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.     

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.     

Genetic Diversity of Buckwheat Cultivars (<Emphasis Type="Italic">Fagopyrum tartaricum</Emphasis> Gaertn.) Assessed with SSR Markers Developed from Genome Survey Sequences

Tartary buckwheat is an important edible crop as well as medicinal plant in China. More and more research is being focused on this minor grain crop because of its medicinal functions, but there is a paucity of molecular markers for tartary buckwheat due to the lack of genomics. In this study, a genome survey was carried out in tartary buckwheat, from which SSR markers were developed for analysis of genetic diversity. The survey generated 21.9 Gb raw sequence reads which were assembled into 348.34 Mb genome sequences included 204,340 contigs. The genome size was estimated to be about 497 Mb based on K-mer analysis. In total, 24,505 SSR motifs were identified and characterised from this genomic survey sequence. Most of the SSR motifs were di-nucleotide (67.14 %) and tri-nucleotide (26.05 %) repeats. AT/AT repeat motifs were the most abundant, accounting for 78.60 % of di-nucleotide repeat motifs. SSR fingerprinting of 64 accessions yielded 49.71 effective allele loci from a total of 63 with the 23 polymorphic SSR primer combinations. Analyses of the population genetic structure using SSR data strongly suggested that the 64 accessions of tartary buckwheat clustered into two separate subgroups. One group was mainly distributed in Nepal, Bhutan and the Yunnan-Guizhou Plateau regions of China; the other group was mainly derived from the Loess Plateau regions, Hunan and Hubei of China and USA. The cluster analysis of these accession’s genetic similarity coefficient by UPMGA methods strongly supported the two subgroup interpretation. However accessions from Qinghai of China could be grouped into either of the two subgroups depending on which classification method was used. This region is at the intersection of the two geographical regions associated with the two subgroups. These results and information could be used to identify and utilize germplasm resources for improving tartary buckwheat breeding.     

Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek)

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard’s similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.     

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

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

Assessment of molecular diversity and evolutionary relationship of kenaf (Hibiscus cannabinus L.), roselle (H. sabdariffa L.) and their wild relatives

Kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) are valuable fibre crop species with diverse end use. Phylogenetic relationship of 73 accessions of kenaf, roselle and their wild relatives from 15 countries was assessed using 44 inter-simple sequence repeat (ISSR) and jute (Corchorus olitorius L.) specific simple sequence repeats (SSR) markers. A total of 113 alleles were identified of which 61.95 % were polymorphic. Jute specific SSR markers exhibited high polymorphism and resolving power in kenaf, although ISSR markers exhibited higher resolving power than SSR markers. Number of polymorphic alleles varied from 1 to 5 for ISSR and 1 to 6 for SSR markers. Cultivated species exhibited higher allele polymorphism (57 %) than the wild species (35 %), but the improved cultivars exhibited lower genetic diversity compared to germplasm accessions. Accessions with common genetic lineage and geographical distribution clustered together. Indian kenaf varieties were distinct from cultivars bred in other countries and shared more genetic homology with African accessions. High genetic diversity was observed in the Indian (J = 0.35–0.74) and exotic kenaf germplasm collections (J = 0.38–0.79), suggesting kenaf might have been introduced in India from Africa through Central Asia during early domestication. Genetic similarity-based cluster analysis was in close accordance with taxonomic classification of Hibiscus.     

Discovery and experimental analysis of microsatellites in an oil woody plant Camellia chekiangoleosa

Oil camellia trees are important woody plants for the production of high-quality cooking oil. On the contrary to their economic importance, their genetic and genomic resources are very limited, which greatly hamper the genetic studies on oil camellia trees. Microsatellites or simple sequence repeats (SSRs) have great value in many aspects of genetic analyses due to their high polymorphism and codominant inheritance. In this study, we report the large-scale development and characterization of SSR markers derived from genomic sequences of Camellia chekiangoleosa by high-throughput pyrosequencing technology. A total of 1,091,393 genomic shotgun reads were generated using Roche 454 FLX sequencer, the average read length was 319 bp, and the total sequence throughput was 347.9 Mb. These sequences were assembled into 35,315 contigs with total length of 14.8 Mb and the N50 contig size of 770 bp. By analyzing with microsatellite (MISA), a total of 5,844 perfect microsatellites were detected from the assembled sequences. Among them, tetranucleotide repeats were found to be the most frequent microsatellites in the genome of C. chekiangoleosa, and all the dominant repeat motifs for different types of SSRs were detected to be rich in A/T. Experimental analysis with 900 SSR primer pairs revealed that 66 % of them succeeded in PCR amplification. Further investigation with 345 SSR primer pairs showed that a relatively high percentage of primers amplified polymorphic loci (31.9 %). Experimental data also revealed that, overall, long microsatellite repeats (>20 bp) were more variable than the short ones (<20 bp) in the genome of oil camellia tree.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae)

? Premise of the study: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. ? Methods and Results: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. ? Conclusions: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

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.     

