Construction of a SNP and SSR linkage map in autotetraploid blueberry using genotyping by sequencing

The construction of the first genetic map in autotetraploid blueberry has been made possible by the development of new SNP markers developed using genotyping by sequencing in a mapping population created from a cross between two key highbush blueberry cultivars, Draper × Jewel (Vaccinium corymbosum). The novel SNP markers were supplemented with existing SSR markers to enable the alignment of parental maps.  In total, 1794 single nucleotide polymorphic (SNP) markers and 233 simple sequence repeat (SSR) markers exhibited segregation patterns consistent with a random chromosomal segregation model for meiosis in an autotetraploid. Of these, 700 SNPs and 85 SSRs were utilized for construction of the ‘Draper’ genetic map, and 450 SNPs and 86 SSRs for the ‘Jewel’ map.  The ‘Draper’ map comprises 12  linkage groups (LG), associated with the haploid chromosome number for blueberry, and totals 1621 cM while the ‘Jewel’ map comprises 20 linkage groups totalling 1610 cM. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents.     

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.     

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.     

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 faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.     

Characterization of masson pine (Pinus massoniana Lamb.) microsatellite DNA by 454 genome shotgun sequencing

Microsatellites (simple sequence repeats, SSRs) are important genetic markers in tree breeding and conservation. Here we utilized high-throughput 454 sequencing technology to mine microsatellites from masson pine (MP) genomic DNA. First, we analyzed the characteristics of SSRs in all nonredundant MP reads (genome survey sequences, GSSs) and compared them with loblolly pine (LP) GSSs and BACs (bacterial artificial chromosome clone sequences), and three other nonconiferous species GSSs. Second, a set of MP GSS–SSR primer pairs were designed. There were extremely low overall GSS–SSR densities (28 SSR/Mb) in MP when compared with LP (48 SSR/Mb) and the other species. AT, AAT, AAAT, and AAAAAT were the richest motifs in di-, tri-, tetra-, and hexanucleotides, respectively. Two hundred forty GSS–SSR primer pairs were designed in total, and 20 novel polymorphic markers were identified using three populations (two natural and one clonal seed orchard) as evaluating samples. These markers should be useful for future MP population genetics studies.     

Newly developed SSR markers reveal genetic diversity and geographical clustering in spinach (<Emphasis Type="Italic">Spinacia oleracea</Emphasis>)

Spinach is a popular leafy green vegetable due to its nutritional composition. It contains high concentrations of vitamins A, E, C, and K, and folic acid. Development of genetic markers for spinach is important for diversity and breeding studies. In this work, Next Generation Sequencing (NGS) technology was used to develop genomic simple sequence repeat (SSR) markers. After cleaning and contig assembly, the sequence encompassed 2.5% of the 980 Mb spinach genome. The contigs were mined for SSRs. A total of 3852 SSRs were detected. Of these, 100 primer pairs were tested and 85% were found to yield clear, reproducible amplicons. These 85 markers were then applied to 48 spinach accessions from worldwide origins, resulting in 389 alleles with 89% polymorphism. The average gene diversity (GD) value of the markers (based on a GD calculation that ranges from 0 to 0.5) was 0.25. Our results demonstrated that the newly developed SSR markers are suitable for assessing genetic diversity and population structure of spinach germplasm. The markers also revealed clustering of the accessions based on geographical origin with clear separation of Far Eastern accessions which had the overall highest genetic diversity when compared with accessions from Persia, Turkey, Europe, and the USA. Thus, the SSR markers have good potential to provide valuable information for spinach breeding and germplasm management. Also they will be helpful for genome mapping and core collection establishment.     

Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.     

