SSR Motifs Research Articles

Cross genera transferability of microsatellite markers from other members of family Bignoniaceae to Tecomella undulata (Sm.) Seem

Transferability of SSR markers from related species/genera is a rapid and cost-effective method to enhance genomic database of genetically neglected crops. Monotypic genus Tecomella undulata, a pharmacologically important endangered timber tree, found in hyper arid regions is one such genetic resource limited species, wherein neither SSR identification nor cross transferability studies have been initiated. Transferability of 69 cross-genera SSR primers selected from other members of family bignoniaceae (Incarvillea sinensis, I. mairei, Jacaranda copaia, Tabebuia aurea and Arrabidaea chica) showed 40.58% (28 primers) transferability amplifying 1–10 amplicons ranging in the size from 80to 600 bp in 24 accessions of T. undulata. Per cent marker transferability varied from 66.67% (A. chica) to 38.89% (J. copaia) among the genera tested while no transferable markers could be identified from I. sinensis. Transferability of simple di-/tri-nucleotide repeat (34.2–37.5%) and complex nucleotide sequence (60.0%) based SSR motifs were higher as compared to penta-/hexa-nucleotide based repeat motifs. Within the 28 transferable markers, 26 (92.86%) were polymorphic while 2 (7.14%) were monomorphic. Average number of alleles per primer was 5.39 while average polymorphic alleles per locus were 5.11. The PIC values of SSR markers ranged from 0.23 to 0.85 with an average of 0.59. Efficiency of the identified cross species SSR markers was tested for diversity analysis using a set of 24 trees (8 each of yellow, orange and red flower colour morphotypes). The 24 samples clustered into six groups with similarity coefficient ranging from 0.29 to 0.67, while six accessions formed distinct out groups. Distribution of accessions in the SSR dendrogram showed no correlation with flower colour. More diversity (99%) was observed within populations compared to that existing between populations (1%). The present study can enrich the genomic background of T. undulata by identifying suitable polymorphic markers from confamiliar species that can be applied for genetic diversity studies, mapping of QTLs and cultivar identification as well.

Genome Survey Sequence and the Development of Simple Sequence Repeat (SSR) Markers in Erianthus arundinaceus

Erianthus arundinaceus possesses high biomass, vigor, ratoonability and resistance to biotic and abiotic stresses and is considered to be an ideal species for improving sugarcane. In this study, a genome survey sequence was performed in E. arundinaceus, and SSR markers were developed based on a genome survey sequence. A total of 167.36 Gb raw data were obtained and were assembled into 3161 Mb genome sequences, including 15,238,738 scaffolds. The genome size was estimated to be approximately 3.23 Gp based on kmer analysis. In total, 36,616 SSR motifs were identified and characterized, including 60.69% dinucleotide, 34.27% trinucleotide, 3.34% tetranucleotide, 0.98% pentanucleotide and 0.71% hexanucleotide repeats. AG/CT repeat motifs were the most abundant, and GC/CG repeat motifs were somewhat rare, accounting for 54.16% and 2.69% of dinucleotide repeat motifs, respectively. A total of 365 SSR loci were obtained by 20 SSR primer pairs amplified in 20 clones of E. arundinaceus and its related species, and 188 SSR loci were specific to E. arundinaceus. Genetic similarity coefficient analysis by UPMGA methods clustered 20 clones of E. arundinaceus and its related species into two groups. Five clones of E. arundinaceus clustered separately with the remaining clones. These results may establish a foundation for further research investigating the E. arundinaceus genome and identifying genuine hybrids between sugarcane and E. arundinaceus.

Grapevine Phenological Quantitative Trait SSR Genotyping Using High-Throughput HRM-PCR Analysis

Discrimination among grapevine varieties based on quantitative traits, such as flowering, veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs. These traits are under complex genetic control for which 6 linked SSR loci (VVS2, VVIn16, VMC7G3, VrZAG29, VMC5G7, and VVIB23) have been identified. Using these markers in HRM-PCR analysis, we assessed genetic diversity among a large collection of 192 grapevine varieties. The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties, 3 prominent foreign varieties and 11 varieties of Palestinian origin. The SSR markers used were highly polymorphic, displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis. This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism. Hence, HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis. The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties, signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.

