Johannes M. M. Engels

Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA

PREMISE OF STUDY To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). METHODS We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. KEY RESULTS All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. CONCLUSIONS Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi‐domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re‐arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH‐psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.

Origins of food crops connect countries worldwide

Research into the origins of food plants has led to the recognition that specific geographical regions around the world have been of particular importance to the development of agricultural crops. Yet the relative contributions of these different regions in the context of current food systems have not been quantified. Here we determine the origins (‘primary regions of diversity’) of the crops comprising the food supplies and agricultural production of countries worldwide. We estimate the degree to which countries use crops from regions of diversity other than their own (‘foreign crops’), and quantify changes in this usage over the past 50 years. Countries are highly interconnected with regard to primary regions of diversity of the crops they cultivate and/or consume. Foreign crops are extensively used in food supplies (68.7% of national food supplies as a global mean are derived from foreign crops) and production systems (69.3% of crops grown are foreign). Foreign crop usage has increased significantly over the past 50 years, including in countries with high indigenous crop diversity. The results provide a novel perspective on the ongoing globalization of food systems worldwide, and bolster evidence for the importance of international collaboration on genetic resource conservation and exchange.

Complex origin of Trinitario-type Theobroma cacao (Malvaceae) from Trinidad and Tobago revealed using plastid genomics

Trinidad and Tobago has a long history of producing high-quality cacao (Theobroma cacao L.). Cacao genotypes in Trinidad and Tobago are of a highly distinctive kind, the so-called “Trinitario” cultivar group, widely considered to be of elite quality. The origin of Trinitario cacao is unclear, although it is generally considered to be of hybrid origin. We used massive parallel sequencing to identify polymorphic plastidic single nucleotide polymorphisms (cpSNPs) and polymorphic plastidic simple sequence repeats (cpSSRs) in order to determine the origin of the Trinitario cultivar group by comparing patterns of polymorphism to a reference set of ten completely sequenced chloroplast genomes (nine T. cacao and one outgroup, T. grandiflorum (Willd. ex Spreng.) Schum). Only three cpSNP haplotypes were present in the Trinitario cultivars sampled, each highly distinctive and corresponding to reference genotypes for the Criollo (CRI), Upper Amazon Forastero (UAF) and Lower Amazon Forastero (LAF) varietal groups. These three cpSNP haplotypes likely represent the founding lineages of cacao to Trinidad and Tobago. The cpSSRs were more variable with eight haplotypes, but these clustered into three groups corresponding to the three cpSNP haplotypes. The most common haplotype found in farms of Trinidad and Tobago was LAF, followed by UAF and then CRI. We conclude that the Trinitario cultivar group is of complex hybrid origin and has derived from at least three original introduction events.

Optimization of the composition of crop collections for ex situ conservation

Many crop genetic resources collections have been established without a clearly defined conservation goal or mandate, which has resulted in collections of considerable size, unbalanced composition and high levels of duplication. Attempts to improve the composition of collections are hampered by the fact that conceptual views to optimize collection composition are very rare. An optimization strategy is proposed herein, which largely builds on the concepts of core collection and core selection. The proposed strategy relies on hierarchically structuring the crop gene pool and assigning a relative importance to each of its different components. Comparison of the resulting optimized distribution of the number of accessions with the actual distribution allows identification of under- and over-representation within a collection. Application of this strategy is illustrated by an example using potato. The proposed optimization strategy is applicable not only to individual genebanks, but also to consortia of cooperating genebanks, which makes it relevant for ongoing activities within projects that aim at sharing responsibilities among institutions on the basis of rational conservation, such as a European genebank integrated system and the global cacao genetic resources network CacaoNet.

Accession management strategies: splitting and lumping.

van Hintum, Th. J. L., Sackville Hamilton, N. R., Engels, J. M. M., van Treuren, R. (2002). Accession management strategies: splitting and lumping. IPGRI 2002. Managing Plant Genetic Resources, Engels, J. M. M.Rao, V. R.Brown, A. H. D.Jackson, M. T. CABI Publishing, ISBN:0851995225, 113-120 Main Contents: The applications of genomic sciences for a better understanding of genepools - Technologies and strategies for ex situ conservation - The deployment and management of genetic diversity in agroecosystems - The role of bioinformatics in conservation and use - In situ conservation of wild species - Indicators for sustainable management of genetic resources - Germplasm enhancement and pre-breeding - Exploring underused species-diverse options - Implications of gene transformation techniques for ex situ conservation choices - GIS applications for genetic resources management - The economics of managing genetic resources and the role of private and public sectors

Geography of Genetic Structure in Barley Wild Relative Hordeum vulgare subsp. spontaneum in Jordan

Informed collecting, conservation, monitoring and utilization of genetic diversity requires knowledge of the distribution and structure of the variation occurring in a species. Hordeum vulgare subsp. spontaneum (K. Koch) Thell., a primary wild relative of barley, is an important source of genetic diversity for barley improvement and co-occurs with the domesticate within the center of origin. We studied the current distribution of genetic diversity and population structure in H. vulgare subsp. spontaneum in Jordan and investigated whether it is correlated with either spatial or climatic variation inferred from publically available climate layers commonly used in conservation and ecogeographical studies. The genetic structure of 32 populations collected in 2012 was analyzed with 37 SSRs. Three distinct genetic clusters were identified. Populations were characterized by admixture and high allelic richness, and genetic diversity was concentrated in the northern part of the study area. Genetic structure, spatial location and climate were not correlated. This may point out a limitation in using large scale climatic data layers to predict genetic diversity, especially as it is applied to regional genetic resources collections in H. vulgare subsp. spontaneum.

