Simple Sequence Repeat Motifs Research Articles

Development of novel genome‑wide simple sequence repeats (SSR) markers in Bunium persicum

Bunium persicum (Boiss.) Fedtsch or Kala jeera is an economically important medicinal spice that grows in cold temperate regions of Central Asia and Northern India. It is mainly used as spice and condiment in various food preparations. However, its high aroma and richness in essential oils make it industrially important. The bioactivity and pharmacological activities of its phytochemicals have been well elucidated. Due to limited cultivation and huge harvest pressure on natural resources, this species is under severe threat of diversity loss. Further, genetics and molecular breeding studies are lagging for this important crop plant due to a dearth of efficient genome wide molecular markers. In the present study, genomic resources for B. persicum have been generated for the first time through de novo sequencing. In addition, novel genomic SSRs from these resources are identified for their use in future diversity analysis and breeding programs. A total of 177029 perfect and 5915 compound SSR motifs were identified in 212585 assembled sequences. Mononucleotide repeats were the most abundant (55.18%) followed by di- (32.35%), tri- (7.21%), tetra- (3.15%) and hexa- nucleotide (0.76%) repeats. Total 101519 and 2649 primers were successfully designed for sequences containing perfect and compound SSR repeat motifs, respectively. For validation, 88 primer pairs were synthesized for perfect SSR motifs, from which 68 pairs gave scorable amplification. From these, 16 primer pairs were found monomorphic and 52 were polymorphic. These 68 primer pairs altogether yielded 101 alleles with an average of 1.71 alleles per loci. Overall average allele frequency (0.7908), PIC value (0.213), heterozygosity value (0.2995), and gene diversity (0.2680) was recorded for the evaluated Bunium accessions. UPGMA tree constructed based on Nei genetic distances successfully distinguished the evaluated B. persicum accessions. Low genetic diversity recorded in the present study emphasizes the need for restoration and maintenance of effective B. persicum population sizes in situ for its future sustenance.

Genome survey sequencing and characterization of simple sequence repeat (SSR) markers in Platostoma palustre (Blume) A.J.Paton (Chinese mesona)

Platostoma palustre (Blume) A.J.Paton is an annual herbaceous persistent plant of the Labiatae family. However, there is a lack of genomic data for this plant, which severely restricts its genetic improvement. In this study, we performed genome survey sequencing of P. palustre and developed simple sequence repeat (SSR) markers based on the resulting sequence. K-mer analysis revealed that the assembled genome size was approximately 1.21 Gb. A total of 15,498 SSR motifs were identified and characterized in this study; among them, dinucleotide, and hexanucleotide repeats had the highest and lowest, respectively. Among the dinucleotide repeat motifs, AT/TA repeat motifs were the most abundant, and GC/CG repeat motifs were rather rare, accounting for 44.28% and 0.63%, respectively. Genetic similarity coefficient analysis by the UPMGA methods clustered 12 clones, of P. palustre and related species into two subgroups. These results provide helpful information for further research on P. palustre resources and variety improvements.

Development of novel SSR markers derived from genomic and transcriptomic data of Moringa oleifera L. var. PKM1 and their applicability

ABSTRACT Limited genomic resources, especially on breeder-friendly simple sequence repeats (SSR), are available in Moringa, a multi-purpose miracle tree. This study predicted a total of 29913 genomic-SSRs and 1364 genic-SSRs from the omics data obtained from a Moringa cultivar, PKM1. Among the genomic SSR motifs, 1, 414, 2183, 25600, 1106 and 609 SSRs were Mono, Di, Tri, Tetra, Penta, and Hexa repeats, respectively. Higher frequency for AGAG (24.01%) followed by GAGA (16.68) and AAAA (13.18%) was observed in genic-SSRs sequences. Primer pairs for 22,336 genomic- and genic-SSR markers were developed and 117 SSR primer pairs were randomly selected and assayed in four representative Moringa cultivars and ecotypes. Consequently, 94.44% of them have showed high level of DNA polymorphism by generating 1 to 4 alleles per SSR marker (with an average of 1.75) among the investigated Moringa accessions with a maximum polymorphic information content (PIC) of 0.95. Besides using as simple and affordable indicator to differentiate morphologically similar Moringa ecotypes, the SSRs reported in this study will also have several other potential applications such as seed purity analysis, intellectual property protection, identification of introgressions in interspecific crosses, evolutionary studies, germplasm management, gene and/or QTL identification and marker-assisted selection.

Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.).

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage ‘TO1000.’ Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14–0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Capsicum chinense MYB Transcription Factor Genes: Identification, Expression Analysis, and Their Conservation and Diversification With Other Solanaceae Genomes.

Myeloblastosis (MYB) genes are important transcriptional regulators of plant growth, development, and secondary metabolic biosynthesis pathways, such as capsaicinoid biosynthesis in Capsicum. Although MYB genes have been identified in Capsicum annuum, no comprehensive study has been conducted on other Capsicum species. We identified a total of 251 and 240 MYB encoding genes in Capsicum chinense MYBs (CcMYBs) and Capsicum baccatum MYBs (CbMYBs). The observation of twenty tandem and 41 segmental duplication events indicated expansion of the MYB gene family in the C. chinense genome. Five CcMYB genes, i.e., CcMYB101, CcMYB46, CcMYB6, CcPHR8, and CcRVE5, and two CaMYBs, i.e., CaMYB3 and CaHHO1, were found within the previously reported capsaicinoid biosynthesis quantitative trait loci. Based on phylogenetic analysis with tomato MYB proteins, the Capsicum MYBs were classified into 24 subgroups supported by conserved amino acid motifs and gene structures. Also, a total of 241 CcMYBs were homologous with 225 C. annuum, 213 C. baccatum, 125 potato, 79 tomato, and 23 Arabidopsis MYBs. Synteny analysis showed that all 251 CcMYBs were collinear with C. annuum, C. baccatum, tomato, potato, and Arabidopsis MYBs spanning over 717 conserved syntenic segments. Using transcriptome data from three fruit developmental stages, a total of 54 CcMYBs and 81 CaMYBs showed significant differential expression patterns. Furthermore, the expression of 24 CcMYBs from the transcriptome data was validated by quantitative real-time (qRT) PCR analysis. Eight out of the 24 CcMYBs validated by the qRT-PCR were highly expressed in fiery hot C. chinense than in the lowly pungent C. annuum. Furthermore, the co-expression analysis revealed several MYB genes clustered with genes from the capsaicinoid, anthocyanin, phenylpropanoid, carotenoid, and flavonoids biosynthesis pathways, and related to determining fruit shape and size. The homology modeling of 126 R2R3 CcMYBs showed high similarity with that of the Arabidopsis R2R3 MYB domain template, suggesting their potential functional similarity at the proteome level. Furthermore, we have identified simple sequence repeat (SSR) motifs in the CcMYB genes, which could be used in Capsicum breeding programs. The functional roles of the identified CcMYBs could be studied further so that they can be manipulated for Capsicum trait improvement.

Chromosome-specific polymorphic SSR markers in tropical eucalypt species using low coverage whole genome sequences: systematic characterization and validation.

Eucalyptus is one of the major plantation species with wide variety of industrial uses. Polymorphic and informative simple sequence repeats (SSRs) have broad range of applications in genetic analysis. In this study, two individuals of Eucalyptus tereticornis (ET217 and ET86), one individual each from E. camaldulensis (EC17) and E. grandis (EG9) were subjected to whole genome resequencing. Low coverage (10×) genome sequencing was used to find polymorphic SSRs between the individuals. Average number of SSR loci identified was 95,513 and the density of SSRs per Mb was from 157.39 in EG9 to 155.08 in EC17. Among all the SSRs detected, the most abundant repeat motifs were di-nucleotide (59.6%–62.5%), followed by tri- (23.7%–27.2%), tetra- (5.2%–5.6%), penta- (5.0%–5.3%), and hexa-nucleotide (2.7%–2.9%). The predominant SSR motif units were AG/CT and AAG/TTC. Computational genome analysis predicted the SSR length variations between the individuals and identified the gene functions of SSR containing sequences. Selected subset of polymorphic markers was validated in a full-sib family of eucalypts. Additionally, genome-wide characterization of single nucleotide polymorphisms, InDels and transcriptional regulators were carried out. These variations will find their utility in genome-wide association studies as well as understanding of molecular mechanisms involved in key economic traits. The genomic resources generated in this study would provide an impetus to integrate genomics in marker-trait associations and breeding of tropical eucalypts.

Genome-Wide Survey Reveals the Microsatellite Characteristics and Phylogenetic Relationships of Harpadon nehereus.