Development and characterization of novel microsatellite markers in Puccinia striiformis f.sp. tritici and their transferability in Puccinia species

AbstractPuccinia striiformis f.sp. tritici (Pst) and P. striiformis f.sp. hordei (Psh) causing stripe rust disease in wheat and barley, respectively, are two devastating phytopathogens. Microsatellite/simple sequence repeat (SSR) markers are increasingly being utilized for analysis of genetic diversity, diagnosis, population structure and possible migratory routes of plant pathogens. In the current study, novel polymorphic SSR markers were designed for Pst using the genomic sequences of PST‐78 isolate. A total of 1,191 SSR motifs, comprising 30% each of di‐ and tri‐nucleotide type of repeats, 17% of penta‐nucleotide, 15% of tetra‐nucleotide and 8% of hexa‐nucleotide repeats, were detected through in silico scanning of PST‐78 genomic sequences. Polymorphism was detected by nine of the 50 designed SSRs (PsSSRs) in seven stripe rust pathotypes of wheat and barley. The mean number of alleles per SSR locus, mean polymorphism information content (PIC), mean heterozygosity, mean major allele frequency (MAF) and mean gene diversity were 2.33, 0.34, 0.33, 0.71 and 0.40, respectively. The dendrogram analysis suggested that newly developed PsSSR markers could distinguish stripe rust pathotypes based on their virulence phenotype. Further, the cross‐genera and cross‐species amplification test of these markers in 14 different rust pathotypes revealed that 9 PsSSRs are capable of amplification in Pst species infecting wild grass, followed by 6 PsSSRs in Pt, 3 PsSSRs in Pgt, 1 PsSSRs in Puccinia species on barberry and Melampsora lini. Thus, the transferability of PsSSRs to other species reduced with increasing genetic distance of target species. These newly designed SSR markers expand the available Pst SSR marker resources and allow better genetic studies.

Development of SSR markers via de novo transcriptome assembly in Akebia trifoliata (Thunb.) Koidz.

Owing to its high nutritive, economic, and medicinal values, Akebia trifoliata has received increased attention, making worthy of being used as a new fruit crop for further domestication and commercialization in China. However, molecular research of A. trifoliata has lagged as investigations of its genomic resources and molecular markers are rare. In this study, a cDNA library of A. trifoliata leaves was sequenced using the Illumina NovaSeq. 6000 sequencing system. In total, 101 417 transcripts, 63 757 unigenes, and 9494 simple sequence repeats were assembled and identified from the transcriptome datasets. The majority of the SSRs were di- and trinucleotide repeats. Length and number of SSR motifs ranged from 15 to 66, and 5 to 48 bp, respectively. Of which, the A/T mononucleotide motif and AG/TC and CT/GA dinucleotide motifs were the most abundant. Furthermore, 100 SSR primers were randomly selected to validate amplification and polymorphism, and 88 A. trifoliata accessions were definitively distinguished by 49 primers. With the Qinling mountains and Huaihe River line as the boundaries, the northern and southern accessions were clustered into different groups, but no clear geographical patterns (city or origin) were observed in the southern accessions. These newly identified molecular markers may provide a foundation for the genetic identification and diversity analysis and marker-assisted selection breeding in species of Akebia.

Whole genome re-sequencing and development of SSR markers in oriental melon

The objective of this study was to use ‘Danta PR’, NGS (Next Generation Sequencing) technology for genome resequencing to develop polymorphic makers between Chinese oriental melon, ‘Hyangseo 1’ and Korean oriental melon. From the resequencing data that covered about 81 times of the genome size, 104,357 of SSR motifs and Indel, and 1,092,436 of SNPs were identified. 299 SSR and 307 Indel markers were chosen to cover each chromosome with 25 markers. These markers were subsequently used to identify genotypes of ‘Danta PR’ BC1 (F1 x ‘Danta PR’) population and a genetic linkage map was constructed. SSR, Indel, and SNPs identified in this study would be useful as a breeding tool to develop new oriental melon varieties.

EST-SSR marker development based on RNA-sequencing of E. sibiricus and its application for phylogenetic relationships analysis of seventeen Elymus species

BackgroundElymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development.ResultsWe obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins.ConclusionsThis study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.

Transcriptome sequencing of Himalayan Raspberry (Rubus ellipticus) and development of simple sequence repeat markers.

