Development Of New Microsatellite Markers Research Articles

Development of new microsatellite markers for DNA fingerprinting pattern of black gram (Vigna mungo L. Hepper) and green gram (Vigna radiate L. Wilckzek)

SSR markers are considered to be the most ideal marker for genetic studies because they are multi-allelic, abundant, randomly and widely distributed throughout the genome, co-dominant that could differentiate plants with homozygous or heterozygous alleles, simple to assay, highly reliable, reproducible. Microsatellite markers are highly polymorphic and informative and could be successfully used for genome analysis in black gram & green gram. Microsatellite markers were used to evaluate genetic diversity in 17 indigenous cultivars of pulse crops (11 cultivars of green gram and 6 cultivars of black gram respectively). They are subjected to variability analysis with 26 microsatellite markers for identification efficient primers to conclude the nature of molecular diversity present among the pulses. The SSR primer G228 showed 63.63% of polymorphism followed by MB-SSR 238 (45.45%) and G006 (36.36%). The 12 microsatellite markers produced 15.90 % polymorphism with banding ranged up to 7 with an average of 2.3 polymorphic banding patterns per SSR primer. Similarly for black gram, three random microsatellite primers G006 (50%) and G166 and G204 (33.33%) revealed considerable DNA polymorphism. The 14 random SSR primers produced 8.33% of polymorphism with banding ranged up to three with an average of 1.28 polymorphic banding pattern per SSR primer. The Distinguish Power (D), Polymorphism Information Content (PIC) value and Marker Index (MI) values revealed some SSR primers like G006, G204 and G166 can alone amplified distinct banding pattern, where as a combination of (G228+G006), (G228+G304) for green gram and the combination (G006+G166) can be used for black gram for ascertaining genetic diversity at any stage of crop growth period for green gram or black gram. From the present study we can conclude that selective microsatellite markers are highly polymorphic, informative and easily reproducible, which can be successfully used either as single or with combination for molecular characterization of crop species belonging to Vigna species.

Development of microsatellite genetic markers in Siberian larch (Larix sibirica Ledeb.) based on the de novo whole genome sequencing

This special issue of the journal is devoted to the outstanding population geneticist Yuri Petrovich Altukhov, who paid much attention in his research to the development of molecular genetic markers for population studies. Over the past time markers and methods of their development have undergone significant change. Thanks to modern methods of whole genome sequencing, it has become possible to develop markers of very different types—selectively neutral, as well as functional. Among them, microsatellite markers remain the most informative, convenient, reproducible, relatively inexpensive, and polymorphic. Whole genome sequencing greatly facilitates their discovery and development. This paper is devoted to the development of new microsatellite markers for a very important species of boreal forest—Siberian larch (Larix sibirica Ledeb.). Using a draft assembly of the larch genome, several thousand contigs containing microsatellite loci with di-, tri, tetra-, and pentanucleotide motifs were selected. A total of 59 pairs of PCR primers were tested for loci with dinucleotide motifs as the most variable. From them, 11 pairs were finally selected for 11 loci with dinucleotide repeats, which showed a high level of polymorphism and can be used in various population genetic studies and to identify the origin of wood and plant material. This study was done at the Laboratory of Forest Genomics of the Genome Research and Education Center of the Siberian Federal University with the support of the Department of Forest Genetics and Forest Tree Breeding of the Georg-August University of Gottingen, the Department for Monitoring of Forest Genetic Resources of the Forest Protection Center of the Krasnoyarsk Territory, and the Laboratory of Forest Genetics and Selection of the Sukachev Institute of Forest of the Siberian Branch of the Russian Academy of Sciences within the framework of the project “Genomics of the Key Boreal Forest Conifer Species and Their Major Phytopathogens in the Russian Federation” funded by the Government of the Russian Federation (grant no. 14.Y26.31.0004).

