Development Of Simple Sequence Repeat Markers Research Articles

Development of SSR Markers Based on Transcriptome Sequencing and Verification of Their Conservation across Species of Ornamental Pennisetum Rich. (Poaceae)

Pennisetum species have importance in foraging, agriculture, energy-production, the environment, and landscaping. To promote the preservation and utilization of ornamental Pennisetum resources, we developed simple sequence repeat (SSR) markers from the Pennisetum setaceum cv. ‘Rubrum’ transcriptome and verified their conservation in 38 sources. Our transcriptome sequencing efforts generated 58.91 Gb of clean data containing 55,627 unigenes. We functionally annotated 30,930 unigenes, with functions enriched in translation and ribosomal structure and biogenesis. Database comparisons indicated that the closest relative of P. setaceum cv. ‘Rubrum’ is Setaria italica. Over five thousand SSR markers were detected in the transcriptomic data. We selected 38 pairs of highly polymorphic SSR markers from 50 randomly selected SSR markers. Based on genetic diversity analysis of 38 ornamental Pennisetum sources, we obtained 312 polymorphic bands, with an average of 8.21 alleles per primer. Principal coordinate analyses and generation of a, which proved that Pennisetum has moderate genetic diversity. In addition, fingerprint maps were constructed to improve Pennisetum identification. The transcriptome data generated by our study enhances the transcriptional information available for P. setaceum. This study lays the foundation for the collection and utilization of ornamental Pennisetum resources and provides a basis for future breeding projects using this species.

Transferability of Simple Sequence Repeat Markers from other Members of Family Fabaceae to Chickpea (Cicer arietinum L.)

Background: Limited genetic variation exists within chickpea (Cicer arietinum L.) owing to its narrow genetic base. Consequently, the DNA-based markers i.e. simple sequence repeat (SSR) markers that show considerable polymorphism in other crops reveal limited polymorphism in chickpea. Development of saturated linkage maps, marker assisted selection and gene cloning needs larger number of polymorphic markers which necessitates development of additional SSR markers for chickpea. Microsatellite marker development is costly, requires a great research expertise and effort. The cross-genera transferability of pre-developed SSR markers is a good alternative to new SSR marker development. Methods: To generate additional SSR markers for chickpea, a total of 292 SSR markers from horsegram (Macrotyloma uniflorum, 94 SSRs), lentil (Lens culinaris, 66 SSRs) and pea (Pisum sativum, 132 SSRs) were evaluated for cross-transferability to chickpea using a panel of four chickpea genotypes-GPF2, ICC16349, ICC10685 and ICC15614. Result: Lentil SSR markers had highest transferability to chickpea (36.36%) followed by pea (18.18%) and horsegram (14.89%). Limited polymorphism was detected in chickpea; 10.61% by lentil markers, 4.25% by horsegram markers and 3.79% by pea markers. Overall, 62 new SSR markers were added to repository of chickpea SSRs.

Mining and validation of novel simple sequence repeat (SSR) markers derived from coconut (Cocos nucifera L.) genome assembly

BackgroundIn the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers. ResultsA total of 7139 novel SSR markers were derived from the genome assembly of coconut ‘Catigan Green Dwarf’ (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40–60%; minimum ΔG value for hairpin loop, −0.3 kcal/mol; minimum ΔG value for self-dimer, −0.9 kcal/mol; and minimum ΔG value for heterodimer, −0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using ‘Catigan Green Dwarf’ (CATD), ‘Laguna Tall’ (LAGT), ‘West African Tall’ (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes. ConclusionThe development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.