Rubus ellipticus is a wild crop having less number of EST-SSR markers. First attempt was made towards the transcriptomics data analysis and generation of EST-SSR markers which were used in genetic diversity and transferability studies. Rubus ellipticus is a raspberry with yellow fruits, native to tropical and subtropical India and Asia. Leaves of Rubus ellipticus 'Kumarhatti' collection were utilized for cDNA library construction. More than 15 million sequencing reads were generated using NextSeq500 Illumina RNA-seq technology. The DNASTAR software was used for de novo assembly from which 7777 unigenes with an average length of 500bp was obtained. These unigenes were annotated using public databases, including NCBI non-redundant and gene ontology. De novo assembly of R. ellipticus unigenes found the highest similarity to apple than to other members of Rosaceae. This is the first attempt to use the Illumina platform of RNA sequencing and de novo assembly for R. ellipticus without a reference genome. In this study, unigenes were used for SSR marker development. ESTs containing SSR motifs were extracted using an online Microsatellite Identification Tool (MISA). SSR primers were designed from the SSR containing 704 EST sequences using the Websat software. Total 304 EST-SSRs primers were successfully designed, out of which 68 randomly selected primer pairs were custom synthesized and used for validation. Real-time PCR was also performed to analyze the relationship of transcriptional factors with fruit ripening. Out of 68 primer pairs, 61 were found to be informative in R. ellipticus, whereas 65 primer pairs were informative in the five tested genera of Rosaceae, i.e., pear, peach, apple, rose, and strawberry with 95.3% and 93.5% polymorphism, leading to the conclusion that these marker systems are very efficient to carryout diversity and cross transferability study in Rosaceae genera.

Microsatellite Borders and Micro-sequence Conservation in Juglans

Walnuts (Juglans spp.) are economically important nut and timber species with a worldwide distribution. Using the published Persian walnut genome as a reference for the assembly of short reads from six Juglans species and several interspecific hybrids, we identified simple sequence repeats in 12 Juglans nuclear and organellar genomes. The genome-wide distribution and polymorphisms of nuclear and organellar microsatellites (SSRs) for most Juglans genomes have not been previously studied. We compared the frequency of nuclear SSR motifs and their lengths across Juglans, and identified section-specific chloroplast SSR motifs. Primer pairs were designed for more than 60,000 SSR-containing sequences based on alignment against assembled scaffold sequences. Of the >60,000 loci, 39,000 were validated by e-PCR using unique primer pairs. We identified primers containing 100% sequence identity in multiple species. Across species, sequence identity in the SSR-flanking regions was generally low. Although SSRs are common and highly dispersed in the genome, their flanking sequences are conserved at about 90 to 95% identity within Juglans and within species. In a few rare cases, flanking sequences are identical across species of Juglans. This comprehensive report of nuclear and organellar SSRs in Juglans and the generation of validated SSR primers will be a useful resource for future genetic analyses, walnut breeding programs, high-level taxonomic evaluations, and genomic studies in Juglandaceae.

Characterization of Chloroplast Genomes, Nuclear Ribosomal DNAs, and Polymorphic SSR Markers Using Whole Genome Sequences of Two Euonymus hamiltonianus Phenotypes

Although genomics provides useful tools for crops, most wild resource plants still lack molecular data. To retrieve useful genomic data and thus provide fundamental information for a resource plant, we established a multi-directional approach using two low coverage whole-genome shotgun sequence (WGS) data of Euonymus hamiltonianus, which is a wild resource plant with potential as a medicinal and ornamental plant. We assembled complete chloroplast genome and nuclear ribosomal DNA (nrDNA) sequences and analyzed polymorphic simple sequence repeats (pSSRs) in the nuclear genome based on the comparison of WGS data between two different phenotypes. We developed a bioinformatics pipeline to identify pSSR motifs by systematic comparison of two WGS datasets. The pipeline is composed of multiple steps including end-joining of paired reads, isolation of joined reads harboring SSR motifs derived from unique non-repetitive regions, identification of pSSR via in silico comparison with counterpart WGS reads, design of pSSR primer sets, and validation. The pipeline was applied to WGS data of E. hamiltonianus and identified 161 contigs with pSSR motifs between the two different phenotypes. Based on the pSSR motifs, 20 primer pairs were designed, of which seven were successfully validated as real pSSR markers. We expect this information to be applicable to genomic resources of E. hamiltonianus.