Genome-wide single nucleotide polymorphism discovery and validation in adzuki bean

Adzuki bean, also known as red bean (Vigna angularis), with 2n = 22 chromosomes, is an important legume crop in East Asian countries, including China, Japan, and Korea. For single nucleotide polymorphism (SNP) discovery, we used Vigna accessions, V. angularis IT213134 and its wild relative V. nakashimae IT178530, because of the lack of DNA sequence polymorphism in the cultivated species. Short read sequences of IT213134 and IT178530 of approximately 37 billion and 35 billion bp were produced using the Illumina HiSeq 2000 system to a sequencing depth of 61.5× and 57.7×, respectively. After de novo assembly was carried out with trimmed HiSeq reads from IT213134, 98,441 contigs of various sizes were produced with N50 of 13,755 bp. Using Burrows–Wheeler Aligner software, trimmed short reads of V. nakashimae IT178530 were successfully mapped to IT213134 contigs. All sequence variations at the whole-genome level were examined between the two Vigna species. Of the 1,565,699 SNPs, 59.4 % were transitions and 40.6 % were transversions. A total of 213,758 SNPs, consisting of 122,327 non-synonymous and 91,431 synonymous SNPs, were identified in coding sequences. For SNP validation, 96 SNPs in the genic region were chosen from among IT213134 contigs longer than 10 kb. Of these 96 SNPs, 88 were confirmed by Sanger sequencing of 10 adzuki bean genotypes from various geographic origins as well as IT213134 and its wild relative IT178530. These genome-wide SNP markers will enrich the existing Vigna resources and, specifically, could be of value for constructing a genetic map and evaluating the genetic diversity of adzuki bean.     

Comparison of SSR and cytochrome P-450 markers for estimating genetic diversity in Picrorhiza kurrooa L.

Picrorhiza kurrooa L., a high altitude medicinal plant, is known for its drug content called Kutkin. In the present study, DNA-based molecular marker techniques, viz. simple sequence repeats (SSR) and cytochrome P-450 markers were used to estimate genetic diversity in Picrorhiza kurrooa. Twenty five accessions of Picrorhiza kurrooa, collected from ten different eco-geographical locations were subjected to 22 SSR and eight cytochrome P-450 primer pairs, out of which 13 SSR markers detected mean 5.037 alleles with a mean polymorphic information content (PIC) of 0.7718, whereas eight cytochrome P-450 markers detected mean 5.0 alleles with a mean PIC of 0.7596. Genetic relationship among the accessions was estimated by constructing the dendrograms using SSR and cytochrome P-450 data. There was a clear consistency between SSR and cytochrome P-450 trees in terms of positioning of most Picrorhiza accessions. SSR markers could cluster various Picrorhiza kurrooa accessions based on their geographical locations whereas cytochrome P-450 markers could cluster few accessions as per their geographical locations. The Mantel test between SSR and cytochrome P-450 markers revealed a good fit correlation (r = 0.6405). The dendrogram constructed using the combined data of SSR and cytochrome P-450s depicted two clusters of accessions based on its eco-geographical locations whereas two clusters contained the accessions from mixed eco-geographical locations. Overall, the results of the present study point towards quiet high degree of genetic variation among the accessions of each eco-geographic region.     

用于绿豆种质资源遗传多样性分析的SSR及STS引物的筛选

目前能够用于绿豆(Vigna radiate)种质资源遗传多样性分析的PCR引物极其有限。通过12份农艺性状差异较大的绿豆种质对绿豆以及小豆(Vigna angularis)、豇豆(Vigna unguiculata)、菜豆(Phaseolus vulgaris)等近缘食用豆中的PCR引物进行筛选,结果表明41对绿豆SSR引物中能够有效扩增的有35对,6对有多态性;28对绿豆STS引物中有23对能够有效扩增,2对有多态性;8对小豆SSR引物能够有效扩增的有6对,但均无多态性;27对豇豆SSR引物能够有效扩增的有17对,1对有多态性;24对菜豆SSR引物能够有效扩增的有9对,1对有多态性。这些多态性引物的获得将有助于中国绿豆种质资源的遗传多样性分析。     

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.     