Simple sequence repeat marker development and diversity analysis in buffalograss

AbstractBuffalograss [Buchloë dactyloides (Nutt.) Engelm. syn. Bouteloua dactyloides (Nutt.) Columbus] is a stoloniferous low input turfgrass species native to the central Great Plains of the United States. Several molecular marker techniques have been used to compare relationships among elite buffalograss germplasm, but most rely on random amplification, are transferred from other species, or are developed from limited DNA sequence information. The objectives of this study were to develop buffalograss derived simple sequence repeat markers (SSRs) from buffalograss sequence data using an in‐silico bioinformatics pipeline and use high resolution melt analysis to screen the SSRs based on their melt profiles on buffalograss cultivars and germplasm. Transcriptomes from buffalograss cultivars ‘378’ and ‘Prestige’ were mined for SSRs using the MIcroSAtellite tool. There were 259 conserved SSRs that were polymorphic between 378 and Prestige. Of those, 96 were tested by HRM on a panel of eight genotypes and 24 were selected for producing melt profiles that could distinguish the genotypes. The selected reactions produced 356 distinct melt profiles when tested on a panel of 96 genotypes, including 88 buffalograss germplasm selections, six buffalograss cultivars, and two blue grama [Bouteloua gracilis (H. B. K.) Lag. ex Steud.] selections, with melt profile frequencies ranging from 0.01 to 0.97. A subset of 86 melt profiles segregating among the blue grama and elite buffalograss cultivars distinguished each genotype. The buffalograss‐specific SSR markers presented here will be useful to study buffalograss genetic diversity, line purity maintenance, and for marker supported plant breeding strategies.

Genome-wide mining of simple sequence repeats and development of polymorphic SSR markers in Aquilaria malaccensis

Aquilaria malaccensis is one of the economically important tree species distributed in North-east India which produces oleoresinous heartwood known as Agarwood - the most expensive wood in the world. There are limited genomic resources in this species. Therefore, the publicly available whole-genome sequence of Aquilaria agallocha, which is a synonym of A. malaccensis, was utilized for genome-wide mining of Simple Sequence Repeats (SSR) and to develop polymorphic SSR markers. In total 2,53,155 SSRs were identified from the draft genome. Primers were designed for di-nucleotide and tri-nucleotide repeats and screening of 90 such primers have resulted in the identification of 10 polymorphic markers. The present report is the first one for exhaustive identification of SSRs from the draft genome of the species. The SSR markers identified in the present study may be efficient in genomic analyses and useful in the genetic assessment and conservation of A. malaccensis.

Development of SSR and SNP markers for identifying opium poppy.

Papaver somniferum L. (opium poppy) is the original plant of heroin, which is a major narcotic drug, and this plant has brought great harm to human health. However, the ban on opium poppy cultivation and trafficking is facing great challenges because of abnormal profits. Therefore, rapid and accurate identification is important to address the abovementioned problems. In this study, eleven simple sequence repeats (SSR) markers and two single nucleotide polymorphism (SNP) markers were mined to distinguish opium poppy from other six Papaver species. These molecular markers were further verified through a large number of plant materials of these seven Papaver species. An excellent multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the three of eleven SSR markers was developed, which effectively improves the efficiency and speed of identification. The present research is of great implication for identifying and investigating the illegal cultivation and trafficking of opium poppy.

A pipeline for effectively developing highly polymorphic simple sequence repeats markers based on multi-sample genomic data.

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution, and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next‐generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi‐sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown eared‐pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that 10 individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high‐quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.

Phylogenetic Analysis of Ryegrass (Lolium rigidum) Populations and the Proliferation of ALS Resistance in Saudi Arabia

