Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags

Traditionally, simple sequence repeat (SSR) markers have been developed from libraries of genomic DNA. However, the large, repetitive nature of conifer genomes makes development of robust, single-copy SSR markers from genomic DNA difficult. Expressed sequence tags (ESTs), or sequences of messenger RNA, offer the opportunity to exploit single, low-copy, conserved sequence motifs for SSR development. From a 20,275-unigene spruce EST set, we identified 44 candidate EST-SSR markers. Of these, 25 amplified and were polymorphic in white, Sitka, and black spruce; 20 amplified in all 23 spruce species tested; the remaining five amplified in all except one species. In addition, 101 previously described spruce SSRs (mostly developed from genomic DNA), were tested. Of these, 17 amplified across white, Sitka, and black spruce. The 25 EST-SSRs had approximately 9% less heterozygosity than the 17 genomic-derived SSRs (mean H=0.65 vs 0.72), but appeared to have less null alleles, as evidenced by much lower apparent inbreeding (mean F=0.046 vs 0.126). These robust SSRs are of particular use in comparative studies, and as the EST-SSRs are within the expressed portion of the genome, they are more likely to be associated with a particular gene of interest, improving their utility for quantitative trait loci mapping and allowing detection of selective sweeps at specific genes.     

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. ‘Summer Black’) flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604–GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.     

Assessing genetic diversity of cotton cultivars using genomic and newly developed expressed sequence tag-derived microsatellite markers

Estimations of genetic diversity and of relationships between varieties are crucial for cotton breeding. The genetic diversity of 59 core cotton cultivars, most of which were collected from China's main cotton-growing areas, was analyzed based on genomic and newly developed expressed sequence tag-derived microsatellite markers, using total DNA extracted from fresh leaf tissue. Three hundred and two fragments were detected, of which 255 were polymorphic. The number of amplification products generated by each primer varied from 2 to 14, with a mean of 5.08 bands/primer. The polymorphism information content was 0.50 to 0.90, with a mean of 0.80. The genetic similarity coefficients were calculated and dendrograms were constructed by the unweighted pair group with arithmetic mean method; the resulting distance matrix gave a dendrogram with four main clusters. Some cultivars with similar pedigrees could be clustered. For example, Zhong206 and Shanmian4, both derived from Deltapine15, were clustered. The genetic similarity coefficient of the 59 core cultivars ranged from 0.53 to 0.99, with a mean of 0.72, indicating that there was a relatively high level of genetic variation.     

Development of simple sequence repeat markers for Botryosphaeria spp. with Fusicoccum anamorphs

We report the development of eight sets of microsatellite markers for the ascomycete fungus and tree pathogen, Botryosphaeria parva. The primers were identified after cloning and sequencing of fragments amplified using simple sequence repeat (SSR) primers. Genome walking was used to determine unknown sequences on either side of new SSRs. The primers were tested and proved useful in nine other Botryosphaeria species that all have Fusicoccum anamorphs, similar to B. parva.     

Genetic diversity analysis of Bt cotton genotypes in Pakistan using simple sequence repeat markers

The popularity of genetically modified insect resistant (Bt) cotton has promoted large scale monocultures, which is thought to worsen the problem of crop genetic homogeneity. Information on genetic diversity among Bt cotton varieties is lacking. We evaluated genetic divergence among 19 Bt cotton genotypes using simple sequence repeat (SSR) markers. Thirty-seven of 104 surveyed primers were found informative. Fifty-two primers selected on the basis of reported intra-hirsutum polymorphism in a cotton marker database showed a high degree of polymorphism, 56% compared to 13% for randomly selected primers. A total of 177 loci were amplified, with an average of 1.57 loci per primer, generating 38 markers. The amplicons ranged in size from 98 to 256 bp. The genetic similarities among the 19 genotypes ranged from 0.902 to 0.982, with an average of 0.947, revealing a lack of diversity. Similarities among genotypes from public sector organizations were higher than genotypes developed by private companies. Hybrids were found to be more distant compared to commercial cultivars and advanced breeding lines. Cluster analysis grouped the 19 Bt cotton genotypes into three major clusters and two independent entries. Cultivars IR-3701, Ali Akbar-802 and advanced breeding line VH-259 grouped in subcluster B2, with very narrow genetic distances despite dissimilar parentage. We found a very high level of similarity among Pakistani-bred Bt cotton varieties, which means that genetically diverse recurrent parents should be included to enhance genetic diversity. The intra-hirsutum polymorphic SSRs were found to be highly informative for molecular genetic diversity studies in these cotton varieties.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

