Genomic evaluation for breeding and genetic management in Cordia africana, a multipurpose tropical tree species.

BACKGROUND: Planting tested forest reproductive material is crucial to ensure the increased resilience of intensively managed productive stands for timber and wood product markets under climate change scenarios. Single-step Genomic Best Linear Unbiased Prediction (ssGBLUP) analysis is a cost-effective option for using genomic tools to enhance the accuracy of predicted breeding values and genetic parameter estimation in forest tree species. Here, we tested the efficiency of ssGBLUP in a tropical multipurpose tree species, Cordia africana, by partial population genotyping. A total of 8070 trees from three breeding seedling orchards (BSOs) were phenotyped for height. We genotyped 6.1% of the phenotyped individuals with 4373 single nucleotide polymorphisms. The results of ssGBLUP were compared with pedigree-based best linear unbiased prediction (ABLUP) and genomic best linear unbiased prediction (GBLUP), based on genetic parameters, theoretical accuracy of breeding values, selection candidate ranking, genetic gain, and predictive accuracy and prediction bias. RESULTS: Genotyping a subset of the study population provided insights into the level of relatedness in BSOs, allowing better genetic management. Due to the inbreeding detected within the genotyped provenances, we estimated genetic parameters both with and without accounting for inbreeding. The ssGBLUP model showed improved performance in terms of additive genetic variance and theoretical breeding value accuracy. Similarly, ssGBLUP showed improved predictive accuracy and lower bias than the pedigree-based relationship matrix (ABLUP). CONCLUSIONS: This study of C. africana, a species in decline due to deforestation and selective logging, revealed inbreeding depression. The provenance exhibiting the highest level of inbreeding had the poorest overall performance. The use of different relationship matrices and accounting for inbreeding did not substantially affect the ranking of candidate individuals. This is the first study of this approach in a tropical multipurpose tree species, and the analysed BSOs represent the primary effort to breed C. africana.

The genome of a steinernematid-associated Pseudomonas piscis bacterium encodes the biosynthesis of insect toxins.

Several species of soil-dwelling Steinernema nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this insect-killing trait is known to be augmented by the nematodes' Xenorhabdus endosymbionts, the role of other steinernematid-associated bacterial genera in the nematode lifecycle remains unclear. This genomic study aimed to determine the potential of Pseudomonas piscis to contribute to the entomopathogenicity of its Steinernema host. Insect larvae were infected with three separate Steinernema cultures. From each of the three treatments, the prevalent bacteria in the haemocoel of cadavers, four days post-infection, were isolated. These three bacterial isolates were morphologically characterised. DNA was extracted from each of the three bacterial isolates and used for long-read genome sequencing and assembly. Assemblies were used to delineate species and identify genes that encode insect toxins, antimicrobials, and confer antibiotic resistance. We assembled three complete genomes. Through digital DNA-DNA hybridisation analyses, we ascertained that the haemocoels of insect cadavers previously infected with Steinernema sp. Kalro, Steinernema sp. 75, and Steinernema sp. 97 were dominated by Xenorhabdus griffiniae Kalro, Pseudomonas piscis 75, and X. griffiniae 97, respectively. X. griffiniae Kalro and X. griffiniae 97 formed a subspecies with other X. griffiniae symbionts of steinernematids from Kenya. P. piscis 75 phylogenetically clustered with pseudomonads that are characterised by high insecticidal activity. The P. piscis 75 genome encoded the production pathway of insect toxins such as orfamides and rhizoxins, antifungals such as pyrrolnitrin and pyoluteorin, and the broad-spectrum antimicrobial 2,4-diacetylphloroglucinol. The P. piscis 75 genome encoded resistance to over ten classes of antibiotics, including cationic lipopeptides. Steinernematid-associated P. piscis bacteria hence have the biosynthetic potential to contribute to nematode entomopathogenicity.

The Gynandropsis gynandra genome provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae.

