Cross-species Amplification Of Microsatellite Markers Research Articles

Short Communication: Cross-species amplification of microsatellite markers developed for Jatropha curcas within five species of Jatropha

Abstract. Saptadi D, Heliyanto B, Sudarsono. 2020. Short Communication: Cross-species amplification of microsatellite markers developed for Jatropha curcas within five species of Jatropha. Biodiversitas 21: 5072-5076. The transferability of SSR markers can be used to access the genetic diversity of related species. There are four close relatives of Jatropha curcas L in Indonesia, which can be utilized as a new diversity source through the interspecific crossing. This research was conducted to determine the ability of cross-species amplification of SSR markers developed from J. curcas to other Jatropha species (J. integerrima, J. multifida, J. gossypifolia, J. podagrica). It also investigated the relationship between these species. Out of 28 primers checked,11 primers showed cross-species amplification in all the species tested. Primer pairs EU099519, EU099528, and EU099525 have no transferability to other species. The overall percentage of polymorphism (PP) among all species tested was 95%, with the mean genetic similarity (GS) was 0.34. Least PP (17.35%) and highest GS (0.60) was found between pairs of J. podagrica and J. multifida. The correlation between the PP with GS was relatively high (0.75). The farthest and closest genetic distance was found between J. curcas/J. gossypifolia and J. podagrica/J. multifida, respectively. Further, selected primers from this study can be utilized in species differentiation, molecular identification of interspecific hybrids, and exploiting the genetic resource.

Identification and cross-species amplification of microsatellite markers derived from expressed sequence data of rose species

Genic SSR markers derived from public expressed sequence tags (ESTs) data are valuable and cost effective marker resources for genome mapping and diversity studies. Owing to their derivation from the transcribed regions which often have putative functions, these markers can be easily associated with desired trait. In the present study, 19 novel SSR markers were identified from 450 non redundant unigenes derived from 3,726 public ESTs of two rose species. Among SSRs, tri-repeats (61.3 %) were most abundant followed by di-repeat (29 %). Newly identified EST-SSR markers recorded significant homology with the known/putative proteins of Arabidopsis thaliana. The cross transferability to 12 rose species ranged from 63.2 to 100 %. Novel SSR loci found to be moderately to highly polymorphic with locus wise average number of alleles and polymorphism information content (PIC) were 4.1 and 0.33, respectively. Cloning and sequencing of EST-SSR size variant amplicons of marker locus Rches12 revealed that the variation in the number of SSR repeat-units was the main source of fragment polymorphism. The high polymorphic potential coupled with high cross-transferability rate demonstrates wider applicability of novel SSR markers in genetic diversity, genome mapping and evolutionary studies in various rose species.

Characterization and cross-species amplification of microsatellite markers in African Silverbill (Lonchura cantans)

We tested the cross-amplification of eight microsatellites developed for Bengalese finch in African Silverbill (Lonchura cantans). In order to develop resources for conservation genetic studies in the species L. cantans, we tested the amplification success and polymorphism in eight previously developed microsatellite loci, in L. cantans. All eight microsatellite markers were successfully amplified, of which all were polymorphic, with 3 to 9 alleles and an expected heterozygosity (HE) ranging from 0.606 to 0.718. On average, there were 5.25 alleles/locus and a mean HE of 0.6456. These eight polymorphic markers could be of potential use in studies of genetic variability, population structure, and reproductive strategy of African Silverbills. The markers tested should be useful for population and conservation genetic studies in this genus, and, in particular, for species closely related to the source species, L. cantans.

Cross-species amplification of microsatellite markers in the invasive spiny-cheek crayfish (Orconectes limosus): Assessment and application

The North American spiny-cheek crayfish, Orconectes limosus (Cambaridae), endangered in its native range, is a widespread invasive species in European waters and conservationally important carrier of crayfish plague. However, its population structure is poorly known, and no informative genetic markers for the species are available. We tested cross-species transfer of microsatellite loci to spiny-cheek crayfish from 5 other crayfish species. Variability of 10 successfully amplifying loci derived from 4 species was then tested in 60 individuals of O. limosus originating from 3 natural populations: the river Danube at Bogyiszló in Hungary, a pond in Starý Klíèov, and the brook Eernovický, both in the Czech Republic. The allele number within the populations ranged from 4 to 10 alleles per locus, while heterozygosity levels varied from 0.650 to 0.900 for H(o) and from 0.660 to 0.890 for H(e). No linkage disequilibrium and no null alleles were detected. The selected markers are useful for assessing population structure, intraspecific variation, and paternity studies in spiny-cheek crayfish.

Cross-species amplification of microsatellite markers for the brown sole in the family Pleuronectidae

Amplification of eight microsatellite loci previously developed and characterized in brown sole Pleuronectes herzensteini was attempted in 11 other flatfish species. Two loci, Phz6 and Phz12, were amplified in all the species examined. Cross-species amplification was successful in eight loci of Kareius bicoloratus and Pleuronectes yokohamae, but in only seven loci of Microstomus achne, Pleuronectes punctatissimus, and Pleuronectes schrenki. Three to five loci could be amplified in six other species. In the three species selected, K. bicoloratus, P. punctatissimus, and P. yokohamae, seven to eight cross-amplified loci exhibited polymorphisms comprising 1–22 alleles. Expected heterozygosity (He) ranged from 0.66 to 0.96 in K. bicoloratus, from 0.62 to 0.96 in P. punctatissimus, and from 0.43 to 0.91 in P. yokohamae. Observed heterozygosity (Ho) ranged from 0.65 to 1.00 in K. bicoloratus, from 0.55 to 0.95 in P. punctatissimus, and from 0.40 to 0.95 in P. yokohamae. The Phz2, Phz3, and Phz12 loci significantly deviated in certain or all three species from Hardy–Weinberg equilibrium. The mean values of homology to the flanking-region sequences of brown sole were 93.7% in K. bicoloratus, 91.2% in P. punctatissimus, and 93.9% in P. yokohamae. These results suggest that microsatellite markers for brown sole are applicable for genetic studies in flatfish species including at least these three species.

Detection of multiple paternity and sperm storage in a captive colony of the central Asian tortoise, Testudo horsfieldii

Reproductive success is a critical measure of an organism’s fitness. Determining reproductive success in vertebrates is confounded by the concealed mechanism and timing of fertilization (e.g., sperm competition and storage). To assess the relationship between observed mating behavior and reproductive success in the central Asian tortoise, Testudo horsfieldii Gray, 1844, we determined individual genotypes from a captive colony of adults and their offspring. We constructed a size-selected genomic library from T. horsfieldii and screened for polymorphic microsatellite markers. The screen resulted in identification of two novel microsatellite regions. Cross-species amplification of microsatellite markers using primers developed for the bog turtle, Glyptemys muhlenbergii (Schoepff, 1801), resulted in isolation of three additional polymorphic microsatellites for T. horsfieldii. The five loci, which have between 5 and 17 alleles and observed heterozygosities between 0.44 and 0.90, were used to determine the frequency of multiple paternity in the captive colony. We found evidence for multiple paternity in 27% of the clutches examined, as well as evidence for overwinter sperm storage and variance in adult male reproductive success. These data indicate that ample opportunity exists for sperm competition and female mate choice in T. horsfieldii.

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation.

Analysis of tetraploidElymusspecies using wheat microsatellite markers and RAPD markers

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.