Development and evaluation of expressed sequence tag-derived microsatellite markers for hop genotyping

The use of expressed sequence tag-simple sequence repeat (EST-SSR) markers might reflect the better relationship among species or cultivars than markers previously used. The first set of 30 EST-SSR was developed in hop (Humulus lupulus L.). They represent 25 gene loci with total of 1268 EST sequences. They were used for characterization of 11 hop samples and cross-amplification in Humulus japonicus Sieb. et Zucc. The number of alleles per locus ranged from two to nine. The observed and expected heterozygosities ranged from 0.182 to 0.956 and from 0.233 to 0.775, respectively. We used EST-SSR markers for cluster analysis of hop genotypes. Dendrogram well matched with genealogical and geographical data for hop genotypes.     

A blackberry (Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers

Background

The recent development of novel repeat-fruiting types of blackberry (Rubus L.) cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST) library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR), and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars.

Results

A cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products.

Conclusion

This rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry.     

Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.)

Efficient and robust molecular markers are essential for molecular breeding in plant. Compared to dominant and bi-allelic markers, multiple alleles of simple sequence repeat (SSR) markers are particularly informative and superior in genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The objective of this study was to enrich SSR markers directly from alfalfa expressed sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the National Center for Biotechnology Information. Total 774 SSR-containing ESTs were identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type, followed by di—(26.1 %), tetra—(11.5 %), penta—(9.7 %), and hexanucleotide (3.9 %). One hundred EST–SSR primer pairs were successfully designed and 29 exhibited polymorphism among 28 alfalfa accessions. The allele number per marker ranged from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896 with an average of 0.608, indicating a high level of polymorphism of the EST–SSR markers. Based on the 29 EST–SSR markers, assessment of genetic diversity was conducted and found that Medicago sativa ssp. sativa was clearly different from the other subspecies. The high transferability of those EST–SSR markers was also found for relative species.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Development of 16 polymorphic simple sequence repeat markers for Lycoris longituba from expressed sequence tags

Lycoris longituba is a tulip-like ornamental plant in China. We report on the data mining of L. longituba expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers. Eighteen EST-SSRs were isolated and validated for 32 individuals. These markers will be valuable for studying the genetic structure and diversity of populations for L. longituba.     

Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag

Oregano (Origanum vulgare) and marjoram (Origanum majorana) are two sensorial distinct spices within the genus Origanum (Lamiaceae). Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) of essential oil glands of O. vulgare. Thirteen EST-SSR loci were evaluated using 20 individual plants of O. vulgare and 19 plants of Origanum majorana. The number of alleles per locus ranged from one to four. All loci developed from O. vulgare successfully cross-amplified in O. majorana.     

Development of simple sequence repeat markers for bermudagrass from its expressed sequence tag sequences and preexisting sorghum SSR markers

Bermudagrass (Cynodon spp.) is extensively cultivated for forage and turf in the the southern United States and in parts of Asia, Africa, southern Europe, Australia and South America. However, few simple sequence repeat (SSR) markers are available for bermudagrass genetics research. Accordingly, the objective of this study was to develop SSR markers in bermudagrass by transferring sorghum genomic SSR primers and by exploring bermudagrass expressed sequence tags (ESTs) from the National Center for Biotechnology Information (NCBI) database. The transferability of 354 tested sorghum SSRs was 57% to C. transvaalensis T577 (2n = 2x = 18), 27% to C. dactylon Tifton 10 (2n = 6x = 54) and 22% to Zebra (2n = 4x = 36). Among the transferred SSRs, 65 primer pairs generated reproducible SSR bands across the three genotypes. From 20,237 Cynodon ESTs at NCBI, 303 designed SSR primer pairs amplified target bands in at least one of C. dactylon var. aridus (2n = 2x = 18), C. transvaalensis T577, C. dactylon cv. Tifton 10, and C. dactylon var. dactylon Zebra. Of the effective EST SSRs, 230 primer pairs produced reproducible bands in all four genotypes. The study demonstrated that EST sequences and sorghum SSR primers are useful sources for the development of SSR markers for bermudagrass. The developed SSR markers will make a valuable contribution to the molecular identification of commercial cultivars, construction of genetic maps, and marker-assisted breeding in bermudagrass.     