Morphological and simple sequence repeat (SSR) approaches were used to determine the genetic diversity of 29 ryegrass (Lolium rigidum) genotypes belonging to eight populations collected from several regions in Saudi Arabia. In this study, 50 in Silico-developed SSR markers derived from genomic and expressed sequence tag (EST) microsatellites were examined. Analysis of variance showed highly significant differences in all studied traits. Cluster analysis based on the morphological data of the 29 Lolium genotypes and using PAST (paleontological statistics) software was performed. According to the results, clustering was based mostly on genotype location. The sensitive genotypes for herbicide were clustered in one group. In addition, using EST-SSR markers, we observed the existence of a considerable number of genetic variations among Lolium genotypes. From these markers, only 31 produced reasonable amplification products. The results showed that 23 SSR markers revealed that 74.19% were polymorphic. The number of alleles detected per primer ranged from one to five in the primer LTC SSR1. The tested primers amplified 1434 bands across eight populations, with an average of 46.26 bands per primer. The polymorphism information content (PIC) values ranged from 0.11 to 0.76 for the primers LT EST-SSR5 and LTC SSR1. The unweighted pair group method with arithmetic average (UPGMA) clustering of the 29 genotypes representing eight populations was based essentially on their locations and herbicide-tolerance levels. Most of the populations formed into four clusters, together representing genotypes. Moreover, the tolerant populations were distinguished from the sensitive ones. The relationship between the genetic diversity and geographical source of Lolium rigidum populations of Saudi Arabia was revealed through this study. The results showed that the efficiency of developed SSR markers are transferable across species. They have been helpful to assess the genetic diversity of the ryegrass population as this could be applied to differentiate between tolerant and sensitive populations of ryegrass.

Genome wide simple sequence repeats development and their application in genetic diversity analysis in wax gourd (Benincasa hispida)

AbstractWax gourd is one of the most important winter vegetables of the Cucurbitaceae family. There are only limited markers available for this crop and the draft genome of wax gourd provides a powerful tool for simple sequence repeats (SSR) marker development. In this study, we developed genome‐wide SSR markers from wax gourd genome and characterized their distribution and frequency of different motifs and repeats. A total of 52,431 microsatellites from wax gourd genome were identified, of which 39,319 SSR markers were developed. The 1152 non‐wax gourd SSR markers were selected from cucumber, melon, watermelon and pumpkin to test their transferability in wax gourd. The 580 SSR markers could be transferable in wax gourd, and 42 of them were detected with polymorphic in 11 tested accessions of wax gourd. In addition, 11 good polymorphic transferrable SSR markers and 21 SSR markers of wax gourd were selected to investigate the genetic diversity and population structure of 129 wax gourd accessions. One hundred twelve alleles were detected by these 32 SSR markers. The result of population structure showed that the 129 wax gourd accessions were divided into two main populations, and the genetic diversity analysis separated them into two clusters. The large number of wax gourd SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker‐assisted selection (MAS) in wax gourd.

Genome-wide development and utilization of Simple Sequence Repeats in Chinese cabbage (<i>Brassica rapa</i> L. ssp. <i>pekinensis</i>)

With the complementation of whole-genome sequencing of Chinese cabbage, it is necessary to develop genome-wide Simple Sequence Repeat (SSR) markers and analyse their characteristics, which will bring a revolution in the molecular marker-assisted breeding of Chinese cabbage. In this study distribution and characteristics of SSR loci in the genome of Chinese cabbage 'Chiifu' was analyzed. Finally, a total of 173,892 SSR markers that occurred in 10 chromosomes were identified, and Perfect Repeat Motifs (P type) which is a main form of SSR loci with a mean distance of 1.89 kb/SSR was identified. Among them, mono-, di-, and tri-nucleotide repeat motifs were equally distributed across chromosomes compared with tetra-, penta-, and hexa-nucleotide repeat motifs. The largest proportion of SSR loci consisted of mono- and dinucleotide repeat motifs, which accounted for 93.04% of the total SSR loci. A/T and AT/TA were the most abundant motifs, accounting for 97.61% and 64.05% of the mono- and dinucleotide repeats, respectively. Furthermore, 200 SSR markers were used for the amplification of 12 Chinese cabbage germplasm, in which 30 were highly polymorphic. These SSR markers were further used for the association analyses of flowering time of 190 Chinese cabbage accessions. The results revealed that 9 and 10 alleles were associated with bolting and flowering time, respectivley. The results show that development of SSR markers is feasible and useful in marker assisted selection of Chinese cabbage.