Genome-Wide Comparative Analyses of Microsatellites in Papaya

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic and universally distributed in eukaryotes. SSRs have been used extensively as sequence tagged markers in genetic studies. Recently, the functional and evolutionary importance of SSRs has received considerable attention. Here we report the mining and characterization of the SSRs in papaya genome. We analyzed SSRs from 277.4 Mb of whole genome shotgun (WGS) sequences, 51.2 Mb bacterial artificial chromosome (BAC) end sequences (BES), and 13.4 Mb expressed sequence tag (EST) sequences. The papaya SSR density was one SSR per 0.7 kb of DNA sequence in the WGS, which was higher than that in BES and EST sequences. SSR abundance was dramatically reduced as the repeat length increased. According to SSR motif length, dinucleotide repeats were the most common motif in class I, whereas hexanucleotides were the most copious in class II SSRs. The tri- and hexanucleotide repeats of both classes were greater in EST sequences compared to genomic sequences. In class I SSR, AT and AAT were the most frequent motifs in BES and WGS sequences. By contrast, AG and AAG were the most abundant in EST sequences. For SSR marker development, 9,860 primer pairs were surveyed for amplification and polymorphism. Successful amplification and polymorphic rates were 66.6% and 17.6%, respectively. The highest polymorphic rates were achieved by AT, AG, and ATG motifs. The genome wide analysis of microsatellites revealed their frequency and distribution in papaya genome, which varies among plant genomes. This complete set of SSRs markers throughout the genome will assist diverse genetic studies in papaya and related species.     

Characterization and utilization of microsatellites in the <Emphasis Type="Italic">Coffea canephora</Emphasis> genome to assess genetic association between wild species in Kenya and cultivated coffee

Coffee is an important beverage crop in the world and has a significant contribution to Kenya’s economy. Here, we analyzed the genome-wide distribution of microsatellites in the Coffea canephora genome. A total of 159,041 SSRs were identified, with an overall density of 308 SSRs per Mb. Tetra-nucleotide repeats are the most abundant, accounting for 32 % of the total SSRs. AT-rich motifs are dominant across all SSR repeat units, while GC-rich motifs were generally rare. A set of 100 SSRs was selected to amplify 96 coffee accessions, including 10 wild accessions collected from Mt. Marsabit (Kenya). Of these SSRs, 33 % generated clear polymorphic bands among all tested accessions, with an average of 3.9 alleles per SSR locus. Wild coffee species from Mt. Marsabit showed a close genetic similarity with cultivated accessions in Kenya, suggesting that the wild species in Mt. Marsabit played an important role in the domestication of cultivated coffee in Kenya. Significantly low pairwise genetic divergence was observed between cultivated and wild accessions in Kenya, suggesting a relatively narrow level of genetic basis among coffee germplasm in Kenya. In addition, cultivated and wild coffee accessions in Kenya show a great divergence from those in other countries. Our results not only provide molecular tools for genetic studies in coffee but are also helpful for conservation and coffee breeding programs in Kenya.     

Development of EST-SSR markers and their utility in revealing cryptic diversity in safflower (Carthamus tinctorius L.)

With the aim of developing additional genomic resources in safflower, a set of 41,011 ESTs of safflower were mined for the presence of SSRs. 18,773 SSR containing ESTs (SSR-ESTs) were identified and were analyzed to remove redundant sequences leading to identification of 8,810 non-redundant SSR-ESTs (categorized into 6104 singletons and 2,706 contigs) having 13,085 non-redundant SSRs. The average number of non-redundant SSRs per EST was 0.32 and they predominantly consisted of dinucleotide (57.7 %), and trinucleotide (37.7 %) repeat motifs. 500 primer pairs were designed for the non-redundant EST-SSRs of which, 151 were tested. 60 markers which gave robust amplicons, were validated in a set of 19 Carthamus lines. A subset of EST-SSR markers, having average polymorphism information content (PIC) ≥0.4 could precisely elucidate the pedigree relatedness among these lines. Further, these markers exhibited high cross-species transferability to five other wild species of Carthamus. The markers reported here would be a valuable addition to existing safflower marker resources aiding in hastening its improvement.     

Development of microsatellite markers in autopolyploid sugarcane and comparative analysis of conserved microsatellites in sorghum and sugarcane

Sugarcane has become an increasingly important first-generation biofuel crop in tropical and subtropical regions. It has a large, complex, polyploid genome that has hindered the progress of genomic research and marker-assisted selection. Genetic mapping and ultimately genome sequence assembly require a large number of DNA markers. Simple sequence repeats (SSRs) are widely used in genetic mapping because of their abundance, high rates of polymorphism, and ease of use. The objectives of this study were to develop SSR markers for construction of a saturated genetic map and to characterize the frequency and distribution of SSRs in a polyploid genome. SSR markers were mined from expressed sequence tag (EST), reduced representation library genomic sequences, and bacterial artificial chromosome (BAC) sequences. A total of 5,675 SSR markers were surveyed in a segregating population. The overall successful amplification and polymorphic rates were 87.9 and 16.4%, respectively. The trinucleotide repeat motifs were most abundant, with tri- and hexanucleotide motifs being the most abundant for the ESTs. BAC and genomic SSRs were mostly AT-rich while the ESTs were relatively GC-rich due to codon bias. These markers were also aligned to the sorghum genome, resulting in 1,203 markers mapped in the sorghum genome. This set of SSRs conserved in sugarcane and sorghum would be the most informative for mapping quantitative trait loci in sugarcane and for comparative genomic analyses. This large collection of SSR markers is a valuable resource for sugarcane genomic research and crop improvement.     

