Dataset Information

Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner).

ABSTRACT: Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic-/genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite markers (EST-SSRs) were developed using the publicly available dataset of 13,175 unigene ESTs, which showed a distribution of 1 SSR/3.4 kb of coffee transcriptome. Genomic SSRs, on the other hand, were developed from an SSR-enriched small-insert partial genomic library of robusta coffee. In total, 69 new SSRs (44 EST-SSRs and 25 genomic SSRs) were developed and validated as suitable genetic markers. Diversity analysis of selected coffee genotypes revealed these to be highly informative in terms of allelic diversity and PIC values, and eighteen of these markers (? 27%) could be mapped on a robusta linkage map. Notably, the markers described here also revealed a very high cross-species transferability. In addition to the validated markers, we have also designed primer pairs for 270 putative EST-SSRs, which are expected to provide another ca. 200 useful genetic markers considering the high success rate (88%) of marker conversion of similar pairs tested/validated in this study.

SUBMITTER: Hendre PS

PROVIDER: S-EPMC4252042 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner).

Hendre Prasad S PS Aggarwal Ramesh K RK

PloS one 20141202 12

Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic-/genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite mark ...[more]

PMID: 25461752

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Project description:BackgroundSpecies-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa.ResultsA small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee.ConclusionThe conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.

Project description:Wild genetic resources and their ability to adapt to environmental change are critically important in light of the projected climate change, while constituting the foundation of agricultural sustainability. To address the expected negative effects of climate change on Robusta coffee trees (Coffea canephora), collecting missions were conducted to explore its current native distribution in Uganda over a broad climatic range. Wild material from seven forests could thus be collected. We used 19 microsatellite (SSR) markers to assess genetic diversity and structure of this material as well as material from two ex-situ collections and a feral population. The Ugandan C. canephora diversity was then positioned relative to the species' global diversity structure. Twenty-two climatic variables were used to explore variations in climatic zones across the sampled forests. Overall, Uganda's native C. canephora diversity differs from other known genetic groups of this species. In northwestern (NW) Uganda, four distinct genetic clusters were distinguished being from Zoka, Budongo, Itwara and Kibale forests A large southern-central (SC) cluster included Malabigambo, Mabira, and Kalangala forest accessions, as well as feral and cultivated accessions, suggesting similarity in genetic origin and strong gene flow between wild and cultivated compartments. We also confirmed the introduction of Congolese varieties into the SC region where most Robusta coffee production takes place. Identified populations occurred in divergent environmental conditions and 12 environmental variables significantly explained 16.3% of the total allelic variation across populations. The substantial genetic variation within and between Ugandan populations with different climatic envelopes might contain adaptive diversity to cope with climate change. The accessions that we collected have substantially enriched the diversity hosted in the Ugandan collections and thus contribute to ex situ conservation of this vital genetic resource. However, there is an urgent need to develop strategies to enhance complementary in-situ conservation of Coffea canephora in native forests in northwestern Uganda.

Project description:BackgroundCoffea canephora is the commonly cultivated coffee species in the world along with Coffea arabica. Different pests and pathogens affect the production and quality of the coffee. Jasmonic acid (JA) is a plant hormone which plays an important role in plants growth, development, and defense mechanisms, particularly against insect pests. The key enzymes involved in the production of JA are lipoxygenase, allene oxide synthase, allene oxide cyclase, and 12-oxo-phytodienoic reductase. There is no report on the genes involved in JA pathway in coffee plants.ObjectiveWe made an attempt to identify and analyze the genes coding for these enzymes in C. canephora.Materials and methodsFirst, protein sequences of jasmonate pathway genes from model plant Arabidopsis thaliana were identified in the National Center for Biotechnology Information (NCBI) database. These protein sequences were used to search the web-based database Coffee Genome Hub to identify homologous protein sequences in C. canephora genome using Basic Local Alignment Search Tool (BLAST).ResultsHomologous protein sequences for key genes were identified in the C. canephora genome database. Protein sequences of the top matches were in turn used to search in NCBI database using BLAST tool to confirm the identity of the selected proteins and to identify closely related genes in species. The protein sequences from C. canephora database and the top matches in NCBI were aligned, and phylogenetic trees were constructed using MEGA6 software and identified the genetic distance of the respective genes. The study identified the four key genes of JA pathway in C. canephora, confirming the conserved nature of the pathway in coffee. The study expected to be useful to further explore the defense mechanisms of coffee plants.ConclusionJA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC, and OPR are identified in C. canephora (robusta coffee) by bioinformatic approaches confirming the conserved nature of the pathway in coffee. The findings are useful to understand the defense mechanisms of C. canephora and coffee breeding in the long run.SummaryJA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC and OPR were identified and analyzed in C. canephora (robusta coffee) by in silico approach. The study has confirmed the conserved nature of JA pathway in coffee; the findings are useful to further explore the defense mechanisms of coffee plants. Abbreviations used:C. canephora: Coffea canephora; C. arabica: Coffea arabica; JA: Jasmonic acid; CGH: Coffee Genome Hub; NCBI: National Centre for Biotechnology Information; BLAST: Basic Local Alignment Search Tool; A. thaliana: Arabidopsis thaliana; LOX: Lipoxygenase, AOS: Allene oxide synthase; AOC: Allene oxide cyclase; OPR: 12 oxo phytodienoic reductase.

Project description:Objective This study was aimed to investigate RGCBE extract as antioxidant and anti-peri-implantitis bacteria through in vitro study and its potential as antioxidant, antibacterial, anti-inflammatory, antibone resorption, and proosteogenic through in silico study.MaterialsAND METHODS: Absorption, distribution, metabolism, excretion and toxicity prediction, molecular docking simulation, and visualization of chlorogenic acid (CGA) and coumaric acid (CA) as anti-inflammatory, antioxidant, and antibacterial were investigated in silico. Inhibition zone by diffusion method, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of RGCBE extract against Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), and Prevotella intermedia (Pi) were done.Statistical analysisthe analysis of variance (ANOVA) difference test, and the post-hoc Tukey's Honest Significant Different (HSD) with a different significance value of p<0.05 RESULTS: GCA and CA compounds are good drug molecules and it has low toxicity. Chlorogenic acid have higher binding activity than coumaric acid to tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, receptor activation NF-κB (RANK) and its ligand (RANKL), interleukin (IL)-6, IL-10, runt related transcription factor (RUNX2), receptor activator nuclear Kappa beta Ligand-osteoprotegrin osteocalcin (RANKL-OPG), osteocalcin, nuclear factor associated T-cell 1 (NFATc1), tartate resistant acid phosphatase (TRAP), peptidoglycan, flagellin, dectin, Hsp70, and Hsp10 protein. RGCB ethanol extract has high antioxidant ability and it has MIC, MBC, and inhibit the growth of Aa, Pg, Fn, and Pi at 50% concentration with significantly different (p=0.0001 and<0.05).Conclusion RGCB ethanol extract has high antioxidant ability and 50% RGCB ethanol extract may act as strong anti-peri-implantitis bacteria in vitro. In addition, CGA in RGCB potential as antioxidant, antibacterial, anti-inflammatory, antibone resorption, and proosteogenic in silico.

Dataset Information

Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner).

Publications

Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner).

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