. Growth traits investigated were height at age five (HT05) and six years (HT06), diameter at breast height at age five (DBH05) and six years (DBH06) and annual growth from age five to six years of height (AGHT) and diameter at breast height (AGDBH). Kruskal-Wallis and interval mapping approaches were used to estimate levels of significance, the number of QTLs, the percentage of the phenotypic variation explained by each of QTLs and their positions on the genetic linkage maps. Twenty six QTLs with significance levels p < 0.05 were detected on the parental maps for the six growth traits, which explained more than 15% of the phenotypic variation, suggesting an existence of major-effect genes. Several QTLs had the pleiotropy controlling more than one single growth trait. Overall, the proportion of phenotypic variation explained by QTLs ranged from 5.9% to 40.6% for HT05 and HT06, from 6.6% to 42.0% for DBH05 and DBH06 and from 5.8% to 22.3% for AGHT and AGDBH. The results from this study provide a basis for marker-aided selection (MAS) in PEE × PCH hybrids.

Development and integration of EST–SSR markers into an established linkage map in switchgrass

Switchgrass (Panicum virgatum L.) is a model cellulosic biofuel crop in the United States. Simple sequence repeat (SSR) markers are valuable resources for genetic mapping and molecular breeding. A large number of expressed sequence tags (ESTs) of switchgrass are recently available in our sequencing project. The objectives of this study were to develop new SSR markers from the switchgrass EST sequences and to integrate them into an existing linkage map. More than 750 unique primer pairs (PPs) were designed from 243,600 EST contigs and tested for PCR amplifications, resulting in 538 PPs effectively producing amplicons of expected sizes. Of the effective PPs, 481 amplifying informative bands in NL94 were screened for polymorphisms in a panel consisting of NL94 and its seven first-generation selfed (S1) progeny. This led to the selection of 117 polymorphic EST–SSRs to genotype a mapping population encompassing 139 S1 individuals of NL94. Of 83 markers demonstrating clearly scorable alleles in the mapping population, 79 were integrated into a published linkage map, with three linked to accessory loci and one unlinked. The newly identified EST–SSR loci were distributed in 17 of 18 linkage groups with 27 (32.5 %) exhibiting distorted segregations. The integration of EST–SSRs aided in reducing the average marker interval (cM) to 3.7 from 4.2, and reduced the number of gaps (each >15 cM) to 10 from 23. Developing new EST–SSRs and constructing a higher density linkage map will facilitate quantitative trait locus mapping and provide a firm footing for marker-assisted breeding in switchgrass.

Constructing genetic linkage maps for Pinus elliottii var. elliottii and Pinus caribaea var. hondurensis using SRAP, SSR, EST and ISSR markers

A total of 122 F1 individuals from a single full-sib interspecific hybrid family crossed between Pinus elliottii var. elliottii (PEE) and P. caribaea var. hondurensis (PCH) were used to construct a detailed genetic linkage maps using four types of molecular markers: sequence-related amplified polymorphism (SRAP), microsatellite (SSR), expressed sequence tag polymorphism (ESTP) and inter-simple sequence repeat (ISSR). There were 381 SRAP, 108 SSR, 25 ESTP and 32 ISSR loci, segregating in the interspecific F1 hybrid individuals. Framework maps were constructed at a LOD score threshold of 4.0 using the JoinMap® 3.0. The map for the male parent (PCH) had 108 markers in 16 linkage groups (LGs), with a total length of 1,065.9 cM (Kosambi) and an average marker interval of 9.87 cM. The map for the female parent (PEE) contained 93 markers in 19 LGs, with a total length of 1,006.7 cM (Kosambi) and an average marker interval of 10.82 cM. The maps for PCH and PEE covered 56.5 and 70.3 % of their respective genomes. Based on the position of 36 loci segregating in both parents, 8 homologous LGs between PEE and PCH were identified.

Development and characterization of 109 polymorphic EST‐SSRs derived from the Chinese bayberry (Myrica rubra, Myricaceae) transcriptome

In Chinese bayberry (Myrica rubra), the available simple sequence repeat (SSR) markers are insufficient to meet the developing demand for genetic and molecular breeding research. This study was aimed at developing a large number of polymorphic expressed sequence tag (EST)-SSRs from the transcriptome of Chinese bayberry. • Five hundred ninety-four compound EST-SSRs and 5557 noncompound ones were identified from 41239 unigene sequences generated from the transcriptome of M. rubra cv. Biqi. Using 10 Chinese bayberry cultivars, 109 polymorphic EST-SSRs were screened from 412 selected. In total, they generated 389 alleles, with a polymorphism ratio of 93.8%. In addition, it was observed that the polymorphism levels of compound EST-SSRs were somewhat lower than those of noncompound ones. • The 109 polymorphic EST-SSRs developed from the Chinese bayberry transcriptome should greatly promote the development of genetic and molecular breeding studies in this as well as other Myricaceae species.