Harpadon nehereus forms one of the most important commercial fisheries along the Bay of Bengal and the southeast coast of China. In this study, the genome-wide survey dataset first produced using next-generation sequencing (NGS) was used to provide general information on the genome size, heterozygosity and repeat sequence ratio of H. nehereus. About 68.74 GB of high-quality sequence data were obtained in total and the genome size was estimated to be 1315 Mb with the 17-mer frequency distribution. The sequence repeat ratio and heterozygosity were calculated to be 52.49% and 0.67%, respectively. A total of 1,027,651 microsatellite motifs were identified and dinucleotide repeat was the most dominant simple sequence repeat (SSR) motif with a frequency of 54.35%. As a by-product of whole genome sequencing, the mitochondrial genome is a powerful tool to investigate the evolutionary relationships between H. nehereus and its relatives. The maximum likelihood (ML) phylogenetic tree was constructed according to the concatenated matrix of amino acids translated from the 13 protein-coding genes (PCGs). Monophyly of two species of the genus Harpadon was revealed in the present study and they formed a monophyletic clade with Saurida with a high bootstrap value of 100%. The results would help to push back the frontiers of genomics and open the doors of molecular diversity as well as conservation genetics studies on this species.

Genome survey and microsatellite motif identification of Pogonophryne albipinna.

The genus Pogonophryne is a speciose group that includes 28 species inhabiting the coastal or deep waters of the Antarctic Southern Ocean. The genus has been divided into five species groups, among which the P. albipinna group is the most deep-living group and is characterized by a lack of spots on the top of the head. Here, we carried out genome survey sequencing of P. albipinna using the Illumina HiSeq platform to estimate the genomic characteristics and identify genome-wide microsatellite motifs. The genome size was predicted to be ∼883.8 Mb by K-mer analysis (K = 25), and the heterozygosity and repeat ratio were 0.289 and 39.03%, respectively. The genome sequences were assembled into 571624 contigs, covering a total length of ∼819.3 Mb with an N50 of 2867 bp. A total of 2217422 simple sequence repeat (SSR) motifs were identified from the assembly data, and the number of repeats decreased as the length and number of repeats increased. These data will provide a useful foundation for the development of new molecular markers for the P. albipinna group as well as for further whole-genome sequencing of P. albipinna.

Development of high conserved cross-species microsatellite markers from cucumber genome and their applicability in genetic diversity and comparative mapping

One of the most widely applicable and used markers considered in plant research is simple sequence repeats (SSRs). The Gy14 cucumber genome provided information for the development of SSR marker in this species which might be used in other related species additionally. We, therefore, developed genome-wide SSR markers from cucumber, which were used for genetic diversity analysis in 16 different cucurbits species and comparative mapping with melon and watermelon. A total of 2171 SSR motifs were identified from seven cucumber chromosomes. Out of them, 1749 markers belong to melon while 442 markers to watermelon. We experimentally validated 70 SSR markers to check their cross-species transferability among 16 different cucurbits species belongs to six genera. Maximum% polymorphism has been recorded for Momordica charantia (82%) followed by Momordica cochinchinesis (78.18%) and Luffa harmaphrodita (73.68%) while Benincasa hispida (49.21%) exhibited minimum% polymorphism. The large number of cucumber SSR markers developed in the present study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in cucumber. Furthermore, the cross-species transferable SSR markers could also be useful in various molecular markers related studies in other closely related species in the Cucurbitaceae family in which draft genomes are not yet available.

Full-length SMRT transcriptome sequencing and microsatellite characterization in Paulownia catalpifolia