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.

Development and characterization of Simple Sequence Repeats (SSRs) markers in durian kura-kura (Durio testudinarius Becc.) using NGS data

Durian Kura-kura (Durio testudinarius Becc.) belongs to the Malvaceae family and is an endemic species of Borneo. Recently, genomic-based next-generation sequencing (NGS) approaches have been carried out for germplasm conservation and plant breeding programs. The NGS technologies allow plant genomes to be sequenced quickly and inexpensively and enable the efficient development of SSR markers through the in-silico approaches. This study aimed to develop and characterize simple sequence repeats (SSRs) from the assembled genome. The 1203929 scaffolds of the assembled genome were produced from the Ray assembler. The SSRs were identified and extracted using the MISA program produced 4315 sequences containing SSRs. The six motif repeats of SSRs were identified; consist of 431 sequences of dinucleotide (the most motif is AT), 3257 sequences of trinucleotide (the most motif is TTA), 516 sequences of tetranucleotide (the most motif is AAAT), 89 sequences of pentanucleotide (the most motif is ATTTT), 18 sequences of hexanucleotide and four sequences of heptanucleotide. The new SSRs markers will be used in further studies of genetic population of D. testudinarius and plant breeding programs.

Genetic diversity and population structure analysis in a large collection of Vicia amoena in China with newly developed SSR markers

Vicia amoena is a high-nutritional quality forage similar to alfalfa. However, studies on the genetic background of V. amoena are scarce. In the present study, the genetic variation of 24 V. amoena populations was assessed with newly developed simple sequence repeat (SSR) markers. A total of 8799 SSRs were identified in the V. amoena genomic-enriched sequences, and the most abundant repeat number was four. A total of 569 sampled individuals were assayed to evaluate the genetic diversity of the V. amoena populations based on 21 polymorphic SSR primers. The polymorphism information content (PIC) ranged from 0.896 to 0.968, with an average of 0.931, which indicated that the markers were highly informative. Based on analysis of molecular variance, 88% of the variance occurred within populations, and the remaining 12% of the variance occurred among populations. The high degree of gene flow (Nm= 4.958) also showed slight differentiation among the V. amoena populations. The V. amoena populations were mainly clustered by steppe and mountain habitats based on principal coordinate analysis (PCoA) and STRUCTURE analysis. This indicated that the elevation and special habitat of geographical origins may be important factors affecting the clustered pattern of V. amoena populations. Neighbour-joining (NJ) analysis did not separate the populations well by geographical origin, which indicated that the genetic structure of V. amoena was complex and needs further study. Overall, our results showed that the newly developed SSR markers could benefit the V. amoena research community by providing genetic background information to help establish a foundation for breeding improvement and germplasm resource conservation.

Development and preliminary application of novel genomewide SSR markers for genetic diversity analysis of an economically important bio-control agent Platygaster robiniae (Hymenoptera: Platygastridae)

Platygaster robiniae Buhl and Duso (Hymenoptera: Platygastridae) is an egg-larvae parasitoid of the black locust gall midge (Obolodiplosis robiniae) (Haldeman) (Diptera: Cecidomyiidae) which is a serious invasive pest in China, where it attacks an important hardwood species, the black locust tree, Robini pseudoacacia L. (Fabales: Fabaceae). Despite the use of P. robiniae as an effective biocontrol agent, the absence of sequence data and other molecular markers have limited its genetic applications for pest management in forests. Simple-sequence repeats (SSRs) are valuable molecular markers for population genetic structure studies. In the present study, we identified 14,123 SSRs, of which 7799 SSR primer pairs were successfully designed. Subsequently, 240 SSR were chosen and tested with 48 P. robiniae accessions from two geographically separated populations in north and south China. Of these, 34 were polymorphic, with an average of three alleles (Na) and four genotypes (NG) each. The average values of observed heterozygosity (Ho) was 0.3514, expected heterozygosity (He) 0.4167, Shannon's information index (I) 0.7143, and polymorphism information content (PIC) 0.3558, respectively. Neighbour joining analysis (bootstrap 1000) revealed that Chengdu (CD) and Dangdong (DD) popluations clustered into two main divisions, and some individuals from two popluations clustered together as the third devision, which indicated the gene flow and genetic differentiation were present between two populations. Our finding indicates that these SSR markers will be useful for further studies on the genotype identification and genetic mapping of the genus Platygaster.