Development of New Microsatellite Markers for Salvia officinalis L. and Its Potential Use in Conservation-Genetic Studies of Narrow Endemic Salvia brachyodon Vandas

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (HE) was compared to the expected genetic diversity at mutation-drift equilibrium (HEQ) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

Development of new microsatellite markers derived from expressed sequence tags for the great scallop (Pecten maximus)

Nine microsatellites were developed from expressed sequence tags sequences for the great scallop (Pecten maximus), and were tested in three natural populations. These markers displayed low to moderate levels of variability. On average across all populations, the number of alleles per locus ranged from 1.333 to 8.667, and the observed and expected heterozygosities ranged from 0.029 to 0.703 and 0.028 to 0.711, respectively. All loci conformed to Hardy–Weinberg equilibrium, except PmGC20 in one single population. These new loci might be very useful for the genetic monitoring of the possible impact of aquaculture on the genetic diversity of native scallop populations.

A panel of polymorphic microsatellite markers in Himalayan monal Lophophorus impejanus developed by cross-species amplification and their applicability in other Galliformes

Isolation and development of new microsatellite markers for any species is still labour-intensive and requires substantial inputs of time, money and expertise. Therefore, cross-species microsatellite amplification can be an effec- tive way in obtaining microsatellite loci for closely related taxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellite markers developed for chicken were cross-amplified in Himalayan monal. All the tested 15 microsatellite markers were polymorphic, with mean (± s.e.) allelic number of 4± 1.51, ranging 2-7 per locus. The observed heterozygosity in the population ranged between 0.285 and 0.714, with mean (± s.e.) of 0.499±0.125, indicating considerable genetic variation in this population. While 12 loci conformed to Hardy-Weinberg equilibrium (P >0.05), 3l oci, i.e. MCW0295, MCW0081, MCW0330 deviated from it (P< 0.05). No evidence for linkage disequilibrium was observed among pair of loci. Our study show that these 15 micro- satellites loci could be employed in population genetic studies for Himalayan monal and their applicability in Jungle Bush Quail, Grey francolin and Kalij pheasant.

Development of new microsatellite markers from an enriched genomic library of date palm (Phoenix dactylifera L.)

SummaryDate palm (Phoenix dactylifera L) has been cultivated since early times. The high nutritional, economic, and social values of date palm culture are well known. In this study, we report on the isolation and characterisation of nine novel polymorphic nuclear microsatellite loci from a repeat-enriched genomic library of P. dactylifera L. The genetic diversity of these loci was estimated in 50 accessions of P. dactylifera L. The number of alleles per locus ranged from two-to-four, with an average of 2.5 alleles per locus. The observed (Ho) and expected (He) heterozygosities ranged from 0.11 – 1.00 and from 0.10 – 0.74, respectively. These microsatellite markers could be useful for studies on the genetic diversity and conservation genetics of date palm.

Development of new microsatellite markers for molecular diversity analysis of Citrus species

SummaryThirty microsatellite loci for further genetic analysis of Citrus species were developed by constructing a microsatellite-enriched library using capture with streptavidin-coated magnetic beads and used to assess genetic diversity in 40 Citrus accessions. In total, 150 alleles were detected, with an average of five alleles per locus. The average gene diversity and polymorphism information content values were 0.58 and 0.52, and ranged from 0.35 – 0.74 and from 0.32 – 0.70, respectively. Values for the observed and expected heterozygosities ranged from 0.13 – 1.00 and from 0.36 – 0.75, respectively. Fifteen loci deviated from the Hardy-Weinberg equilibrium (P < 0.05). The mean similarity coefficient among accessions was 0.5906. Based on the UPGMA algorithm, two main groups were successfully identified. These new microsatellite markers can be used to further investigate the genetics of, and phylogenic relationships in Citrus spp.

Development of new microsatellite markers and their application in the analysis of genetic diversity in lentils

This paper reports the development of new microsatellite markers for lentil (Lens culinaris subsp. culinaris) and their use for genetic diversity analysis of a lentil core collection developed at ICARDA (Aleppo-Syria). Fourteen new markers were developed from microsatellite flanking sequences of a genomic library from a cultivated lentil accession ILL5588. The core collection used comprises 109 accessions from 15 countries representing 57 cultigens (including 18 breeding lines) from 8 countries and 52 wild types of germplasm (L. culinaris subsp. orientalis, L. culinaris subsp. tomentosus and L. culinaris subsp. odemensis) from 11 countries. Total number of alleles detected across all microsatellite loci was 182, with a mean of 13 alleles per locus. The wild accessions were rich in alleles (151 alleles) compared to cultigens (114 alleles). The genetic diversity index for the microsatellite loci in the wild accessions ranged from 0.16 (for locus SSR28 in L. culinaris subsp. odemensis) to 0.93 (for locus SSR66 in L. culinaris subsp. orientalis) with a mean of 0.66, while in the cultigens genetic diversity varied between 0.03 (locus SSR28) and 0.87 (locus SSR207) with a mean of 0.65. The cluster analysis indicated two major clusters, mainly one with the cultigens and the other with the wild accessions.