Paulownia catalpifolia is an important, fast-growing timber species known for its high density, color and texture. However, few transcriptomic and genetic studies have been conducted in P. catalpifolia. In this study, single-molecule real-time sequencing technology was applied to obtain the full-length transcriptome of P. catalpifolia leaves treated with varying degrees of drought stress. The sequencing data were then used to search for microsatellites, or simple sequence repeats (SSRs). A total of 28.83 Gb data were generated, 25,969 high-quality (HQ) transcripts with an average length of 1624 bp were acquired after removing the redundant reads, and 25,602 HQ transcripts (98.59%) were annotated using public databases. Among the HQ transcripts, 16,722 intact coding sequences, 149 long non-coding RNAs and 179 alternative splicing events were predicted, respectively. A total of 7367 SSR loci were distributed throughout 6293 HQ transcripts, of which 763 complex SSRs and 6604 complete SSRs. The SSR appearance frequency was 28.37%, and the average distribution distance was 5.59 kb. Among the 6604 complete SSR loci, 1–3 nucleotide repeats were dominant, occupying 97.85% of the total SSR loci, of which mono-, di- and tri-nucleotide repeats were 44.68%, 33.86% and 19.31%, respectively. We detected 112 repeat motifs, of which A/T (42.64%), AG/CT (12.22%), GA/TC (9.63%), GAA/TTC (1.57%) and CCA/TGG (1.54%) were most common in mono-, di- and tri-nucleotide repeats, respectively. The length of the repeat SSR motifs was 10–88 bp, and 4997 (75.67%) were ≤ 20 bp. This study provides a novel full-length transcriptome reference for P. catalpifolia and will facilitate the identification of germplasm resources and breeding of new drought-resistant P. catalpifolia varieties.

Genome-wide identification of microsatellite and development of polymorphic SSR markers for spotted sea bass (Lateolabrax maculatus)

The spotted sea bass (Lateolabrax maculatus) is a valuable commercial mariculture fish in China and has huge potential for future genetic improvement. However, only limited number of SSR (simple sequence repeat) markers have been developed for L. maculatus. In this study, a total of 220,709 SSRs were identified from the L. maculatus genome. The total length, density and frequency of SSRs were 5,119,809 bp, 9898.28 bp/Mb and 425.06 loci/Mb, respectively. Dinucleotide repeats were predominant among SSRs (85.00 %) and AC was the most abundant SSR motifs. Both the number and length of SSRs in each chromosome were positively associated with the chromosome length. A total of 155,905 SSR markers were successfully designed (with an average of 6496 SSRs per chromosome). The frequency of SSR markers varied from 245.63 per Mb to 346.60 per Mb. 14 polymorphic SSR primers were used to evaluate the genetic diversity of L. maculatus wild population from Zhuhai. A total of 74 alleles were detected and most SSR markers showed high polymorphism information content (PIC). The cross-species transferability of 11 out of 67 tested loci were proven successful in largemouth bass Micropterus salmoides. The genome-wide SSRs and newly developed SSR markers made available in this study will facilitate future genetic breeding and genomic studies of spotted sea bass.

Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome.

The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

SSRgenotyper: A simple sequence repeat genotyping application for whole-genome resequencing and reduced representational sequencing projects.

PREMISEMany programs can identify simple sequence repeat (SSR) motifs in genomic data. SSRgenotyper extends SSR identification to en masse genotyping from resequencing data for diversity panels and linkage mapping populations.METHODS AND RESULTSSSRgenotyper will find and genotype SSRs from SAM files and an SSR reference FASTA. Several outputs are possible, including a simple table with the SSR marker name, position, and SSR alleles, defined by the repeat number of the repeat motif. Specific output files include a GENEPOP‐formatted file for downstream genetic diversity analyses and a traditional A, H, B mapping file output that is phased to the parents of the population for biparental linkage map construction. Linkage maps produced using SSRgenotyper genotypes were highly collinear with physical maps and correctly inferred known phylogenies.CONCLUSIONSSSRgenotyper provides an easy‐to‐use, accurate, and scalable SSR genotyping platform for whole‐genome resequencing data. SSRgenotyper is freely available at https://github.com/dlewis27/SSRgenotyper.

Genetic Diversity and Population Structure of Cannabis Based on the Genome-Wide Development of Simple Sequence Repeat Markers.

Cannabis has been used as a source of nutrition, medicine, and fiber. However, lack of genomic simple sequence repeat (SSR) markers had limited the genetic research on Cannabis species. In the present study, 92,409 motifs were identified, and 63,707 complementary SSR primer pairs were developed. The most abundant SSR motifs had six repeat units (36.60%). The most abundant type of motif was dinucleotides (70.90%), followed by trinucleotides, tetranucleotides, and pentanucleotides. We randomly selected 80 pairs of genomic SSR markers, of which 69 (86.25%) were amplified successfully; 59 (73.75%) of these were polymorphic. Genetic diversity and population structure were estimated using the 59 (72 loci) validated polymorphic SSRs and three phenotypic markers. Three hundred ten alleles were identified, and the major allele frequency ranged from 0.26 to 0.85 (average: 0.56), Nei’s genetic diversity ranged from 0.28 to 0.82 (average: 0.56), and the expected heterozygosity ranged from 0.28 to 0.81 (average: 0.56). The polymorphism information content ranged from 0.25 to 0.79 (average: 0.50), the observed number of alleles ranged from 2 to 8 (average: 4.13), and the effective number of alleles ranged from 0.28 to 0.81 (average: 0.5). The Cannabis population did not show mutation-drift equilibrium following analysis via the infinite allele model. A cluster analysis was performed using the unweighted pair group method using arithmetic means based on genetic distances. Population structure analysis was used to divide the germplasms into two subgroups. These results provide guidance for the molecular breeding and further investigation of Cannabis.