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to C3 Arabidopsis (Arabidopsis thaliana). Here, we present the genome sequence of G. gynandra, anchored onto 17 main pseudomolecules with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. Our analyses revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-α), then an addition of a third genome (Th-α, +1×) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the polyploidy history, gene duplication and retention, as well as their impact on the evolution of C4 photosynthesis in Cleomaceae.

Chromosome evolution and the genetic basis of agronomically important traits in greater yam.

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

'Systems approach' plant breeding illustrated by trees.

The linkage in new and creative ways of existing plant breeding methods responsive to different global trends and values provides a 'systems approach' to address a broad set of global production challenges more effectively. Here, we illustrate such an approach through its application to trees, chosen because of their extensive diversity in features, uses, users, production contexts, and domestication pathways. We coin the resulting strategy 'tree diversity breeding' and consider it with reference to trends and values related to participation, environment, biotechnology, and markets as examples. Features of the approach for trees are applicable to plant breeding more widely, as we seek to address complex problems through strategic biodiversity use.

Genomic Resources to Guide Improvement of the Shea Tree.

A defining component of agroforestry parklands across Sahelo-Sudanian Africa (SSA), the shea tree (Vitellaria paradoxa) is central to sustaining local livelihoods and the farming environments of rural communities. Despite its economic and cultural value, however, not to mention the ecological roles it plays as a dominant parkland species, shea remains semi-domesticated with virtually no history of systematic genetic improvement. In truth, shea's extended juvenile period makes traditional breeding approaches untenable; but the opportunity for genome-assisted breeding is immense, provided the foundational resources are available. Here we report the development and public release of such resources. Using the FALCON-Phase workflow, 162.6 Gb of long-read PacBio sequence data were assembled into a 658.7 Mbp, chromosome-scale reference genome annotated with 38,505 coding genes. Whole genome duplication (WGD) analysis based on this gene space revealed clear signatures of two ancient WGD events in shea's evolutionary past, one prior to the Astrid-Rosid divergence (116-126 Mya) and the other at the root of the order Ericales (65-90 Mya). In a first genome-wide look at the suite of fatty acid (FA) biosynthesis genes that likely govern stearin content, the primary determinant of shea butter quality, relatively high copy numbers of six key enzymes were found (KASI, KASIII, FATB, FAD2, FAD3, and FAX2), some likely originating in shea's more recent WGD event. To help translate these findings into practical tools for characterization, selection, and genome-wide association studies (GWAS), resequencing data from a shea diversity panel was used to develop a database of more than 3.5 million functionally annotated, physically anchored SNPs. Two smaller, more curated sets of suggested SNPs, one for GWAS (104,211 SNPs) and the other targeting FA biosynthesis genes (90 SNPs), are also presented. With these resources, the hope is to support national programs across the shea belt in the strategic, genome-enabled conservation and long-term improvement of the shea tree for SSA.

Draft Genomes of Two Artocarpus Plants, Jackfruit (A. heterophyllus) and Breadfruit (A. altilis).

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism.

Draft genome sequence of Solanum aethiopicum provides insights into disease resistance, drought tolerance, and the evolution of the genome.

BACKGROUND: The African eggplant (Solanum aethiopicum) is a nutritious traditional vegetable used in many African countries, including Uganda and Nigeria. It is thought to have been domesticated in Africa from its wild relative, Solanum anguivi. S. aethiopicum has been routinely used as a source of disease resistance genes for several Solanaceae crops, including Solanum melongena. A lack of genomic resources has meant that breeding of S. aethiopicum has lagged behind other vegetable crops. RESULTS: We assembled a 1.02-Gb draft genome of S. aethiopicum, which contained predominantly repetitive sequences (78.9%). We annotated 37,681 gene models, including 34,906 protein-coding genes. Expansion of disease resistance genes was observed via 2 rounds of amplification of long terminal repeat retrotransposons, which may have occurred ∼1.25 and 3.5 million years ago, respectively. By resequencing 65 S. aethiopicum and S. anguivi genotypes, 18,614,838 single-nucleotide polymorphisms were identified, of which 34,171 were located within disease resistance genes. Analysis of domestication and demographic history revealed active selection for genes involved in drought tolerance in both "Gilo" and "Shum" groups. A pan-genome of S. aethiopicum was assembled, containing 51,351 protein-coding genes; 7,069 of these genes were missing from the reference genome. CONCLUSIONS: The genome sequence of S. aethiopicum enhances our understanding of its biotic and abiotic resistance. The single-nucleotide polymorphisms identified are immediately available for use by breeders. The information provided here will accelerate selection and breeding of the African eggplant, as well as other crops within the Solanaceae family.