Genome-Wide Comparative Analysis of Microsatellites in Pineapple

Pineapple (Ananas comosus (L.) Merrill) is the second most important tropical fruit in term of international trade. The availability of whole genomic sequences and expressed sequence tags (ESTs) offers an opportunity to identify and characterize microsatellite or simple sequence repeat (SSR) markers in pineapple. A total of 278,245 SSRs and 41,962 SSRs with an overall density of 728.57 SSRs/Mb and 619.37 SSRs/Mb were mined from genomic and ESTs sequences, respectively. 5′-untranslated regions (5′-UTRs) had the greatest amount of SSRs, 3.6–5.2 fold higher SSR density than other regions. For repeat length, 12 bp was the predominant repeat length in both assembled genome and ESTs. Class I SSRs were underrepresented compared with class II SSRs. For motif length, dinucleotide repeats were the most abundant in genomic sequences, whereas trinucleotides were the most common motif in ESTs. Tri- and hexanucleotides of total SSRs were more prevalent in ESTs than in the whole genome. The SSR frequency decreased dramatically as repeat times increased. AT was the most frequent single motif across the entire genome while AG was the most abundant motif in ESTs. Across six examined plant species, the pineapple genome displayed the highest density, substantially more than the second-place cucumber. Annotation and expression analyses were also conducted for genes containing SSRs. This thorough analysis of SSR markers in pineapple provided valuable information on the frequency and distribution of SSRs in the pineapple genome. This genomic resource will expedite genomic research and pineapple improvement.     

Development of SSR markers to study diversity in the genus Cymbidium

Cymbidium spp. are important potted flowers with extremely high ornamental and economic value. The present study reports the development of 14 new simple sequence repeat (SSR) markers through the construction of an enriched Cymbidium goeringii library and cross-amplification in Cymbidium sinensis and Cymbidium hybridium. Of 525, 322 (61.33%) clones had SSR motifs and among motifs di-nucleotides were predominant and followed by tri-nucleotide and tetra-nucleotide type. In polymorphic analysis using 14 newly developed SSRs, a total of 201 alleles across 96 Cymbidium accessions were detected with an average of 14.4 per locus. The average heterozygosity was 0.394. The average gene diversity and polymorphism information content values were 0.394 and 0.639, respectively. The mean genetic similarity coefficient was 0.4297, indicating a wide genetic variation among the Cymbidium accessions. These newly developed SSRs will be useful tools for genotype identification, germplasm conservation, molecular breeding, and assessments of genetic diversity and population structure in Cymbidium.     

Mapping quantitative trait loci for important agronomic traits in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis>) mini core collection with genomic and genic SSR markers

Allele identification for agro-morphological traits and stress resistance is a major concern across the globe for improving productivity of finger millet. Here, we used 46 genomic and 58 genic simple sequence repeats (SSRs) markers in a set of 66 accessions used to constitute a global mini-core collection for analysing their genetic structure as a population and establishing association among markers and twenty morphological traits including resistance to finger blast. Phenotypic data revealed a wide range of variation for all traits except flag leaf width and flag leaf sheath width. We got amplification of 81 alleles by the 31 genomic SSRs at an average of 2.61 alleles per locus. Polymorphism information content (PIC) values varied from 0.21 to 0.75 and average gene diversity was 0.49. Structure analysis of the population using the genomic SSR data divided the accessions into two clusters where Indian and exotic accessions were grouped in separate clusters. Genic SSRs which were associated with blast resistance genes, amplified 36 alleles at an average of 2 alleles per locus. PIC values ranged from 0.32 to 0.37 and average gene diversity was 0.45. Population structure analysis using data from these SSRs grouped the accessions into three clusters, which broadly correspond to their reaction to blast disease. Twenty-two significant associations were found using the GLM approach for 20 agro-morphological traits both in 2012 and 2014, while, 7 and 5 significant marker-trait associations were identified using MLM in 2012 and 2014 respectively. The SSR markers FMBLEST35 and FMBLEST36 designed from the Pi21 gene sequence of rice were found to be associated with blast disease resistance in finger millet indicating that the gene homologues play a significant role in an important role for neck blast resistance.     