Identification, analysis and development of salt responsive candidate gene based SSR markers in wheat

BackgroundSalinity severely limits wheat production in many parts of the world. Development of salt tolerant varieties represents the most practical option for enhancing wheat production from these areas. Application of marker assisted selection may assist in fast tracking development of salt tolerant wheat varieties. However, SSR markers available in the public domain are not specifically targeted to functional regions of wheat genome, therefore large numbers of these need to be analysed for identification of markers associated with traits of interest. With the availability of a fully annotated wheat genome assembly, it is possible to develop SSR markers specifically targeted to genic regions. We performed extensive analysis to identify candidate gene based SSRs and assessed their utility in characterizing molecular diversity in a panel of wheat genotypes.ResultsOur analysis revealed, 161 SSR motifs in 94 salt tolerance candidate genes of wheat. These SSR motifs were nearly equally distributed on the three wheat sub-genomes; 29.8% in A, 35.7% in B and 34.4% in D sub-genome. The maximum number of SSR motifs was present in exons (31.1%) followed by promoters (29.8%), 5’UTRs (21.1%), introns (14.3%) and 3’UTRs (3.7%). Out of the 65 candidate gene based SSR markers selected for validation, 30 were found polymorphic based on initial screening and employed for characterizing genetic diversity in a panel of wheat genotypes including salt tolerant and susceptible lines. These markers generated an average of 2.83 alleles/locus. Phylogenetic analysis revealed four clusters. Salt susceptible genotypes were mainly represented in clusters I and III, whereas high and moderate salt tolerant genotypes were distributed in the remaining two clusters. Population structure analysis revealed two sub-populations, sub-population 1 contained the majority of salt tolerant whereas sub-population 2 contained majority of susceptible genotypes. Moreover, we observed reasonably higher transferability of SSR markers to related wheat species.ConclusionWe have developed salt responsive gene based SSRs in wheat for the first time. These were highly useful in unravelling functional diversity among wheat genotypes with varying responses to salt stress. The identified gene based SSR markers will be valuable genomic resources for genetic/association mapping of salinity tolerance in wheat.

Comparative genome-wide characterization leading to simple sequence repeat marker development for Nicotiana

BackgroundSimple sequence repeats (SSRs) are tandem repeats of DNA that have been used to develop robust genetic markers. These molecular markers are powerful tools for basic and applied studies such as molecular breeding. In the model plants in Nicotiana genus e.g. N. benthamiana, a comprehensive assessment of SSR content has become possible now because several Nicotiana genomes have been sequenced. We conducted a genome-wide SSR characterization and marker development across seven Nicotiana genomes.ResultsHere, we initially characterized 2,483,032 SSRs (repeat units of 1–10 bp) from seven genomic sequences of Nicotiana and developed SSR markers using the GMATA® software package. Of investigated repeat units, mono-, di- and tri-nucleotide SSRs account for 98% of all SSRs in Nicotiana. More complex SSR motifs, although rare, are highly variable between Nicotiana genomes. A total of 1,224,048 non-redundant Nicotiana (NIX) markers were developed, of which 99.98% are novel. An efficient and uniform genotyping protocol for NIX markers was developed and validated. We created a web-based database of NIX marker information including amplicon sizes of alleles in each genome for downloading and online analysis.ConclusionsThe present work constitutes the first deep characterization of SSRs in seven genomes of Nicotiana, and the development of NIX markers for these SSRs. Our online marker database and an efficient genotyping protocol facilitate the application of these markers. The NIX markers greatly expand Nicotiana marker resources, thus providing a useful tool for future research and breeding. We demonstrate a novel protocol for SSR marker development and utilization at the whole genome scale that can be applied to any lineage of organisms.The Tobacco Markers & Primers Database (TMPD) is available at http://biodb.sdau.edu.cn/tmpd/index.html

Comparative assessment of genetic diversity in Albanian olive (Olea europaea L.) using SSRs from anonymous and transcribed genomic regions

A total of 560,578 sequencing reads obtained from the de novo NGS transcriptome sequencing of Olea europaea were searched for the presence of tri- and tetra-nucleotide repeat motifs, which resulted in 375 tri-nucleotide microsatellites and 252 tetra-nucleotides. Twelve expressed sequence tags (EST)-derived SSRs that gave a reproducible pattern, enabling easy scoring and allele size determination, were further tested for polymorphism on 165 cultivars O. europaea subsp. sativa and on 18 oleasters (O. europaea subsp. sylvestris). The main diversity parameters of EST-SSRs were compared to the genomic SSRs that have been most frequently used so far in olive genotyping and mapping studies. Diversity parameters were also compared for different lengths of repeat motifs (di-nucleotide, tri-nucleotide, and tetra-nucleotide microsatellites). The occurrence of null alleles was only observed in anonymous SSRs, while their frequencies were negative for all EST-SSR loci. Eight out of 12 EST-SSRs were classified as highly informative, indicating their applicability in assessing molecular diversity and genetic relatedness in olive. The 12 novel EST-SSR markers selected as a subset of all identified loci containing SSR motifs showed their suitability for evaluation of genetic diversity and population structure and for genetic mapping. They were also linked to genes and, as shown by annotation analysis, four of them have potential for interpretation of allelic variability related to disease resistance and can be evaluated as a potential diagnostic marker for disease resistance. The development of new EST-SSRs provides a valuable tool in molecular breeding programs for both cultivated and wild olives.

