Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

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

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Development of simple sequence repeat markers for Japanese isolates of Magnaporthe grisea

We developed nine simple sequence repeat (SSR) markers useful for differentiating Japanese isolates of Magnaporthe grisea through a bioinformatic approach. Repeat sequences in the genome of M. grisea were identified by a Tandem Repeat Finding program. Length polymorphisms at 28 loci were examined, nine of which were selected on the basis of detected polymorphisms. These nine SSR markers showed a Nei's gene diversity ranging from 0.23 to 0.91 among 48 field isolates of two natural populations. These SSR markers are well suited for M. grisea epidemiology and population genetics.     

Development and characterization of simple sequence repeat DNA markers for Zelkova serrata

We developed 14 microsatellite loci from an enriched genomic DNA library of a broad‐leaved deciduous tree, Zelkova serrata. Of 198 clones from the library, 112 contained microsatellite repeat regions. The M13‐tailed primer method was used for economy. Sequence‐specific primer pairs were designed for 58 of 76 candidate clones. Fourteen of these primer pairs successfully amplified polymorphic single loci among 34 individuals collected from the Kanto breeding region in Japan. The expected heterozygosity for the 14 microsatellite markers ranged from 0.378 to 0.876, suggesting that these will prove valuable for breeding and ecological studies on Z. serrata.     

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology

? Premise of the study: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. ? Methods and Results: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. ? Conclusions: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.     

Development of 16 polymorphic simple sequence repeat markers for Lycoris longituba from expressed sequence tags

Lycoris longituba is a tulip-like ornamental plant in China. We report on the data mining of L. longituba expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers. Eighteen EST-SSRs were isolated and validated for 32 individuals. These markers will be valuable for studying the genetic structure and diversity of populations for L. longituba.     

Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat.

Expressed sequence tags (ESTs) are a valuable source of molecular markers. To enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in hexaploid wheat (Triticum aestivum L.), over 260,000 ESTs from 5 different grass species were analyzed and 5418 SSR-containing sequences were identified. Using sequence similarity analysis, 156 cross-species superclusters and 138 singletons were used to develop primer pairs, which were then tested on the genomic DNA of barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and wheat. Three-hundred sixty-eight primer pairs produced PCR amplicons from at least one species and 227 primer pairs amplified DNA from two or more species. EST-SSR sequences containing dinucleotide motifs were significantly more polymorphic (74%) than those containing trinucleotides (56%), and polymorphism was similar for markers in both coding and 5' untranslated (UTR) regions. Out of 112 EST-SSR markers, 90 identified 149 loci that were integrated into a reference wheat genetic map. These loci were distributed on 19 of the 21 wheat chromosomes and were clustered in the distal chromosomal regions. Multiple-loci were detected by 39% of the primer pairs. Of the 90 mapped ESTs, putative functions for 22 were identified using BLASTX queries. In addition, 80 EST-SSR markers (104 loci) were located to chromosomes using nullisomic-tetrasomic lines. The enhanced map from this study provides a basis for comparative mapping using orthologous and PCR-based markers and for identification of expressed genes possibly affecting important traits in wheat.     

Development of simple sequence repeat markers for Botryosphaeria spp. with Fusicoccum anamorphs

We report the development of eight sets of microsatellite markers for the ascomycete fungus and tree pathogen, Botryosphaeria parva. The primers were identified after cloning and sequencing of fragments amplified using simple sequence repeat (SSR) primers. Genome walking was used to determine unknown sequences on either side of new SSRs. The primers were tested and proved useful in nine other Botryosphaeria species that all have Fusicoccum anamorphs, similar to B. parva.     

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.     

Development of simple sequence repeat markers for bermudagrass from its expressed sequence tag sequences and preexisting sorghum SSR markers

Bermudagrass (Cynodon spp.) is extensively cultivated for forage and turf in the the southern United States and in parts of Asia, Africa, southern Europe, Australia and South America. However, few simple sequence repeat (SSR) markers are available for bermudagrass genetics research. Accordingly, the objective of this study was to develop SSR markers in bermudagrass by transferring sorghum genomic SSR primers and by exploring bermudagrass expressed sequence tags (ESTs) from the National Center for Biotechnology Information (NCBI) database. The transferability of 354 tested sorghum SSRs was 57% to C. transvaalensis T577 (2n = 2x = 18), 27% to C. dactylon Tifton 10 (2n = 6x = 54) and 22% to Zebra (2n = 4x = 36). Among the transferred SSRs, 65 primer pairs generated reproducible SSR bands across the three genotypes. From 20,237 Cynodon ESTs at NCBI, 303 designed SSR primer pairs amplified target bands in at least one of C. dactylon var. aridus (2n = 2x = 18), C. transvaalensis T577, C. dactylon cv. Tifton 10, and C. dactylon var. dactylon Zebra. Of the effective EST SSRs, 230 primer pairs produced reproducible bands in all four genotypes. The study demonstrated that EST sequences and sorghum SSR primers are useful sources for the development of SSR markers for bermudagrass. The developed SSR markers will make a valuable contribution to the molecular identification of commercial cultivars, construction of genetic maps, and marker-assisted breeding in bermudagrass.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Development of simple sequence repeat markers from bacterial artificial chromosomes without subcloning

Simple sequence repeats (SSRs) were isolated from pearl millet bacterial artificial clones (BACs) without any subcloning steps. SSR sequences were targeted using 3' end-anchored SSR primers. Flanking sequences were isolated by suppression PCR. In this pilot study, 25 SSR markers have been developed from 40 BAC pools, comprising a total of 384 clones. This novel way to develop new markers has the added advantage that mapping the SSR markers will anchor individual BACs to the genetic maps and, thus, facilitate the construction of BAC contigs.     