Genetic structure of wild emmer wheat populations as reflected by transcribed versus anonymous SSR markers.

Simple sequence repeat (SSR) markers have become a major tool in population genetic analyses. The anonymous genomic SSRs (gSSRs) have been recently supplemented with expressed sequence tag (EST) derived SSRs (eSSRs), which represent the transcribed regions of the genome. In the present study, we used 8 populations of wild emmer wheat (Triticum turgidum subsp. dicoccoides) to compare the usefulness of the two types of SSR markers in assessing allelic diversity and population structure. gSSRs revealed significantly higher diversity than eSSRs in terms of average number of alleles (14.92 vs. 7.4, respectively), polymorphic information content (0.87 vs. 0.68, respectively), and gene diversity (He; 0.55 vs. 0.38, respectively). Despite the overall differences in the level of diversity, Mantel tests for correlations between eSSR and gSSR pairwise genetic distances were found to be significant for each population as well as for all accessions jointly (RM=0.54, p=0.01). Various genetic structure analyses (AMOVA, PCoA, STRUCTURE, unrooted UPGMA tree) revealed a better capacity of eSSRs to distinguish between populations, while gSSRs showed a higher proportion of intrapopulation (among accessions) diversity. We conclude that eSSR and gSSR markers should be employed in conjunction to obtain a high inter- and intra-specific (or inter- and intra-varietal) distinctness.     

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.     

Selection of generally applicable SSR markers for evaluation of genetic diversity and identity in Lilium

There are approximately 100 species and 10,000 cultivars of Lilium and in general their phylogeny is understood. Difficulties remain, however, in understanding the breeding relationships of cultivars and commercial hybrids. One solution to this problem is to identify a selection of validated and transferable SSR markers for use in genotyping. Although over 100 Lilium SSRs have been developed, they have not been validated for use with broad populations. Here, were-evaluated 112 SSRs with 69 lily accessions from different sources, and selected 70 SSRs as easy to score, transferable and polymorphic in all accessions tested. Based on the marker data from 70 SSRs, two main clusters were established for 69 accessions using TREECON, one includes Asiatic hybrids, Longiflorum × Asiatic hybrids and Asiatic local landraces (Lilium brownii, L. brownii var. giganteum, Lilium pumilum, Lilium davidii var. unicolor and Lilium lancifolium), the other is composed primarily of Oriental hybrids and Oriental × Trumpet hybrids, which is in agreement with previous studies and the breeding pedigree. The utility of the 70 SSR markers for establishing parentage and taxon identity of landraces was validated. Our study offers valuable information and validated markers for Lilium systematic classification and the establishment of identity.     

Genetic diversity analysis of maintainer and restorer accessions in upland cotton (Gossypium hirsutum L.)

Genetic diversity amongst 91 upland cotton accessions (50 maintainer, ‘B’ and 41 restorer ‘R’ lines) and three wild species viz., G. aridum, G. thurberi and G. anomalum was analyzed using SSR and RAPD markers. A total of 53 primers (30 SSR and 23 RAPD) were sampled for screening 94 accessions, of which 26 SSR and 17 RAPD primers were polymorphic. Average polymorphism detected by SSR, RAPD and SSR + RAPD markers was 72.5, 62 and 66.66 per cent, respectively. A unique marker CIR-200₂₆₀ that distinguishes G. thurberi from all upland accessions has been identified. Similarity coefficient values within and between B and R lines ranged from 0.65–0.95, 0.61–0.98 and 0.53–0.93 for SSR and 0.72–0.98, 0.73–0.97 and 0.69–0.98 for RAPD markers. UPGMA cluster analysis was consistent with the pedigree and genotypic background of the accessions. RAPD and SSR matrices showed significant positive product moment correlation (r?=?0.93 and 0.92) with the RAPD + SSR combined data matrix, respectively. The result indicates a moderate level of genetic diversity in B and R accessions of upland cotton. Genetically diverse combinations were identified to further evaluate heterotic performance. The maintainer, AKH-108, AKH-118 and AKH-2173; and restorer AKH-31 and AKH 4943 accession were identified as most distinct and divergent, could be used as candidate parental genotypes in hybrid and varietal development programme and also development of mapping population for trait mapping in cotton.     

Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.