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Background: Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. Methods: The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. Result: In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.

Comparative transcriptomics provides a strategy for phylogenetic analysis and SSR marker development in Chaenomeles

The genus Chaenomeles has long been considered an important ornamental, herbal and cash crop and is widely cultivated in East Asia. Traditional studies of Chaenomeles mainly focus on evolutionary relationships at the phenotypic level. In this study, we conducted RNA-seq on 10 Chaenomeles germplasms supplemented with one outgroup species, Docynia delavayi (D. delavayi), on the Illumina HiSeq2500 platform. After de novo assemblies, we generated from 40,084 to 49,571 unigenes for each germplasm. After pairwise comparison of the orthologous sequences, 9,659 orthologues within the 11 germplasms were obtained, with 6,154 orthologous genes identified as single-copy genes. The phylogenetic tree was visualized to reveal evolutionary relationships for these 11 germplasms. GO and KEGG analyses were performed for these common single-copy genes to compare their functional similarities and differences. Selective pressure analysis based on 6,154 common single-copy genes revealed that 45 genes were under positive selection. Most of these genes are involved in building the plant disease defence system. A total of 292 genes containing simple sequence repeats (SSRs) were used to develop SSR markers and compare their functions in secondary metabolism pathways. Finally, 10 primers were chosen as SSR marker candidates for Chaenomeles germplasms by comprehensive standards. Our research provides a new methodology and reference for future related research in Chaenomeles and is also useful for improvement, breeding and selection projects in other related species.

De Novo Transcriptomic Characterization Enables Novel Microsatellite Identification and Marker Development in Betta splendens.

The wild populations of the commercially valuable ornamental fish species, Betta splendens, and its germplasm resources have long been threatened by habitat degradation and contamination with artificially bred fish. Because of the lack of effective marker resources, population genetics research projects are severely hampered. To generate genetic data for developing polymorphic simple sequence repeat (SSR) markers and identifying functional genes, transcriptomic analysis was performed. Illumina paired-end sequencing yielded 105,505,486 clean reads, which were then de novo assembled into 69,836 unigenes. Of these, 35,751 were annotated in the non-redundant, EuKaryotic Orthologous Group, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. A total of 12,751 SSR loci were identified from the transcripts and 7970 primer pairs were designed. One hundred primer pairs were randomly selected for PCR validation and 53 successfully generated target amplification products. Further validation demonstrated that 36% (n = 19) of the 53 amplified loci were polymorphic. These data could not only enrich the genetic information for the identification of functional genes but also effectively facilitate the development of SSR markers. Such knowledge would accelerate further studies on the genetic variation and evolution, comparative genomics, linkage mapping and molecular breeding in B. splendens.

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.

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers in swamp eel (Monopterus albus).