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane (Saccharum sp. hybrid).

Sugarcane (Saccharum species hybrid) is the major source of sugar (> 80% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvementof sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteinsrelated to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Genome-wide EST-SSR Marker Identification in Red Wiggler Worm Eisenia fetida (Savigny, 1826)

Earthworms belong to the main groups of soil-dwelling invertebrates and are an important element for soil biota. The ecological importance of earthworms on terrestrial ecosystem is mainly attributed to their close synergistic interaction with soil biota. Among earthworms, Eisenia fetida has been considered as preferred for vermiculture and vermicomposting practices due to their reproductive potentials, short life cycle and wide temperature and density pressure tolerances. Although the genome and transcriptome data for E. fetida are available, the EST-SSRs composition and frequency of this species, which used for phylogenetic studies, genus-level taxonomy and population genetics, remain poorly defined. In current study, we mined publicly available transcriptome data and characterised genome-wide EST-SSR markers for this epigeic species. A total of 13,060 EST-SSRs were identified from 162,609 contigs. The most abundant EST-SSR types were found to be trimeric repeats (5,998, 46%) followed by dimeric (4,762, 36%), tetrameric (1,716, 13%), pentameric (478, 4%) and hexameric repeats (106, 1%). For SSR motifs, the most prevalent motifs were AC/GT (14.72%), followed by AT/AT (13.20%), ATC/ATG (13.03%), AAT/ATT (12.30%) and AG/CT (8.46%). Following validation of the species using COI sequences and to test the designed primer pairs, the eight primer pairs were designed, but only six of them were successfully amplified. Although EST-SSR information is scarce for annelids, the EST-SSR patterns of E. fetida seem to be similar to annelids. Overall, the EST-SSR markers help in taxonomic resolution of Eisenia genus from other earthworm genera and in studying the population structure and geographic distributions of E. fetida.

Pursuit for est microsatellites in a tetraploid model from de novo transcriptome sequencing

Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of the established bioinformatics approach by amplifying them in eight different taxa within the genus Centaurium having various ploidy levels (diploids, tetraploids and hexaploids). Nine markers displayed polymorphism and/or transferability among studied taxa. They provided 54 alleles in total, ranging from 2 to 14 alleles per locus. The highest number of alleles was observed in C. erythraea, C. littorale and a hybridogenic taxon C. pannonicum. The developed markers are qualified to be used in genetic population studies on declining natural populations of Centaurium species, thus providing valuable information to evolutionary and conservation biologists. The developed cost-effective methodology provides abundant de novo assembled short contigs and holds great promise to mine numerous additional EST-SSR-containing markers for possible use in genetics population studies of tetraploid taxa within the genus Centaurium.

Development of Simple-Sequence Repeats Markers from Durian (Durio zibethinus Murr. cultv. Matahari) Genomic Library

Simple sequence repeats have been proved as powerful markers and widely used in molecular breeding to reduce cycles and cost efective. The availability of the marker is, however, very limited in durian. This research aimed to develop SSR markers from durian genomic library. Genomic DNA was isolated from durian shoot leaf, whilst SSR motifs were isolated using membrane-based oligonucleotide enrichment hybridization protocol. Annotation made on the library found 527 unique motifs from 354 durian libraries which form 425 loci. The SSR motifs obtained were generally short repeats which reached 89.6 %, whilst longer repeats were found consisted of compound motifs. Eleven loci were selected as representative for further test to prove their informativity. A number of unique allels were successfully amplified from 17 durian genomes. The analysis showed the polymorphic information content (PIC) values ranged from 0.000 to 0.662 with an average of 0.390. The SSR loci also showed their ability to be used for durian diversity analysis as the evident that the loci could be used as genetic markers for assisting further durian breeding program.

