Microsatellite Simple Sequence Repeat Research Articles

Assessment of in silico BAC-based simple sequence repeat (SSR) marker development for tomato (Solanum lycopersicum L.)

Tomato landraces are less sensitive to environmental stresses and grown mainly under rain fed conditions. They are still grown in small farms due to quality and special demand of consumers. These landraces are valuable sources of genetic traits, and plant breeders can use breeding programs for crop improvement. One of the primary needs of the crop improvement is the estimation of genetic diversity. Development of microsatellite simple sequence repeat (SSR) markers from map-referenced bacterial artificial chromosomes (BAC) clones is a very effective means of targeting markers to marker scarce positions in the genome. This study was aimed at developing a set of functional SSR markers via in silico analysis of publicly available tomato DNA sequences. As a result, 17 SSR markers were developed and tested on one tomato commercial cultivar and eight local landraces. 12 loci (27 alleles) were scored and showed 100% polymorphic patterns. The calculated polymorphism information content (PIC) values for the SSR markers developed ranged from 0.62 to 0.97 (mean 0.89). The SSR motifs CT(26) AT(27) and TTC(6) TTA(4) had the highest PIC value (0.97), while CAA(5)A(8) had the lowest PIC value (0.62). According to tomato expressed sequence tag (EST) analysis, some of these developed SSR markers, such as mono and di-nucleotide are related to some genes. The T(16) motif is related to hydroxyproline-rich glycoprotein, which is a family protein from Arabidopsis thaliana . On the other hand, the SSR with tri-nucleotide repeat motif AAC(4)A(11) was related to a putative homologous protein to A7Q2S4 from Vitis vinifera . Keywords: Tomato landraces, in silico simple sequence repeat (SSR) markers, DNA markers, genetic diversity

Outcrossing rate in olive assessed by microsatellite and inter simple sequence repeat (ISSR) markers

Olive is known to be an allogamous species. The aim of this study was to estimate the magnitude of cross-pollination rate using microsatellite simple sequence repeats (SSR) and inter simple sequence repeats (ISSR) molecular markers in olive genotypes. The DNA from maternal plants and 23 progenies of two different accessions, Ascolano USA and MGS GRAP541 were extracted and screened with two microsatellite and ten ISSR markers. The outcrossing rate and other related parameters were analyzed using the MLTR application. The set of estimates, individually and collectively, support the hypothesis of frequent allogamy in both olive genotypes evaluated, with high rates of outcrossing in both markers. Keywords: Olea europaea L., outcrossing rate, zygotic embryos, mating system, molecular markers

Generation and Characterization of Interspecific Hybrids of Lotus uliginosus × Lotus corniculatus

ABSTRACTA method is described for obtaining interspecific hybrids between commercial lines of Lotus uliginosus Schkuhr and Lotus corniculatus L. Hybridization was possible between these species using embryo rescue. Two strategies were used to confirm 40 F1 hybrids, that is, flow cytometry and microsatellite simple sequence repeats (SSRs). The L. uliginosus and L. corniculatus parents have significantly different genome size (2.5 ± 0.02 and 2.22 ± 0.02 pg, respectively) and the offspring from the cross had intermediate values. Two SSR primers were polymorphic between the genotypes tested, TM1150 and EH380069. Maternal effect was detected in L. uliginosus genotypes, as determined by greater frequency of F1 hybrids. Recombinant traits were observed in the F1 hybrid progeny. Ninety F1 hybrids resulting from L. uliginosus × L. corniculatus crosses showed different levels of fertility in the polycross; <3% F1 plants produced more than 1000 seeds without embryo rescue while 10% of plants did not flower the first year. The F1 hybrid plants grown under greenhouse conditions exhibited shoot phenotypes similar to L. uliginosus (maternal) whereas F2 phenotypes were similar to L. corniculatus (paternal) in field trials. Rhizome presence was observed in 60% of F2 hybrid plants. The diameters of the root crowns of these plants, however, were similar to that of L. corniculatus.