Swamp eel is one model species for sexual reversion and an aquaculture fish in China. One local strain with deep yellow and big spots of Monopterus albus has been selected for consecutive selective breeding. The objectives of this study were characterizing the Simple Sequence Repeats (SSRs) of M. albus in the assembled genome obtained recently, and developing polymorphic SSRs for future breeding programs. The genome wide SSRs were mined by using MISA software, and their types and genomic distribution patterns were investigated. Based on the available flanking sequences, primer pairs were batched developed, and Polymorphic SSRs were identified by using Polymorphic SSR Retrieval tool. The obtained polymorphic SSRs were validated by using e-PCR and capillary electrophoresis, then they were used to investigate genetic diversity of one breeding population. A total of 364,802 SSRs were identified in assembled M. albus genome. The total length, density and frequency of SSRs were 8,204,641 bp, 10,259 bp/Mb, and 456.16 loci/Mb, respectively. Mononucleotide repeats were predominant among SSRs (33.33%), and AC and AAT repeats were the most abundant di- and tri-nucleotide repeats motifs. A total of 287,189 primer pairs were designed, and a high-density physical map was constructed (359.11 markers per Mb). A total of 871 polymorphic SSRs were identified, and 38 SSRs of 101 randomly selected ones were validated by using e-PCR and capillary electrophoresis. Using these 38 polymorphic SSRs, 201 alleles were detected and genetic diversity level (Na, PIC, HO, and He) was evaluated. The genome-wide SSRs and newly developed SSR markers will provide a useful tool for genetic mapping, diversity analysis studies in swamp eel in the future. The high level of genetic diversity (Na = 5.29, PIC = 0.5068, HO = 0.4665, He = 0.5525) but excess of homozygotes (FIS = 0.155) in one breeding population provide baseline information for future breeding program.

New Simple Sequence Repeat Markers on Linkage Groups 2 and 7, and Investigation of New Sources of Eastern Filbert Blight Resistance in Hazelnut

Eastern filbert blight (EFB), caused by Anisogramma anomala, is a fungal disease threatening the european hazelnut (Corylus avellana) industry in the Willamette Valley of Oregon. The pathogen is endemic to the eastern United States where it causes little damage to the wild Corylus americana but causes severe cankers on most cultivars of the commercially important european hazelnut. The host genetic resistance in ‘Gasaway’ is conferred by a dominant allele at a single locus on linkage group 6 (LG6), and resistance from several other sources has been mapped to the same region. Some fungal isolates can overcome ‘Gasaway’ resistance, prompting a search for other sources of resistance. Resistance from other sources has been mapped to LG2 and LG7, for which additional simple sequence repeat (SSR) markers would facilitate marker-assisted selection (MAS). In this study, an in silico approach was used to develop new polymorphic SSR markers in the EFB resistance regions on LG2 and LG7. Starting with a search of 17 contigs of the ‘Jefferson’ genome sequence, 45 new polymorphic SSR markers were developed, characterized, and placed on the linkage map. The new SSR markers had an average of 10.18 alleles per locus, and average values for expected heterozygosity, observed heterozygosity, polymorphism information content, and frequency of null alleles of 0.72, 0.65, 0.68, and 0.068, respectively. Of the 42 new polymorphic SSRs segregating in the mapping population, 24 were on LG2, 12 were on LG7, and six were placed on other LGs. The new and previously developed SSR markers were used to study six new sources of EFB resistance, four from Russia and two from Crimea. Six resistant selections were crossed with susceptible selections, resulting in 7 progenies. Phenotyping for disease response revealed that segregation in progenies of the two Moscow selections (#2 and #27), one Russian selection (OSU 1187.101), and one Crimean selection (H3R12P62) fit the 1:1 segregation ratio expected for control of resistance by a dominant allele at a single locus; but in progenies of the other Russian selection (OSU 1166.123) and the other Crimean selection (H3R07P11), there was an excess of resistant seedlings. Correlation of disease scores and alleles at SSR loci indicated that resistance from three Russian selections (Moscow selections #2 and #27 and OSU 1166.123) and the Crimean selection H3R12P62 was on LG7, while resistance from Russian selection OSU 1187.101 was on LG2. Resistance from Crimean selection H3R07P11 was not correlated with markers on LG6, or LG2, or LG7. These sources and new SSR markers will be useful in MAS and the pyramiding of resistance genes in the breeding of new EFB-resistant cultivars.