The role of genetics in mainstreaming the production of new and orphan crops to diversify food systems and support human nutrition.

Especially in low-income nations, new and orphan crops provide important opportunities to improve diet quality and the sustainability of food production, being rich in nutrients, capable of fitting into multiple niches in production systems, and relatively adapted to low-input conditions. The evolving space for these crops in production systems presents particular genetic improvement requirements that extensive gene pools are able to accommodate. Particular needs for genetic development identified in part with plant breeders relate to three areas of fundamental importance for addressing food production and human demographic trends and associated challenges, namely: facilitating integration into production systems; improving the processability of crop products; and reducing farm labour requirements. Here, we relate diverse involved target genes and crop development techniques. These techniques include transgressive methods that involve defining exemplar crop models for effective new and orphan crop improvement pathways. Research on new and orphan crops not only supports the genetic improvement of these crops, but they serve as important models for understanding crop evolutionary processes more broadly, guiding further major crop evolution. The bridging position of orphan crops between new and major crops provides unique opportunities for investigating genetic approaches for de novo domestications and major crop 'rewildings'.

African Orphan Crops Consortium (AOCC): status of developing genomic resources for African orphan crops.

MAIN CONCLUSION: The African Orphan Crops Consortium (AOCC) successfully initiated the ambitious genome sequencing project of 101 African orphan crops/trees with 6 genomes sequenced, 6 near completion, and 20 currently in progress. Addressing stunting, malnutrition, and hidden hunger through nutritious, economic, and resilient agri-food system is one of the major agricultural challenges of this century. As sub-Saharan Africa harbors a large portion of the severely malnourished population, the African Orphan Crops Consortium (AOCC) was established in 2011 with an aim to reduce stunting and malnutrition by providing nutritional security through improving locally adapted nutritious, but neglected, under-researched or orphan African food crops. Foods from these indigenous or naturalized crops and trees are rich in minerals, vitamins, and antioxidant, and are an integral part of the dietary portfolio and cultural, social, and economic milieu of African farmers. Through stakeholder consultations supported by the African Union, 101 African orphan and under-researched crop species were prioritized to mainstream into African agri-food systems. The AOCC, through a network of international-regional-public-private partnerships and collaborations, is generating genomic resources of three types, i.e., reference genome sequence, transcriptome sequence, and re-sequencing 100 accessions/species, using next-generation sequencing (NGS) technology. Furthermore, the University of California Davis African Plant Breeding Academy under the AOCC banner is training 150 lead African scientists to breed high yielding, nutritious, and climate-resilient (biotic and abiotic stress tolerant) crop varieties that meet African farmer and consumer needs. To date, one or more forms of sequence data have been produced for 60 crops. Reference genome sequences for six species have already been published, 6 are almost near completion, and 19 are in progress.

The draft genomes of five agriculturally important African orphan crops.

BACKGROUND: The expanding world population is expected to double the worldwide demand for food by 2050. Eighty-eight percent of countries currently face a serious burden of malnutrition, especially in Africa and south and southeast Asia. About 95% of the food energy needs of humans are fulfilled by just 30 species, of which wheat, maize, and rice provide the majority of calories. Therefore, to diversify and stabilize the global food supply, enhance agricultural productivity, and tackle malnutrition, greater use of neglected or underutilized local plants (so-called orphan crops, but also including a few plants of special significance to agriculture, agroforestry, and nutrition) could be a partial solution. RESULTS: Here, we present draft genome information for five agriculturally, biologically, medicinally, and economically important underutilized plants native to Africa: Vigna subterranea, Lablab purpureus, Faidherbia albida, Sclerocarya birrea, and Moringa oleifera. Assembled genomes range in size from 217 to 654 Mb. In V. subterranea, L. purpureus, F. albida, S. birrea, and M. oleifera, we have predicted 31,707, 20,946, 28,979, 18,937, and 18,451 protein-coding genes, respectively. By further analyzing the expansion and contraction of selected gene families, we have characterized root nodule symbiosis genes, transcription factors, and starch biosynthesis-related genes in these genomes. CONCLUSIONS: These genome data will be useful to identify and characterize agronomically important genes and understand their modes of action, enabling genomics-based, evolutionary studies, and breeding strategies to design faster, more focused, and predictable crop improvement programs.

Use of microsatellite markers for the assessment of bambara groundnut breeding system and varietal purity before genome sequencing.

Maximizing the research output from a limited investment is often the major challenge for minor and underutilized crops. However, such crops may be tolerant to biotic and abiotic stresses and are adapted to local, marginal, and low-input environments. Their development through breeding will provide an important resource for future agricultural system resilience and diversification in the context of changing climates and the need to achieve food security. The African Orphan Crops Consortium recognizes the values of genomic resources in facilitating the improvement of such crops. Prior to beginning genome sequencing there is a need for an assessment of line varietal purity and to estimate any residual heterozygosity. Here we present an example from bambara groundnut (Vigna subterranea (L.) Verdc.), an underutilized drought tolerant African legume. Two released varieties from Zimbabwe, identified as potential genotypes for whole genome sequencing (WGS), were genotyped with 20 species-specific SSR markers. The results indicate that the cultivars are actually a mix of related inbred genotypes, and the analysis allowed a strategy of single plant selection to be used to generate non-heterogeneous DNA for WGS. The markers also confirmed very low levels of heterozygosity within individual plants. The application of a pre-screen using co-dominant microsatellite markers is expected to substantially improve the genome assembly, compared to a cultivar bulking approach that could have been adopted.

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

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.

High-throughput and parallel SNP discovery in selected candidate genes in Eucalyptus camaldulensis using Illumina NGS platform.

Next generation sequencing (NGS) technologies have revolutionized the pace and scale of genomics- and transcriptomics-based SNP discovery across different plant and animal species. Herein, 72-base paired-end Illumina sequencing was employed for high-throughput, parallel and large-scale SNP discovery in 41 growth-related candidate genes in Eucalyptus camaldulensis. Approximately 100 kb of genome from 96 individuals was amplified and sequenced using a hierarchical DNA/PCR pooling strategy and assembled over corresponding E. grandis reference. A total of 1191 SNPs (minimum 5% other allele frequency) were identified with an average frequency of 1 SNP/83.9 bp, whereas in exons and introns, it was 1 SNP/108.4 bp and 1 SNP/65.6 bp, respectively. A total of 75 insertions and 89 deletions were detected of which approximately 15% were exonic. Transitions (Tr) were in excess than transversions (Tv) (Tr/Tv: 1.89), but exceeded in exons (Tr/Tv: 2.73). In exons, synonymous SNPs (Ka) prevailed over the non-synonymous SNPs (Ks; average Ka/Ks ratio: 0.72, range: 0-3.00 across genes). Many of the exonic SNPs/indels had potential to change amino acid sequence of respective genes. Transcription factors appeared more conserved, whereas enzyme coding genes appeared under relaxed control. Further, 541 SNPs were classified into 196 'equal frequency' (EF) blocks with almost similar minor allele frequencies to facilitate selection of one tag-SNP/EF-block. There were 241 (approximately 20%) 'zero-SNP' blocks with absence of SNPs in surrounding ±60 bp windows. The data thus indicated enormous extant and unexplored diversity in E. camaldulensis in the studied genes with potential applications for marker-trait associations.