ANALYSIS OF GENETIC DIVERSITY AND STRUCTURE OF SOME HISTORIC OLIVE (OLEA EUROPAEA L.) GERMPLASM IN THE UNITED STATES USING MICROSATELLITE MARKERS

Fifteen microsatellite simple-sequence repeat (SSR) loci were used to genotype 58 accessions of cultivated olive, Olea europaea L. ssp. europaea var. europaea, from two collections of 19th to 20th century plantings in Arizona and California, in order to examine genetic diversity and structure within and among collections. These collections are Lotusland Gardens in Santa Barbara, CA, and the Arboretum of the University of Arizona, Tucson. Genotypes in the olive collection at the USDA-ARS National Clonal Germplasm Repository for Fruit & Nut Crops (NCGR) in Davis, California were compared with the historic collections to potentially establish cultivar identity. Mean number of alleles per locus was 5.0 for Lotusland and 3.5 for the University of Arizona. Mean observed heterozygosity per collection was 0.650 for Lotusland and 0.598 for the University of Arizona. Up to 90% of the samples per collection were found to be synonymous with named, primarily Spanish or Italian, cultivars, yet these historic collections also contain significant genetic diversity, including genotypes and alleles not present in the NCGR germplasm collection. The cluster analysis placed the unknown genotypes within clades containing cultivars from the Eastern Mediterranean or from throughout the Mediterranean region, and in some cases produced relatively distinct branches.

Simple Sequence Repeat Marker Development and Mapping Targeted to Previously Unmapped Regions of the Strawberry Genome Sequence

The genome sequence of the woodland strawberry (Fragaria vesca L.) is an important resource providing a reference for comparative genomics studies and future sequenced rosaceous species and has great utility as a model for the development of markers for mapping in the cultivated strawberry Fragaria ×ananassa Duchesne ex Rozier. A set of 152 microsatellite simple sequence repeat (SSR) primer pairs was developed and mapped, along with 42 previously published but unmapped SSRs, permitting the precise assignment of 28.2 Mbp of previously unanchored genome sequence scaffolds (13% of the F. vesca genome sequence). The original ordering of F. vesca sequence scaffolds was performed without a physical map, using predominantly SSR markers to order scaffolds via anchoring to a comprehensive linkage map. This report complements and expands resolution of the Fragaria spp. reference map and refines the scaffold ordering of the F. vesca genome sequence using newly devised tools. The results of this study provide two significant resources: (i) the concurrent validation of a substantial set of SSRs associated with these previously unmapped regions of the Fragaria spp. genome and (ii) the precise placement of previously orphaned genomic sequence. Together, these resources improve the resolution and completeness of the strawberry genome sequence, making it a better resource for downstream studies in Fragaria spp. and the family Rosaceae.

Sequence‐characterized amplified region and simple sequence repeat markers for identifying the major quantitative trait locus responsible for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

With 1 figure and 2 tablesAbstractDowny mildew caused by the oomycete Hyaloperonospora parasitica Constant. (Pers. Ex Fr.) is a serious threat to Brassicaceae family. Previously, a major quantitative trait locus (QTL) for seedling resistance (BrDW) and its flanking markers, K14‐1030 and phosphoglucomutase, were identified in the Chinese cabbage. In order to establish the marker‐assisted selection (MAS) technique, K14‐1030 was successfully converted into a sequence‐characterized amplified region marker SCK14‐825, and a bacterial artificial chromosome (BAC) with sequence homology to K14‐1030 was identified. On the basis of the homologous and the linked BAC sequences, two microsatellite simple sequence repeat markers, kbrb058m10‐1 and kbrb006c05‐2, were designed and mapped on the confidence intervals of BrDW. These three markers could explain the QTL effect to a considerable extent and yield relatively high selection accuracy, which would be helpful in MAS for breeding downy mildew‐resistant Brassica rapa ssp. pekinensis varieties.