Development of EST-SSR markers and their application in the genetic diversity of persimmon (Diospyros kaki Thunb.)

SSRs related to parent selection of D. kaki were validated by transcriptome sequencing, and genetic diversity of these markers among 76 persimmon germplasm resources was additionally investigated. The selection of hybrid parents is critical in cross-breeding plants for germplasm innovation and cultivating improved varieties. This study used a published RNA-Seq database of a persimmon cultivar ‘Zenjimaru’ to develop simple sequence repeat (SSR) markers to assist efficiently selection of appropriate persimmon cultivars for cross-breeding. A total of 154,741 unigenes with 41,999 SSR loci at a frequency of one per 2.30 kb were identified. Of the different SSR motif types, mononucleotide repeats (47.63%) were the most abundant, followed by dinucleotide repeats (38.23%). The main repeat motifs were A/T (42.26%) and AG/CT (24.72%). A total of 17,111 SSR primer pairs were designed, from which 100 selected loci were synthesized and preliminary screened with capillary electrophoresis by testing PCR products from 15 persimmon germplasm resources. The effectiveness of 13 primer pairs that produced products with distinct peaks at the predicted sizes was subsequently determined using 76 persimmon germplasm resources. A total of 111 alleles were identified by using 13 SSR markers, ranging from 2 to 17 with an average of 8.54 bands per locus. Additionally, different hybridized combinations could be selected according to a dendrogram based on these newly developed 13 SSR markers. And two androecious persimmons (“Yunjia-9” and “Luotian Yeshi-1”) were genetically closely related to Chinese pollination-constant non-astringent (C-PCNA) persimmons, which was significant for the cultivation of PCNA offspring. The identified SSR markers will not only promote persimmon cross-breeding programs by increasing the efficiency of parent selection but also assist in genetic resource identification and conservation.

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing

BackgroundLiriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm.ResultsWe performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79.ConclusionsThe genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Genetic variability in Rubus ellipticus collections assessed by morphological traits and EST-SSR markers

Rubus ellipticus Smith. commonly known as ‘Yellow Himalayan raspberry’ is an important member of Rosaceae family with high medicinal importance having antioxidant and antibacterial properties. This study was done with the objective of characterizing 21 R. ellipticus collections from different regions of India, based on morphological descriptors and expressed sequence tags-simple sequence repeat (EST-SSR) markers. Broad phenotypic variability among the R. ellipticus collections was detected by using morphological descriptors. The Euclidian distance matrix discriminated two groups, based on morphological characters. A set of 68 EST-SSR markers were developed based on 7777 unigenes generated by us from ‘Kumarhatti-1’ collection of R. ellipticus using assembled pair—end sequences from Illumina NextSeq 500. The ESTs containing simple sequence repeat motifs were extracted using Microsatellite identification tool and primers were designed. Out of 68 EST-SSRs polymorphism was detected using 61 (89.7%) primer pairs, indicating high genetic variability in the R. ellipticus collections. Effective multiplex ratio, marker index and polymorphism information content were 0.43, 0.24 and 0.50, respectively. The unweighted pair group method with arithmetic mean coincided with the bootstrap analysis and two groups were formed, separating the collections Uttrakhand from the other. Morphological descriptors along with EST-SSR markers proved efficient in detecting the levels of genetic variability among the collections maintained in the field. These results can be used as an additional source of exploitation in R. ellipticus breeding programs.

Genome-wide characterization and development of simple sequence repeat markers for genetic studies in pomegranate (Punica granatum L.)