A review of factors that influence the production of quality seed for long-term conservation in genebanks

Seed quality is a critical aspect in agriculture as well as in the long-term conservation of plant genetic resources in genebanks. Since potential seed longevity depends on initial quality, genebank curators need to be aware of the best management practices that contribute to the production of high quality seed during routine germplasm regeneration/multiplication. Among the factors influencing initial seed quality, those related to crop management, including plant nutrient and water supply during crop growth, climatic conditions during seed development and maturation, as well as the harvest and drying practices are of considerable significance. Seeds of high quality can be obtained by planting in suitable areas/fields and at appropriate times, applying good crop management practices, adoption of proper harvesting and drying techniques, careful handling and processing to minimize mechanical injuries and unwanted seed mixing with other accessions, and ensuring minimum deterioration before reaching the designated storage. However, seed production and post-harvest handling highly depend on the biology and agronomy of the species. As germplasm collections contain a wide range of diversity for morphological and agronomic characters and that there might well be critical gaps in knowledge among genebank staff or about the species in question, genebanks may also need to embark on research to gain crop specific knowledge on optimal seed production procedures to improve seed quality.

Changes in barley (Hordeum vulgare L. subsp. vulgare) genetic diversity and structure in Jordan over a period of 31 years

In many regions of the world, the cultivation of landraces is still common, in particular in centres of crop diversity. Significant effort has been put into ex situ conservation of landraces but limited data exist on the changes in genetic diversity that occur over time in farmers’ fields. We assessed temporal changes in barley landrace diversity in Jordan using seed samples collected in 1981 and 2012 from the same locations. We did not observe significant changes in the amount of genetic diversity, but samples collected in 2012 were more homogenous and less locally distinct. In two sites, we observed replacement of the old material. We observed a change in phenotype, and phenotypes were found to be more homogeneous among sites in 2012. Climate changed significantly over the study period, becoming hotter and dryer, but we did not identify any correlation between the changes in climate and genetic and phenotypic variations. While the amount of genetic diversity in terms of allelic richness and number of multi-locus genotypes has been maintained, local distinctiveness among landrace barley populations in Jordan was reduced.

Genetic Diversity and Erosion—A Global Perspective

Biodiversity is continually declining, according to global biodiversity indicators (Butchart et al. in Science 328:1164–1168, 2010). Population trends, habitat extent, habitat condition, and composition of species communities—indicators of the state of diversity—are declining, while at the same time pressures on biodiversity posed by resource consumption, invasive alien species, pollution, overexploitation, and climate change are increasing. The rate of current loss of species is reported to be 100–1000 times the natural background rate (Chivian and Berstein in Sustaining life on earth. How human health depends on biodiversity. Oxford University Press, New York, 2008, Chivian and Berstein in How our health depends on biodiversity. Center for Health and the global environment. Harvard medical school, Boston, 2010; Pimm et al. in Science 344, 2014). Dramatic though that figure is, it underestimates the full loss of diversity because it ignores loss at both genetic and population level (Myers in Seeds and sovereignty. The use and control of plant genetic resources. Duke University Press, Durham, 1988; Mendenhall et al. in Biol Conserv 151:32–34, 2012). One of the first publications alerting the world about the losses of genetic diversity within species, later termed “genetic erosion,” was published in 1914 (Baur in Die Bedeutung der primitiven Kulturrassen und der wilden Verwandten unserer Kulturpflanzen fuer die Pflanzenzuechtung; Jahrbuch Deutsche Landwirt, 1914). The first concern about loss of diversity regarded agriculturally important species, as these are of direct and daily use to people. One hundred years later, genetic erosion is addressed at the global level in international agendas that set targets and propose actions to reduce the loss of genetic diversity, such as the Global Plan of Action (GPA) for Plant Genetic Resources for Food and Agriculture (PGRFA) of the FAO Commission on Genetic Resources for Food and Agriculture (CGRFA) and the Aichi biodiversity targets of the Convention on Biodiversity (CBD). The fact that genetic erosion today is addressed at global level implies that the crucial importance of genetic diversity for sustaining life on earth has been recognized. Strategies and actions to reduce the ongoing loss of genetic diversity are now in place. However, these measures have been found only partially successful as only few significant reductions in rates of decline were observed (Butchart et al. in Science 328:1164–1168, 2010), and global estimates of the extent of genetic erosion are still lacking. This chapter focuses on the importance of genetic diversity in PGRFA, how diversity of PGRFA is affected by genetic erosion, development of activities undertaken by international bodies to address genetic erosion, options to improve knowledge about the underlying processes that lead to genetic erosion, and the need for systematic monitoring of genetic diversity to better safeguard, conserve, and use PGRFA.