Development and characterization of 21 EST‐derived microsatellite markers in Vicia faba (fava bean)

Expressed sequence tag (EST)-derived microsatellite simple sequence repeat (SSR) markers were developed in Vicia faba L. by screening the NCBI database. Markers were validated and explored for size polymorphism among fava bean accessions. Twenty-one EST-SSR primer pairs were identified, and the loci characterized were size polymorphic among 32 fava bean genotypes from diverse geographical locations. The number of alleles (N(a)) per locus ranged from 2 to 9, and expected heterozygosity (H(E)) ranged from 0.0476 to 0.8304, respectively. These markers will be useful to study genetic diversity, genetic mapping, and molecular breeding in fava bean.

Evaluation of genetic identity and variation in cultivars of Pyrus pyrifolia (Burm.f.) Nakai from China using microsatellite markers

SummaryOne-hundred and six accessions of Pyrus, including 87 local Chinese accessions of P. pyrifolia, were identified genetically using nine microsatellite (simple sequence repeat) loci developed from apple and pear. A total of 147 putative alleles were obtained. Among the 106 accessions, 100 could be differentiated successfully, except for three pairs of synonymous accessions or mutants. For the 87 accessions of Chinese P. pyrifolia, the number of putative alleles per locus ranged from ten to 21, with an average of 15.3. The observed heterozygosity and polymorphic information content were 0.60 and 0.75, respectively. A phenogram based on the SSR genotypes was obtained. The 87 accessions of Chinese P. pyrifolia clustered into seven groups, based on their geographical origin. Most P. bretschneideri cultivars grouped together with the P. pyrifolia accessions from the same geographical areas. Some Japanese pear cultivars (i.e., ‘Choujuurou’, ‘Housui’, ‘Niitaka’, ‘Okusankichi’, and ‘Shinseiki’) grouped together with some accessions of P. pyrifolia from the Yunnan, Jiangxi, and Zhejiang Provinces of China. The similarity coefficient between ‘Jiangwantangli’ from Jiangxi Province and a Japanese pear cultivar, ‘Choujuurou’, was 0.80. Cultivars of P. communis and P. ussuriensis showed distant relationships with those of P. pyrifolia.

Analysis of unigene derived microsatellite markers in family solanaceae.

The family Solanaceae is the source of several economically important plants. The aim of this study was to trace and characterize simple sequence repeat (SSR) markers from unigene sequences of Solanum lycopersicum, an important member of family Solanaceae. 18,228 unigene sequences of Solanum lycopersicum was taken in order to develop SSR markers and analyzed for the in-silico design of PCR primers. A total of 12,090 (66.32 %) unigenes containing 17,524 SSRs (microsatellites) were identified. The average frequency of microsatellites in unigenes was one in every 1.3 kb of sequence. The analysis revealed that trinucleotide motifs, coding for Glutamic acid (GAA) and AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking sequences of the SSRs generated 877 primers with forward and reverse strands. Functional categorization of SSRs containing unigenes was done through gene ontology terms like Biological process, Cellular component and Molecular function.

A new electrophoresis technique to separate microsatellite alleles.

Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other electrophoresis methods. Individual PCR reactions consisting of DNA from several Cornus florida L. (flowering dogwood) cultivars and two SSR primer pairs were assembled for analysis using the following three methods: agarose gel, polyacrylamide gel and QIAxcel System. Two separate PCR reactions consisting of the same components plus a fluorescent-labeled primer were set up for analyses using the CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. These five electrophoretic methods were assessed for advantages and disadvantages. Polyacrylamide gels had highest resolution of alleles, whereas agarose gels had the lowest. However, with both separation media, it was difficult to score the size of alleles. Capillary electrophoresis with the CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer easily separated products and determined allelic size, but was more expensive than electrophoresis using either agarose or polyacrlamide gels. The QIAxcel System had lower resolution than CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. However, QIAxcel System was rapid and cost effective compared to the two widely used capillary sequencers, and also provided a computer generated gel image. For researchers in small to intermediate-sized laboratories, the QIAxcel System using a twelve channel, sieving-gel cartridge is an affordable device for SSR assays used for mapping and population diversity analysis.

Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean

With 2 figures and 2 tables Abstract In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F2 population derived from a single F1 plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.

Microsatellite-Based Genetic Diversity Assessment in Grape (Vitis vinifera L) Germplasm and its Relationship with Agronomic Traits

The population of grape (Vitis vinifera L), commonly cultivated in India, shows a wide range of ripening periods and fruit quality and is an unexploited resource for breeding programs. The main purpose of this study was to fingerprint these accessions and to construct a molecular database including the cultivars commonly grown in India. A total of 42 genotypes were analyzed using seven microsatellite simple sequence repeat (SSR) markers and their main morphological and agronomic characteristics were compared. A total of 45 alleles in all 42 genotypes were obtained with 7 primers with an average number of 6.4 alleles per locus. The dissimilarity matrix showed that a maximum of 110 units was obtained between the genotypes “Convent Large Black” and “Arka Hans,” while a minimum dissimilarity if 37 units were obtained between the genotypes “Anab-e-Shahi” and “Dilkhush.” In the dendrogram the microsatellite segregated the genotypes into two clusters (A and B) at 476 units with two subclusters each. The subclusters grouped genotypes predominantly as A1 with seeded fruits, A2 with seedless fruits, B1 with pigmented and seedless fruits, and B2 with colorless and seeded fruits. The use of seven polymorphic microsatellite markers and the level of genetic variability detected within Indian grapevine germplasm suggested that this is a reliable, efficient, and effective marker system that can be used for diversity analysis and subsequently in crop improvement programs.

Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

Downy mildew caused by the fungus Peronospora parisitica is a serious threat to members of the Brassicaceae family. Annually, a substantial loss of yield is caused by the widespread presence of this disease in warm and humid climates. The aim of this study was to localize the genetic factors affecting downy mildew resistance in Chinese cabbage (Brassica rapa ssp. pekinensis). To achieve this goal, we improved a preexisting genetic map of a doubled-haploid population derived from a cross between two diverse Chinese cabbage lines, 91-112 and T12-19, via microspore culture. Microsatellite simple sequence repeat (SSR) markers, isozyme markers, sequence-related amplified polymorphism markers, sequence-characterized amplified region markers and sequence-tagged-site markers were integrated into the previously published map to construct a composite Chinese cabbage map. In this way, the identities of linkage groups corresponding to the Brassica A genome reference map were established. The new map contains 519 markers and covers a total length of 1,070 cM, with an average distance between markers of 2.06 cM. All markers were designated as A1–A10 through alignment and orientation using 55 markers anchored to previously published B. rapa or B. napus reference maps. Of the 89 SSR markers mapped, 15 were newly developed from express sequence tags in Genbank. The phenotypic assay indicated that a single major gene controls seedling resistance to downy mildew, and that a major QTL was detected on linkage group A8 by both interval and MQM mapping methods. The RAPD marker K14-1030 and isozyme marker PGM flanked this major QTL in a region spanning 2.9 cM, and the SSR marker Ol12G04 was linked to this QTL by a distance of 4.36 cM. This study identified a potential chromosomal segment and tightly linked markers for use in marker-assisted selection to improve downy mildew resistance in Chinese cabbage.

Development and characterization of 11 microsatellite markers in Lithocarpus edulis

Eleven microsatellite (simple sequence repeat) markers were isolated from Lithocarpus edulis, an evergreen broadleaved tree species in Japan, based on an inter-compound microsatellite method and a single-strand DNA extension adaptor ligation method. There were 2–17 alleles per locus, and the observed and expected heterozygosities ranged from 0.1 to 0.9 and from 0.185 to 0.949, respectively.