Development of new microsatellite markers from a salt‐marsh sedge Carex rugulosa by compound simple sequence repeat–polymerase chain reaction

We developed 12 polymorphic microsatellite markers from a salt-marsh sedge Carex rugulosa. The number of alleles per locus ranged from two to four, with an average of 2.75. The observed and expected heterozygosities ranged from 0.067 to 0.600 and from 0.128 to 0.620, respectively. These simple sequence repeat markers will allow the identification of genets and evaluation of the genetic diversity of C. rugulosa.

Tomato genome sequencing: deciphering the euchromatin region of the chromosome 8

As a member of the Solanaceae Genomics Network International Tomato Sequencing Project, we launched on sequencing of the chromosome 8. Our task is to sequence the euchromatin region of the chromosome, the estimated size of which is 17 megabases. BAC-by-BAC strategy is adopted for sequencing. We initially received BAC clone candidates anchored to overgo probes developed from 33 markers mapped on chromosome 8. For confirmation of the BAC candidates, we analyzed the sequence of PCR product amplified from the BAC DNA with primers designed on the marker sequence. Twenty-five BAC clones were verified and subjected to shotgun sequencing. As of Nov. 2006, we finished 40 clones to Phase 3 (total non-redundant length 4,429,168 bases) of 25 are seed and 15 are extended clones. In order to find additional seed points, we performed PCR screening of markers against 3D DNA-pool of LE-HBa, SL-MboI and SL-EcoRI BAC libraries, and succeeded in obtaining 9 new seeds. We report the current status of our project and future perspectives toward the final goal, which include development of new microsatellite markers from tomato EST sequences and Selected BAC Mixture shotgun sequencing for the gap filling.

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass, Morone saxatilis, and cross‐species amplification in white bass, Morone chrysops, and their hybrid

AbstractTo support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.

Construction of a Microsatellite Linkage Map with Two Sequenced Rice Varieties

Based on the successful development of new microsatellite markers from the data of two whole-sequenced rice varieties, japonica variety Nipponbare and indica variety 9311, an F 2 population of 90 lines, which was derived from a single cross between Nipponbare and 9311, was applied to construct a genetic linkage framework map. The map covered 2 455.7 cM of total genomic length, and consisted of 152 simple sequence repeats (SSRs) loci including 46 pairs of new SSR primers developed by our research institute. The average genetic distance between two markers was 16.16 cM. In addition, markers RM345 and RM494, which have not been mapped on the Temnykh's map et al. (2001) were anchored on the sixth chromosome of this map. We compared this research with maps of Temnykh et al.(2001) and LAN et al. (2003) regarding the aspects of type and size of population, type and quantity of markers, and the marker arrangement order on chromosome, etc. Results indicated that the similarity of marker linear alignment was 93.81% between this map and T-map, Finally, the important significance of using sequenced rice varieties to construct linkage map was also discussed.

Interspecific Utility of Microsatellites in Fish: A Case Study of (CT)(n) and (GT)(n) Markers in the Shanny Lipophrys pholis (Pisces: Blenniidae) and Their Use in Other Blennioidei.

We report the development of new microsatellite markers that can be used for population analyses in the shanny Lipophrys pholis. The procedure involved the construction of a microsatellite-enriched genomic bank. Five (GT)(n) and (CT)(n) microsatellites have been characterized, four of which are polymorphic. The analysis of one population allowed us to verify their usefulness as markers in population studies. Moreover, interspecific amplifications have been performed using primers defined in other species to amplify Lipophrys pholis, or using the primers defined in Lipophrys pholis to amplify other species. We use these results to discuss the hypothesis that microsatellites are highly conserved in fish.