Agronomic characterization, genetic diversity and association analysis of cotton cultivars using simple sequence repeat molecular markers

Cotton is the most important textile plant in the world and is one of the most important crops for the production of oilseed. Because of its worldwide economic importance, new cultivars are constantly being released in the world and consequently in the Greek market, as Greece is the largest producer in Europe. We used simple sequence repeat (SSR) markers for the identification and the phylogenetic analysis of the most widely cultivated cotton cultivars in Greece. Initially, we used 12 pairs of SSR molecular markers for the analysis of 29 cultivars of Gossypium hirsutum and an interspecific hybrid (G. hirsutum x G. barbadense). Of the 12 pairs of SSR primers, 11 amplified polymorphic products, while one pair did not amplify any product. Globally, 17 polymorphic marker loci were identified. Two to four different alleles were amplified at each genomic locus, with a mean of 2.53 alleles per locus. Among the 30 genotypes that we analyzed, the polymorphism information content ranged from 0 to 0.548, with a mean of 0.293. Three main groups were formed among the 30 genotypes when a phylogenetic analysis was performed using UPGMA. Computational analysis of each molecular marker separately showed an association of SSR markers with agronomic traits such as fiber quality. To our knowledge, this is the first in-depth molecular analysis of cotton cultivars grown in Greece using SSR markers. An analysis of association of SSR markers with fiber quality traits of 29 cotton cultivars is reported for the first time.     

Genetic diversity and genetic relationships of Chukrasia spp. (Meliaceae) as revealed by inter simple sequence repeat (ISSR) markers

Key message

ISSR characterization of Chukrasia populations from the natural range revealed two distinct groups of populations consonant with morphological differentiation. Results suggest the current taxonomic classification of the genus should be reviewed.

Abstract

There are different views as to whether the genus Chukrasia (Meliaceae) consists of one species, C. tabularis, or two species C. tabularis and C. velutina. Despite a clear pattern of variation in many morphological characteristics such as leaves and bark, some authors regard the latter merely an ecotype of the former in seasonal forest. In the present study, we used ISSR markers to determine the genetic diversity and population structure among 23 Chukrasia subpopulations from across the natural range in Asia. Molecular analysis clearly differentiated two distinct groups of subpopulations, corresponding to the putative species, as well as well-defined subpopulations corresponding to geographic regions within the two groups. The molecular results are in concordance with morphological differentiation and corresponded to the two recognized taxa. The present study suggests that current taxonomic classification of the genus Chukrasia should be reviewed.     

Development of expressed sequence tag-derived microsatellite markers for the sea urchin Hemicentrotus pulcherrimus

The first set of 15 expressed sequence tag-simple sequence repeat markers was developed in sea urchin Hemicentrotus pulcherrimus. Number of alleles per locus ranged from two to 17. The observed and expected heterozygosities ranged from 0.022 to 0.911 and from 0.022 to 0.916, respectively. These informative marker loci will be useful for the assessment of genetic variation and population structure of this species.     

Use of simple sequence repeat markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp) cultivars resistant and susceptible to red rot

Red rod is an economically important disease of sugarcane caused by the fungus Colletotrichum falcatum. We used a simple sequence repeat (SSR)-based marker system to identify and analyze genetic relationships of red rot resistant and susceptible sugarcane cultivars grown in Pakistan. Twenty-one highly polymorphic SSR markers were used for DNA fingerprinting and genetic diversity analysis of 20 sugarcane cultivars. These SSR markers were found to be highly robust; we identified 144 alleles, with 3-11 alleles per marker and a mean of 6.8. Three SSR markers were able to identify all 20 cultivars. DNAMAN(?)-generated homology tree was used to analyze genetic diversity among these cultivars; all cultivars shared 58% or more similarity. We correlated polymorphism information content and resolving power values with marker effectiveness in the process of sugarcane cultivar identification. We concluded that a small number of SSR-derived DNA markers will allow breeders to identify red rot resistant and susceptible cultivars.     

Isolation, characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)

The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.