Development and validation of EST derived SSR markers with relevance to downy mildew (Sclerospora graminicola Sacc.) resistance in pearl millet [Pennisetum glaucum (L.) R. Br.

Molecular markers associated or linked with a particular trait are immensely essential for marker-assisted selection. Downy mildew is a severe disease in pearl millet causing huge production losses annually. There is a need to develop reliable functional markers associated with downy mildew resistance. In this study, expressed sequence tag-SSR (EST-SSRs) markers were developed from transcriptome data of downy mildew inoculated resistant and susceptible pearl millet genotypes. The high quality pearl millet reads were assembled by Trinity into 26,690 transcripts and 3586 (13.4%) transcripts harbored 4510 SSR motifs with the frequency of one SSR per 5.73 kb. The distribution of SSR motifs in different repeat type classes highlighted tri-nucleotide repeats as the highest (54.6% with CCG/CGG) followed by hexa-nucleotide (16.1% with CCGGCG/CCGGCG), penta-nucleotide (14.5% with CCGCG/CGCGG), tetra-nucleotides (8.0% with AGGG/CCCT) and di-nucleotide (6.8% with AG/CT) repeats. Total of 2419 EST-SSRs markers were developed and 230 markers based on their function in downy mildew-pearl millet interaction were validated in 12 pearl millet genotypes which are parents of mapping population. A total of 33 (20.62%) EST-SSR markers displayed polymorphism with a polymorphism information content (PIC) value ranging from 0.188 to 0.375 with an average of 0.354 and 2.2 alleles. The highest polymorphism was displayed by tri-nucleotide repeat EST-SSR markers followed by hexa-nucleotide, penta-nucleotide and di-nucleotide. The EST-SSR primers designed from eight transcripts coding for peroxidase 16-like, transcription factor myc7e, wound stress protein precursor, peroxidase 16-like, zinc finger ccch domain-containing, wrky transcription factor, beta purothionin were found to be more polymorphic than rest of the transcripts. The newly developed EST-SSR markers have added to the repository of molecular markers for pearl millet and can be employed for downy mildew resistance breeding.

Development and characterization of EST‐SSR markers in Asarum sakawanum var. stellatum and cross‐amplification in related species

EST‐SSR markers were developed for Asarum sakawanum var. stellatum to evaluate the genetic diversity and genetic structure of series Sakawanum and assess their utility in other series in the genus Asarum. A total of 1864 primer pairs, including 1864 tri‐nucleotide SSR motifs, were designed based on transcriptome data. Of the 96 primers tested, 17 loci were successfully amplified and polymorphic in series Sakawanum taxa. Furthermore, all these loci were successfully amplified in A. takaoi, whereas only 10 loci were amplified in the more phylogenetically distant A. pellucidum. For populations of A. sakawanum var. stellatum, the values of observed and expected heterozygosity ranged from 0.261 to 0.957 and 0.317 to 0.881, respectively. These newly developed EST‐SSR markers will be used in population genetic studies of series Sakawanum to infer the evolutionary history and genetic diversity of these series, many of which are endangered in Japan.

Genic and Intergenic SSR Database Generation, SNPs Determination and Pathway Annotations, in Date Palm (Phoenix dactylifera L.).

The present investigation was carried out aiming to use the bioinformatics tools in order to identify and characterize, simple sequence repeats within the third Version of the date palm genome and develop a new SSR primers database. In addition single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions were recognized. Moreover, the pathways for the sequences assigned by SSR primers, the biological functions and gene interaction were determined. A total of 172,075 SSR motifs was identified on date palm genome sequence with a frequency of 450.97 SSRs per Mb. Out of these, 130,014 SSRs (75.6%) were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, only 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. A total of 111,403 of SSR primer pairs were designed, that represents 291.9 SSR primers per Mb. Out of the 111,403, only 31,380 SSR primers were in the genic regions, while 80,023 primers were in the intergenic regions. A number of 250,507 SNPs were recognized in 84,172 SSR flanking regions, which represents 75.55% of the total SSR flanking regions. Out of 12,274 genes only 463 genes comprising 896 SSR primers were mapped onto 111 pathways using KEGG data base. The most abundant enzymes were identified in the pathway related to the biosynthesis of antibiotics. We tested 1031 SSR primers using both publicly available date palm genome sequences as templates in the in silico PCR reactions. Concerning in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars.