cpSSR Primers Research Articles

Chloroplast microsatellite diversity of Pinus brutia Ten. and Pinus halepensis Mill. populations across the Mediterranean basin: Inferences of their distributions

Aim of study: To characterize and compare the genetic resources and gain some insights into the evolutionary history of Aleppo pine (Pinus halepensis Mill.) and Brutia pine (Pinus brutia Ten.) species which are both distributed across more than 8 million hectares of area in the Mediterranean Basin. Study area: Fifty-six populations from eight Mediterranean basin countries where P. halepensis and P. brutia species are located. Materials and methods: We analyzed 1344 seeds belonging to 56 populations using five cpSSR primers (Pt15169, Pt30204, Pt41093, Pt87268, and Pt110048). Main results: The analysis of molecular variance (AMOVA) revealed that the genetic diversity among the Brutia pine populations was slightly higher than that of Aleppo pine (27.06% and 24.27%, respectively). The Aleppo pine populations separately displayed a clear east-west differentiation across the Mediterranean Basin, confirming previous results using other markers. Although the Brutia pine populations showed no spatial genetic pattern, geographically close populations and/or populations from their continual distribution range were genetically closer than the fragmented and/or ecologically marginal populations. Research highlights: The seven Aleppo pine populations from the eastern range (Türkiye, Greece, and Italy) were more than two-fold diverse than the 13 populations from the western range (Spain and Morocco). The eastern range of Aleppo pine and Brutia pine populations had similar levels of genetic diversity parameters. These results suggested that the Eastern Mediterranean Basin is a possible genetic diversity center for the two pine species.

Development and application of chloroplast molecular markers in Panax notoginseng

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.

Genetic Polymorphism of the Wild and In vitro Regenerated Plants of the Medicinal Grass Cymbopogon schoenanthus subsp. proximus

Cymbopogon schoenanthus subsp. proximus grows wild in subtropical Africa, Sudan and Egypt. The species is heavily collected for its use in folk medicine and drug production. A wild population from south Egypt was used to determine preliminary genetic polymorphism within the species, using nineteen ISSR, fourteen RAPD and seven cpSSR primers. Three regeneration systems, somatic embryogenesis (SE), direct organogenesis (D), and indirect organogenesis (ID), were established from seed explants of the same population and polymorphism within regenerated plants was determined. ISSR generated a total of 222 amplified fragments for all genotypes, while RAPDs and cpSSR yielded 139 and 34 fragments, respectively. Wild plants showed an average low polymorphism for all marker types of 45.8%. Regenerated plants polymorphism was also low (SE=44.6, D=44, ID=46.2%). ISSR and cpSSR markers were more sensitive in elucidating polymorphism (51.5 and 46.87%) than RAPD (37.85%). ISSR was the most significant marker in producing unique bands, for the wild genotypes (6), SE (7), D (5) and ID (6). cpSSR followed producing 4 for wild genotypes, 6 for SE, 3 for D and 6 for ID organogenesis. Unweighted pair group with arithmetic average (UPGMA) clustering analysis and Jaccard’s similarity data suggested that wild plants and those regenerated through somatic embryogenesis and direct organogenesis are more similar. The study elucidated low polymorphism within both the wild population and regenerated genotypes, with plants regenerated through somatic embryogenesis and direct organogenesis being more similar to wild plant genotypes, suggesting their future use in studies with genetic transformation and ex-situ conservation of the species.

Chloroplast based genetic diversity among Chinese grapes genotypes

Chloroplast simple sequence repeat (cpSSR) markers previously reported were used to analyze chloroplast diversity of Chinese grapes. Nine cpSSR primer pairs from the chloroplast genomes of citrus and tobacco (Nicotiana tabacum L.) were found useful for analyzing the grapes chloroplast genome (cpDNA). Selected cpSSR primer pairs showed polymorphism among 64 varieties, with polymorphism information content (PIC) values ranging from 0.522 to 0.659, with an average of 0.590. Results depicted that a total number of 43 alleles were detected on nine loci with an average of 4.77 alleles per locus with highest number of six alleles for marker ARCP2 and CCMP10. Gene diversity explained by these cpSSR makers ranged from 4 to 68% with an average of 63.66% per marker. The genetic relationship suggested by UPGMA based dendrogram divided the genotypes into three main clusters and four subgroups, results showed that 2B is the biggest group. Overall Hong Ma Ge and Da LI Mei Gui Xiung 98% similar, while 64-1-19 was the most diverse as compared to rest of genotypes.

Development and Characterization of Chloroplast Simple Sequence Repeat markers in Pinus koraiensis

Novel cpSSR primers were developed based on the sequence information of the Pinus koraiensis chloroplast genome. A total of 30 cpSSR loci were detected in the chloroplast genome, and a total of 30 primer sets flanking those loci were designed. All primer sets were successfully amplified for chloroplast DNA in P. koraiensis. The cross-species transferability of the 30 primer sets was considerably high in P. pumila (100%) and P. paviflora (97%) belonging to the same Subgenus (Strobus) of P. koraiensis. Meanwhile, the transferability was relatively low (73%) in P. densiflora and P. sylvestris belonging to Subgenus Pinus. A total of 13 cpSSR loci out of the 30 loci were polymorphic in the Mt. Jumbong population of P. koraiensis. The mean of haploid diversity(H) was 0.512. The number of haplotypes(N) and the haplotype diversity() were 25 and 0.992, respectively. Of the 25 haplotypes, 22 were unique in the analyzed population. The unique haplotypes differentiated 22 individuals (79%) from the total of 28 individuals. In conclusion, the novel cpSSR primers developed in this study would be applicable to other Pinus species, especially the subgenus Strobus, and provide a high level of polymorphism for the study of genetic variation of P. koraiensis.

Cytoplasmic diversity of Brassica napus L., Brassica oleracea L. and Brassica rapa L. as determined by chloroplast microsatellite markers

Cytoplasmic genomes in most angiosperms are known to be maternally inherited. Oilseed rape (Brassica napus L.) as a natural amphidiploid species hence may carry the B. oleracea L. or the B. rapa L. cytoplasm, depending on the cross direction. The presence of either the B. oleracea or the B. rapa cytoplasm in oilseed rape has been reported to affect agronomically important traits. However, to date little is known about the cytoplasmic composition and genetic diversity of current winter oilseed rape cultivars and breeding material. The aim of this study was to assess the usefulness of 40 previously published chloroplast cpSSR markers from Brassica species and Arabidopsis thaliana (L.) Heynh. for distinguishing the cytoplasms of 49 different genotypes of B. napus and its diploid ancestor species. Results showed that only 14 out of the 40 tested primer combinations were suitable to distinguish the cytoplasms of a test set of 8 Brassica genotypes. With the 14 primer pairs 64 different cpSSR alleles were identified in the set of 49 genotypes. Cluster analysis indicated distinct groups for the cytoplasms of B. napus, B. rapa, and B. oleracea. However, an unambiguous identification and classification of the cytoplasm types was not possible in all cases with the available polymorphic set of cpSSR primer pairs.

Chloroplast Microsatellite Diversity Among and Within Prunus mahaleb L. and P. avium L. Species

Genetic diversity of 58 Mahaleb cherry (Prunus mahaleb) genotypes and six sweet cherry (P. avium) accessions was studied using 25 cpSSR primer pairs. Thirteen out of them demonstrated two to four alleles with an average of 2.46 alleles per primer pair. The mean of PIC value for the primers was 0.32. The average values of expected heterozygosity (He) and Shannon’s information index (I) for all loci were 0.35 and 0.55, respectively. The dendrogram based on cpSSR markers has been illustrated by MEGA4 software with Maximum Composite likelihood model and the Neighbor-joining method, which clustered the genotypes into four groups. Sum of first three principal components analysis (PCAs) could be represented most of (63.07%) the total variation in the original dimensions and confirmed the results of cluster analysis. Based on the AMOVA results, the allele numbers among groups and species studied in this research were more than those observed within them. In this study cpSSR markers provided a good tool for assessment of genetic diversity among and within mahaleb cherry and sweet cherry genotypes.

Short Note: Cross-Species Amplification and Characterization ofPinusChloroplast Microsatellite Markers inCedrus deodaraRoxb.

AbstractThe study reports the transferability of chloroplast microsatellite markers developed forPinusspecies toCedrus deodara. A total of 49 primer pairs (both nuclear and chloroplast) of Pinus species were tested inC. deodaraout of which 21 chloroplast primers showed positive amplification and 20 were found polymorphic. The primers were screened on 100 adult trees of two natural populations ofC. deodara. Using twenty cpSSR primers, a total of 64 variants were found which combined in 70 different haplotypes. The total haplotype diversity in two populations was 0.860 and 0.876 with a mean of 0.868. These sets of markers can further be used for population genetic studies and characterization inC. deodarafor which no cpSSR markers have been reported till date.

Population genetic structure and diversity of high value vulnerable medicinal plant Acorus calamus in India using RAPD and chloroplast microsatellite markers

Acorus calamus is a highly valued medicinal plant with global distribution used in several drugs of health care systems. We evaluated the genetic diversity and population structure of 50 populations of A. calamus from different geographical regions in India through RAPD and chloroplast microsatellite markers. From the total screened 82 RAPD primers and 18 cpSSR primers, 10 RAPD and nine cpSSRs were found polymorphic. The selected 10 RAPD primers produced a total of 96 reproducible bands, out of which 65 were polymorphic (67.70%). Whereas, the selected nine cpSSR markers produced 26 alleles and all of them were polymorphic. The mean genetic diversity (HT) among populations using RAPD (HT = 0.263) and cpSSR (HT = 0.530) markers was higher in comparison to the mean genetic diversity within populations. Mean coefficient of gene differentiation (GST) between the populations was also high for both RAPD (GST = 0.830) and cpSSR markers (GST = 0.735), whereas the estimated gene flow was very low for RAPD (Nm = 0.102) and for cpSSR (Nm = 0.179). AMOVA analysis revealed that more genetic variation resided among the populations than within populations. Significant differences (p < 0.001) were observed between the populations and individuals within the populations. Cluster analysis of RAPD and cpSSR data using UPGMA algorithm based on Nei’s genetic similarity matrix placed the 50 populations into two main clusters. The implication of the results of this study in devising strategy for conservation of A. calamus is discussed.

Study on Mating System of &lt;i&gt;Pinus koraiensis&lt;/i&gt; in Natural Population Based on cpSSR Technology

Chloroplast simple sequence repeat (cpSSR) technique was firstly used to study mating system of natural Pinus koraiensis population. Nine pairs of primers with clear spectrum bands, high stability and polymorphism were selected from 70 pairs of cpSSR primers to analyze the mating systems of 28 individuals. 14 polymorphic loci were detected in the nine pairs of primers. The multi-locus (tm) and single-locus (ts) outcrossing rate were 0.966 (SD=0.000) and 0.939 (SD=0.000), respectively, which were slightly higher than those of other tree species. The constant index (F=-0.035) was below zero, indicating the occurrence of excessive heterozygote. Moreover, inbreeding index (tm-ts=0.027; SD=0.000) was close to zero, indicating an insignificant inbreeding. MLDT analysis showed that multi-locus outcrossing rate differed from single-locus outcrossing rate in mating systems of 28 individuals, but the differences were not statistically significant.

A novel set of highly polymorphic chloroplast microsatellite and ISSR markers for the biofuel crop Jatropha curcas

A novel set of polymorphic ISSR and chloroplast microsatellite markers were identified to mark the genetic relationship among different accessions of Jatropha curcas. A total of 12 polymorphic ISSR primers used, amplified a total of 26 fragments among ten different accessions of J. curcas. The cross-species amplification of microsatellite loci is timesaving as well as a cost-effective approach for developing locus specific markers for new species. Further, in an attempt to reveal a high degree of polymorphism and genetic variation among different accessions of J. curcas chloroplast microsatellite primer pairs developed for Acorus calamus (Acoraceace) were examined for cross-species amplification and validation among them. Out of 18 cpSSRs, six (33.33%) were identified as resulting in good amplifications and polymorphism. The number of amplified alleles per cpSSR primer pair ranged from 2 to 4 with the total number of 18 size variants as whole. The 18 size variants at 6 loci among 10 accessions were combined in 4 different haplotypes. The total chloroplast DNA diversity (HT) and the differentiation (GST) were found as 0.700 and 0.560 respectively. Whereas, Shannon's information index (I), Nei's genetic diversity among different accessions of J. curcas using ISSR and cpSSR markers was estimated at 0.33±0.28; 0.22±0.19 and 0.94±0.35; 0.55±0.18 respectively. The genetic relationship among the accessions was established using UPGMA cluster analysis, which were grouped into two major clusters denoted as cluster-I and cluster-II. The implication of the results in devising the importance of a identified novel set of markers for the genetic relationship and genetic diversity analysis in J. curcas is discussed.

Polymorphic chloroplast microsatellite markers in the octoploid Lepidium meyenii (Brassicaceae) and cross‐species amplification in Lepidium

As a crop and medicinal plant, the octoploid Andean endemic Lepidium meyenii suffers from taxonomic uncertainty. Few molecular markers are available to genotype individuals or track gene flow in wild and cultivated material. • Using available sequence data, eight cpSSR primer pairs were developed for L. meyenii. Levels of polymorphism checked in 56 individual L. meyenii, including cultivated and wild material, revealed that the number of alleles per locus ranged from three to five, and intrapopulation allele frequencies ranged from 0.071 to 1.0. Polymerase-chain-reaction screens using our cpSSR primers in 27 other Lepidium species and three Coronopus species suggested a high degree of interspecific amplification. • These polymorphic cpSSR markers should prove useful in characterizing genetic variation among cultivated and wild L. meyenii. Additionally, interspecific amplifications suggest that these markers will be useful for the study of related taxa.

A PRELIMINARY STUDY ON CHLOROPLAST SIMPLE SEQUENCE REPEATS IN CASTANEA

Ten previously reported chloroplast simple sequence repeats (cpSSRs) primers from Nicotiana tabacum and Arabidopsis thaliana were used to analyze the chloroplast DNA variabilities in three main Castanea species, i.e. C. mollissima, C. crenata and C. henryi. The results showed eight cpSSR primers revealed specific amplification bands, three of which produced polymorphic bands. Eight polymorphic alleles were generated using those three primers, with an average of 2.67 alleles per primer. The general universality of cpSSR primers was further discussed.

Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species

Chloroplast microsatellites, or simple sequence repeats (cpSSRs), are typically mononucleotide tandem repeats. When located in the noncoding regions of the chloroplast genome (cpDNA), they commonly show intraspecific variation in repeat number. Despite the growing number of studies applying cpSSRs, studies of economically important plants and their relatives remain over-represented. Thus, the potential of cpSSRs to offer unique insights into ecological and evolutionary processes in wild plant species has yet to be fully realized. This review provides an overview of the technical resources available to aid cpSSR discovery including a list of cpSSR primer sets available and cpDNA sequencing resources. Our updated analysis of 99 whole chloroplast genomes downloaded from GenBank confirms that potentially variable cpSSRs are abundant in the noncoding cpDNA of plants. Overall variation in the frequency of cpSSRs was extreme, ranging from one to 700 per genome (median=93), while in 81 vascular plants, between 35 and 160 cpSSRs were detected per genome (median=86). We offer five recommendations to aid wider development and application of cpSSRs: (i) When genus-specific cpSSR primers are available, cross-species amplification can often be fruitful. (ii) While potentially useful, universal cpSSR primers at best provide access to only a small number of variable markers. (iii) De novo sequencing of noncoding cpDNA is the most effective and efficient way to develop cpSSR markers in wild species. (iv) DNA sequencing of cpSSR alleles is essential, given the complex nature of the genetic variation associated with hypervariable cpDNA regions. (v) The reliability of cpSSR length based genetic assays need to be validated in all studies.

Development of specific primers for cpSSR analysis in caper, olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.