Isolation and characterization of resistance gene analogues from Psilanthus species that represent wild relatives of cultivated coffee endemic to India.

Biotic or abiotic stress can cause considerable damage to crop plants that can be managed by building disease resistance in the cultivated gene pool through breeding for disease resistance genes (R-genes). R-genes, conferring resistance to diverse pathogens or pests share a high level of similarity at the DNA and protein levels in different plant species. This property of R-genes has been successfully employed to isolate putative resistance gene analogues (RGAs) using a PCR-based approach from new plant sources. Using a similar approach, in the present study, we have successfully amplified putative RGAs having nucleotide-binding-site leucine-rich repeats (NBS-LRR-type RGAs) from seven different sources: two cultivated coffee species (Coffea arabica L. and Coffea canephora Pierre ex. A. Froehner), four related taxa endemic to India (wild tree coffee species: Psilanthus bengalensis (Roem. & Schuttles) J.-F. Leroy, Psilanthus khasiana , Psilanthus travencorensis (Wight & Arn.) J.-F. Leroy, Psilanthus weightiana (Wall. ex Wight & Arn.) J.-F. Leroy), and a cDNA pool originally prepared from light- and drought-stressed Coffea arabica L. leaves. The total PCR amplicons obtained using NBS-LRR-specific primers from each source were cloned and transformed to construct seven independent libraries, from which 434 randomly picked clones were sequenced. In silico analysis of the sequenced clones revealed 27 sequences that contained characteristic RGA motifs, of which 24 had complete uninterrupted open reading frames. Comparisons of these with published RGAs showed several of these to be novel RGA sequences. Interestingly, most of such novel RGAs belonged to the related wild Psilanthus species. The data thus suggest the potential of the secondary gene pool as possible untapped donors of resistance genes to the present day cultivated species of coffee.

Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies.

BACKGROUND: Species-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. RESULTS: A 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. CONCLUSION: The 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.

Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species.

Genic microsatellites or EST-SSRs derived from expressed sequence tags (ESTs) are desired because these are inexpensive to develop, represent transcribed genes, and often a putative function can be assigned to them. In this study we investigated 2,553 coffee ESTs (461 from the public domain and 2,092 in-house generated ESTs) for identification and development of genic microsatellite markers. Of these, 2,458 ESTs (all >100 bp in size) were searched for SSRs using MISA--search module followed by stackPACK clustering that revealed a total of 425 microsatellites in 331 (13.5%) non-redundant ESTs/consensus sequences suggesting an approximate frequency of 1 SSR/2.16 kb of the analysed coffee transcriptome. Identified microsatellites mainly comprised of di-/tri-nucleotide repeats, of which repeat motifs AG and AAG were the most abundant. A total of 224 primer pairs could be designed from the non-redundant SSR-positive ESTs (excluding those with only mononucleotide repeats) for possible use as potential genic markers. Of this set, a total of 24 (10%) primer pairs were tested and 18 could be validated as usable markers. Sixteen of these markers revealed moderate to high polymorphism information content (PIC) across 23 genotypes of C. arabica and C. canephora, while 2 markers were found to be monomorphic. All the markers also showed robust cross-species amplifications across 14 Coffea and 4 Psilanthus species. The apparent broad cross-species/genera transferability was further confirmed by cloning and sequencing of the amplified alleles. Thus, the study provides an insight about the frequency and distribution of SSRs in coffee transcriptome, and also demonstrates the successful development of genic-SSRs. It is expected that the potential markers described here would add to the repertoire of DNA markers needed for genetic studies in cultivated coffee and also related taxa that constitute the important secondary genepool for coffee improvement.