Development and characterization of microsatellite markers in the fungus Antrodia cinnamomea from dbEST database

We developed and characterized 8 polymorphic microsatellite loci or simple sequence repeats from the expressed sequence tags database of the medicinal fungus, Antrodia cinnamomea. Variability was assessed in a panel of 23 strains collected from various regions of Taiwan. The number of alleles per polymorphic locus ranged from two to seven loci with an average of 4.375 alleles per locus. The observed and expected heterozygosity values ranged from 0.130 to 0.783 and from 0.122 to 0.809, respectively. No significant Hardy–Weinberg equilibrium was detected (P < 0.01) at all loci. The EST-SSR markers developed in the present study can be used for strain identification and population genetic studies in A. cinnamomea.

Genetic Diversity and Association Analysis for Salinity Tolerance, Heading Date and Plant Height of Barley Germplasm Using Simple Sequence Repeat Markers

The objective of this study was to investigate the genetic diversity of barley accessions. Additionally, association trait analysis was conducted for grain yield under salinity, heading date and plant height. For this purpose, 48 barley genotypes were analyzed with 22 microsatellite simple sequence repeat (SSR) markers. Four of the 22 markers (Bmac316, scssr03907, HVM67 and Bmag770) were able to differentiate all barley genotypes. Cluster and principal coordinate analysis allowed a clear grouping between countries from the same region. The genotypes used in this study have been evaluated for agronomic performance in different environments. Conducting association analysis for grain yield under salinity conditions using TASSEL software revealed a close association of the marker Bmag749 (2H, bin 13) in two different environments with common significant alleles (175, 177), whereas the HVHOTR1 marker (2H, bin 3) was only significant in Sakhar_Egypt with alleles size being 158 and 161. Heading date also showed an association with scssr03907 through the common significant specific allele 111 and EBmac0415 markers in three different agro climatic locations, whereas HVCMA, scssr00103 and HVM67 were linked to heading date in the Egyptian environment only. The plant height association analysis revealed significant markers Bmag770 via the significant allele 152 and scssr09398.

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.

Development of microsatellite markers in a clonal perennial herb, Convallaria keiskei

AbstractWe developed eight polymorphic microsatellite simple sequence repeat (SSR) loci from genomic DNA of a clonal perennial herb, Convallaria keiskei, using a dual‐suppression‐polymerase chain reaction (PCR) technique and an improved technique. These markers with four to 10 alleles per locus identified 29 genotypes in 82 samples collected from a population in Hokkaido, Japan. The observed and expected heterozygosities ranged from 0.241 to 0.862 and from 0.639 to 0.825, respectively. These SSR markers will be available to identify genets and evaluate genetic diversity of C. keiskei.

Genetic variability of the bollworm, Helicoverpa armigera, occurring on different host plants

The bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a polyphagous pest of worldwide occurrence inflicting annual crop damage in India worth US$ 1billion. In India this insect occurs as a major pest in many economically important crops, including cotton, pigeonpea, chickpea, tomato, okra, and blackgram. Understanding the genetic variation among the H. armigera populations occurring on host plants has become essential to understand the variation in their susceptibility to different insecticides, including Bacillus thuringiensis. This preliminary study uses 10 microsatellite simple sequence repeat (SSR) markers, to provide insight into the genetic variability of H. armigera populations from six different host plants. Nine of the SSR primers indicated high variability across the different host associated populations with polymorphism ranging from 75 to 100 per cent. Using the un-weighted pair-group method analysis, H. armigera collected and reared from cotton stood out as unique in one cluster while the insects collected and reared on all other hosts grouped separately.

Isolation and characterization of eight compound microsatellite markers in a mangrove tree Kandelia candel (L.) Druce

Abstract Kandelia candel is an important mangrove tree species of family Rhizophoraceae. Here we isolated eight codominant compound microsatellite simple sequence repeat (SSR) loci from K. candel. Our isolated loci provided compound SSR markers with polymorphism of three to 11 alleles per locus. The expected and observed heterozygosities ranged from 0.230 to 0.887 and from 0.083 to 1.00, respectively. These markers would be the useful tools for analysing questions concerning population genetic structure and mating system of K. candel.