Development of simple sequence repeat markers for the plant pathogenic rust fungus Puccinia triticina

Eighteen polymorphic di‐ and trinucleotide simple sequence repeat markers were developed for the phytopathogenic rust fungus Puccinia triticina. The allelic diversity varied from two to nine alleles per locus. Levels of observed heterozygosity ranged from 0.095 to 0.952. Seven of the loci deviated significantly from Hardy–Weinberg equilibrium (P < 0.002) with 70% having levels of observed heterozygosity higher than expected heterozygosity. Null allele(s) were observed for locus PtSSR76 with a frequency of 9%. A preliminary screen of other cereal rust fungi (P. coronata, P. graminis, P. recondita and P. striiformis) indicated that these primer pairs are specific to P. triticina.     

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.     

Development of soybean aphid genomic SSR markers using next generation sequencing

Simple sequence repeats (SSRs) or microsatellites are very useful molecular markers, owing to their locus-specific codominant and multiallelic nature, high abundance in the genome, and high rates of transferability across species. The soybean aphid (Aphis glycines Matsumura) has become the most damaging insect pest of soybean (Glycine max (L.) Merr.) in North America, since it was first found in the Midwest of the United States in 2000. Biotypes of the soybean aphid capable of colonizing newly developed aphid-resistant soybean cultivars have been recently discovered. Genetic resources, including molecular markers, to study soybean aphids are severely lacking. Recently developed next generation sequencing platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The objectives of this study were (i) to develop and characterize genomic SSR markers from soybean aphid genomic sequences generated by next generation sequencing technology and (ii) to evaluate the utility of the SSRs for genetic diversity or relationship analyses. In total 128 SSR primer pairs were designed from sequences generated by Illumina GAII from a reduced representation library of A. glycines. Nearly 94% (120) of the primer pairs amplified SSR alleles of expected size and 24 SSR loci were polymorphic among three aphid samples from three populations. The polymorphic SSRs were successfully used to differentiate among 24 soybean aphids from Ohio and South Dakota. Sequencing of PCR products of two SSR markers from four aphid samples revealed that the allelic polymorphism was due to variation in the SSR repeats among the aphids. These markers should be particularly useful for genetic differentiation among aphids collected from soybean fields at different localities and regions. These SSR markers provide the soybean aphid research community with the first set of PCR-based codominant markers developed from the genomic sequences of A. glycines.     

Development of simple sequence repeat markers in rye (Secale cereale L.).

Simple sequence repeats (SSRs), also referred to as microsatellites, represent a PCR-based marker system that has been described in mammalian and plant genomes in recent years. In self-pollinating crop plants they have been shown to be superior to other DNA markers with respect to their level of polymorphism. The technical advantages compared with RFLP markers should also facilitate marker analysis in outcrossing crops like rye. In order to determine the usefulness of SSR markers in rye genetics and breeding, several genomic libraries were screened for (CT/GA)n and (GT/CA)n dinucleotide repeats. It was estimated that these motifs occur at a frequency of one per 268-519 kb. Seventy four out of 182 positive clones were sequenced, and the majority (56.8%) revealed perfect repeats, predominantly of the type (GT/CA)n (61.9%). Fifty seven primer pairs were designed and 27 (47.4%) resulted in specific SSR markers, of which 20 were genetically mapped or assigned to chromosomes or chromosome arms, respectively. The level of polymorphism of four SSR and three RFLP markers was assessed in two open-pollinated rye cultivars. On average, the SSR markers showed larger values of expected heterozygosity (0.62 vs. 0.43) and allele number (5.9 vs. 3.4) than RFLP markers in both cultivars.     

Development of simple sequence repeat markers for the plant pathogenic rust fungus,Puccinia graminis

Twenty‐four dinucleotide simple sequence repeat markers were developed for the phytopathogenic fungus, Puccinia graminis. The identified loci were polymorphic, with allelic diversity ranging from two to 11 alleles. Observed and expected levels of heterozygosity ranged from 0.000 to 0.960 and from 0.113 to 0.846, respectively. Fourteen of the loci deviated significantly from Hardy–Weinberg equilibrium. Null alleles were observed for 10 of the 24 loci with a frequency of 4–16%. A preliminary screen of other Puccinia cereal rust fungi (P. coronata, P. striiformis and P. triticina) indicated that these primer pairs are specific to P. graminis.