Patterns of genetic diversity in the Andean gene pool of common bean reveal a candidate domestication gene

Most studies on the genetic diversity of common bean (Phaseolus vulgaris L.) have focussed on accessions from the Mesoamerican gene pool compared to the Andean gene pool. A deeper knowledge of the genetic structure of Argentinian germplasm would enable researchers to determine how the Andean domestication event affected patterns of genetic diversity in domesticated beans and to identify candidates for genes targeted by selection during the evolution of the cultivated common bean. A collection of 116 wild and domesticated accessions representing the diversity of the Andean bean in Argentina was genotyped by means of 114 simple sequence repeat (SSR) markers. Forty-seven Mesoamerican bean accessions and 16 Andean bean accessions representing the diversity of Andean landraces and wild accessions were also included. Using the Bayesian algorithm implemented in the software STRUCTURE we identified five major groups that correspond to Mesoamerican and Argentinian wild accessions and landraces and a group that corresponds to accessions from different Andean and Mesoamerican countries. The neighbour-joining algorithm and principal coordinate clustering analysis confirmed the genetic relationships among accessions observed with the STRUCTURE analysis. Argentinian accessions showed a substantial genetic variation with a considerable number of unique haplotypes and private alleles, suggesting that they may have played an important role in the evolution of the species. The results of statistical analyses aimed at identifying genomic regions with consistent patterns of variation were significant for 35 loci (~20 % of the SSRs used in the Argentinian accessions). One of these loci mapped in or near the genomic region of the glutamate decarboxylase gene. Our data characterize the population structure of the Argentinian germplasm. This information on its diversity will be very valuable for use in introgressing Argentinian genes into commercial varieties because the majority of present-day common bean varieties are of Andean origin.     

Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris)

Genetic diversity was studied among 21 accessions of lentil using SSR markers and morphological traits in order to assess the diversification of Indian gene-pool of lentil through introgression of exotic genes and introduction of germplasm. Among these , 16 genotypes either had ‘Precoz’ gene, an Argentine line in their pedigree or genes from introduced lines from ICARDA. Sixty five SSR markers and eight phenotypic traits were used to analyse the level of genetic diversity in these genotypes. Forty three SSR markers (66 %) were polymorphic and generated a total of 177 alleles with an average of 4.1 alleles per SSR marker. Alleles per marker ranged from 2 to 6. The polymorphic information content ranged 0.33 to 0.80 with an average of 0.57, suggesting that SSR markers are highly polymorphic among the studied genotypes. Genetic dissimilarity based a dendrogram grouped these accessions into two main clusters (cluster I and cluster II) and it ranged 33 % to 71 %, suggesting high level of genetic diversity among the genotypes. First three components of PCA based morphological traits explained higher variance (95.6 %) compared to PCA components based on SSR markers (42.7 %) of total genetic variance. Thus, more diversity was observed for morphological traits and genotypes in each cluster and sub-cluster showed a range of variability for seed size, earliness, pods/plant and plant height. Molecular and phenotypic diversity analysis thus suggested that use of germplasm of exotic lines have diversified the genetic base of lentil germplasm in India. This diversified gene-pool will be very useful in the development of improved varieties of lentil in order to address the effect of climate change, to adapt in new cropping systems niches such as mixed cropping, relay cropping, etc. and to meet consumers’ preference.     

Analysis of Diversity in Chinese Cultivated Barley with Simple Sequence Repeats: Differences Between Eco-Geographic Populations

The genetic diversity of 116 barley accessions, representing five Chinese eco-geographic populations, was studied using simple sequence repeat (SSR) markers. The 21 SSR loci revealed 128 alleles with an average of 6.1 alleles per locus. The highest values of proportion of polymorphic loci (P) and gene diversity index (He) were obtained in the Northern (P = 1.00; He = 0.60) and the Yangtze River reaches and Southern populations (P = 1.00; He = 0.59). The lowest values were in the populations of the Yellow River reaches (P = 0.86; He = 0.44). The highest average number of alleles per locus (4.52) and number of unique alleles (7) were found in the Qinghai–Tibet plateau population. Cluster analysis revealed that together with the row type, strong eco-geographic variables influenced the classification. Associations of SSR and eco-geographic values were established for 11 SSR loci. Four to six markers were found to discriminate among geographic groups, which may serve as tools for diagnosis of the eco-geographic populations and provide evidence for the adaptive nature of SSR markers.