Universal chloroplast primer pairs for Simple Sequence Repeat analysis in diverse genera of fruit crops

To provide an alternative method to investigate chloroplast genome (cpDNA) diversity in sub-tropical and tropical fruit crops, where cpDNA sequences are unknown, a universal set of chloroplast simple sequence repeat (cpSSR) primers were developed. Thirty-three cpSSR primers from rice (Oryza sativa), pine (Pinus thunbergii), tobacco (Nicotiana tabacum) and Arabidopsis thaliana were applied to 45 fruit species (or varieties) belonging to 27 genera in 20 families. After being screened, the utility of these primers was verified. Experiments revealed that nine out of 14 primer pairs from N. tabacum, and seven out of 11 from A. thaliana, specifically amplified bands in cpDNA from most species tested, and polymorphisms were observed. While four out of the five primers from O. sativa generated intensive bands in agarose gels, but only a smear in polyacrylamide gels, all cpSSR primers from P. thunbergii failed to generate any specific amplified products in polyacrylamide gels. The results illustrate the feasibility of exploring diversity among unknown cpDNA sequences in exotic minor fruit crops by PCR methods.

A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and used to analyze chloroplast diversity of Citrus and closely related genera. Fourteen cpSSR primer pairs from the chloroplast genomes of tobacco (Nicotiana tabacum L.) and Arabidopsis were found useful for analyzing the Citrus chloroplast genome (cpDNA) and recoded with the prefix SPCC (SSR Primers for Citrus Chloroplast). Eleven of the 14 primer pairs revealed some degree of polymorphism among 34 genotypes of Citrus, Fortunella, Poncirus and some of their hybrids, with polymorphism information content (PIC) values ranging from 0.057 to 0.732, and 18 haplotypes were identified. The cpSSR data were analyzed with NTSYS-pc software, and the genetic relationships suggested by the unweighted pair group method based on arithmetic means (UPGMA) dendrogram were congruent with previous taxonomic investigations: the results showed that all samples fell into seven major clusters, i.e., Citrus medica L., Poncirus, Fortunella, C. ichangensis Blanco, C. reticulata Swingle, C. aurantifolia (Christm.) Swingle and C. grandis (L.) Osbeck. The results of previous studies combined with our cpSSR analyses revealed that: (1) Calamondin (C. madurensis Swingle) is the result of hybridization between kumquat (Fortunella) and mandarin (C. reticulata), where kumquat acted as the female parent; (2) Ichang papeda (C. ichangensis) has a unique taxonomic status; and (3) although Bendiguangju mandarin (C. reticulata) and Satsuma mandarin (C. reticulata) are similar in fruit shape and leaf morphology, they have different maternal parents. Bendiguangju mandarin has the same cytoplasm as sweet orange (C. sinensis), whereas Satsuma mandarin has the cytoplasm of C. reticulata. Seventeen PCR products from SPCC1 and 21 from SPCC11 were cloned and sequenced. The results revealed that mononucleotide repeats as well as insertions and deletions of small segments of DNA were associated with SPCC1 polymorphism, whereas polymorphism generated by SPCC11 was essentially due to the variation in length of the mononucleotide repeats.

Diversity and genetic structure in populations of Pseudotsuga menziesii (Pinaceae) at chloroplast microsatellite loci.

Genetic variation was compared between uniparentally-inherited (chloroplast simple sequence repeats, cpSSRs) vs. biparentally-inherited (isozyme and random amplified polymorphic DNA, RAPD) genetic markers in Douglas-fir (Pseudotsuga mensiezii) from British Columbia. Three-hundred twenty-three individuals from 11 populations were assayed. In Douglas-fir, the cpSSR primer sites were well-conserved relative to Pinus thunbergii (11 of 17 loci clearly amplified), but only 3 loci were appreciably polymorphic. At these cpSSR loci, we found an unexpectedly low level of polymorphism within populations, and no genetic differentiation among populations. By contrast, the nuclear markers showed variation typical of conifers, with significant among-population differentiation. This difference is likely the outcome of both historical factors and high pollen dispersal.