Genome-wide characterization and development of first set of polymorphic Class I SSRs markers in pomegranate (Punica granatum L.) through in silico analysis using the draft genome sequence of pomegranate cv. Dabenzi (296 Mb) as reported by Qin et al. (2017). The availability of the draft genome sequence of pomegranate cv. Dabenzi presents unprecedented opportunities for the development of largescale genomic resources, such as DNA markers for genotyping applications. In this study, we identify a new set of highly polymorphic simple sequence repeat (SSR) markers by targeting the SSR motif lengths of ≥ 24 bp. A total of 1,73,633 SSRs were identified in the 296-Mb pomegranate genome assembly, reflecting an average density of 527.97 SSRs/Mb. Of these, 43,853 SSRs belong to Class I category (> 20 bp). Concerning the abundance of repeat types in the current dataset, dinucleotide (NN) repeats (31.19%) were the dominant class among all SSRs identified in the genome, followed by tetranucleotide (NNNN: 20.5%) and trinucleotide repeats (NNN: 16.8%). The top two SSR motifs in NN category were AT/AT (64.90%) and AG/CT (28.51%), whereas AAT/ATT (34.66%) and AAG/CTT (28.91%) were the most abundant among NNN repeats. Primer pairs were designed for a total of 2856 Class I SSRs and 110 primers were then assayed initially on eight pomegranate genotypes for polymorphism survey. Polymorphic fragments were obtained for 82 SSRs (77.36%), of which a subset of 13 informative SSRs was further employed to investigate genetic diversity among 46 pomegranate genotypes. Approaches, such as population structure, cluster and PCA elucidated genetic relationships among 46 diverse pomegranate genotypes. In summary, here we developed the first set of genome-wide SSRs in pomegranate that will serve as a powerful genomic tool for future genetic studies. These SSRs have widespread applications in QTL mapping and marker-assisted selection for breeding.

Genome-wide distribution of simple sequence repeats in pomegranate and their application to the analysis of genetic diversity

Pomegranate (Punica granatum L.) germplasm collections are increasingly appreciated as a repository for the genetic improvement of species, and their genetic analysis is an essential prerequisite for their utilization in pomegranate breeding. Sufficient markers are a prerequisite for genetic analysis. In this study, we identified 36,792 simple sequence repeat (SSR) markers in the pomegranate genome. Dinucleotide repeats are the most abundant SSRs, and of the mono-, di-, tri-, and tetranucleotide repeats, A/T, AT/AT, AAT/ATT, and AAAT/ATTT were the most dominant motif type. There were only 1.80% SSR motifs located in the coding regions, while most (98.20%) SSR motifs were located in noncoding regions. We confirmed the reliability of 50 SSR markers by PCR amplification, from which 11 highly polymorphic SSR markers were selected to study the genetic diversity of 218 pomegranate accessions. A total of 63 alleles was detected for the 11 SSR primers, ranging from 2 (PG152) to 9 (PG093, PG080) across the pomegranate accessions. The polymorphism information content values of each SSR primer pair ranged from 0.22 to 0.70, with a mean value of 0.45. Based on population structure analysis, these accessions were clustered into three main populations and 42 accessions were chosen as the core set. The availability of these SSR markers and the clustering result for the pomegranate germplasm provide new important information for pomegranate genetics and breeding programs.

Genome-wide development of simple sequence repeats database for flax (Linum usitatissimum L.) and its use for genetic diversity assessment

Flax (Linum usitatissimum L.) is an important industrial crop and is widely cultivated in North America, Europe, and China for its fiber and oil. Identifying flax molecular markers is a vital step towards improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers have proven to be the most valuable type of genetic marker for molecular breeding and genetic diversity studies. However, the development of genome-wide SSRs in flax has not yet been reported, which limits their use in molecular breeding. In this study, we screened 25,871 SSR motifs from L. usitatissimum, mainly consisting of trinucleotide (52.8%) and dinucleotide (36.1%) repeats. Based on the 24,375 SSR motifs, 71,184 primers pairs of SSR markers were designed for the flax genome, covering the whole genome at a density of 225.3/Mb. Furthermore, 96 pairs of SSR markers from different chromosomes were used to assess a panel of 26 flax accessions obtained from different parts of the world. The 21 SSR markers exhibited polymorphisms across the 26 flax accessions, and the flax varieties were accurately clustered into two groups and two subgroups corresponding to their type (wild species or cultivar) and use (fiber or oilseed). This study demonstrates the potential value in analyzing genetic diversity in flax, and the large-scale development of SSR markers developed in this study will facilitate future molecular analyses in flax.