Peter Wenzl

Agriculture: Feeding the future

Humanity depends on fewer than a dozen of the approximately 300,000 species of flowering plants for 80% of its caloric intake. And we capitalize on only a fraction of the genetic diversity that resides within each of these species. This is not enough to support our food system in the future. Food availability must double in the next 25 years to keep pace with population and income growth around the world. Already, food-production systems are precarious in the face of intensifying demand, climate change, soil degradation and water and land shortages. Farmers have saved the seeds of hundreds of crop species and hundreds of thousands of ‘primitive’ varieties (local domesticates called landraces), as well as the wild relatives of crop species and modern varieties no longer in use. These are stored in more than 1,700 gene banks worldwide. Maintaining the 11 international gene-bank collections alone costs about US

Low level of genetic diversity in cultivated pigeonpea compared to its wild relatives is revealed by diversity arrays technology

18 million a year.

DArT for high-throughput genotyping of cassava (Manihot esculenta) and its wild relatives

Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies.

DArT markers: diversity analyses and mapping in Sorghum bicolor.

Understanding the distribution of genetic diversity within and among individuals, populations, species and gene pools is crucial for the efficient management of germplasm collections. Molecular markers are playing an increasing role in germplasm characterization, yet their broad application is limited by the availability of markers, the costs and the low throughput of existing technologies. This is particularly true for crops of resource-poor farmers such as cassava, Manihot esculenta. Here we report on the development of Diversity Arrays Technology (DArT) for cassava. DArT uses microarrays to detect DNA polymorphism at several hundred genomic loci in a single assay without relying on DNA sequence information. We tested three complexity reduction methods and selected the two that generated genomic representations with the largest frequency of polymorphic clones (PstI/TaqI: 14.6%, PstI/BstNI: 17.2%) to produce large genotyping arrays. Nearly 1,000 candidate polymorphic clones were detected on the two arrays. The performance of the PstI/TaqI array was validated by typing a group of 38 accessions, 24 of them in duplicate. The average call rate was 98.1%, and the scoring reproducibility was 99.8%. DArT markers displayed fairly high polymorphism information content (PIC) values and revealed genetic relationships among the samples consistent with the information available on these samples. Our study suggests that DArT offers advantages over current technologies in terms of cost and speed of marker discovery and analysis. It can therefore be used to genotype large germplasm collections.

DArT markers for the rye genome - genetic diversity and mapping

BackgroundThe sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm.ResultsA genotyping array was developed representing approximately 12,000 genomic clones using Pst I+Ban II complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM.ConclusionWe have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.

Reconstruction of the Synthetic W7984 × Opata M85 wheat reference population

BackgroundImplementation of molecular breeding in rye (Secale cereale L.) improvement programs depends on the availability of high-density molecular linkage maps. However, the number of sequence-specific PCR-based markers available for the species is limited. Diversity Arrays Technology (DArT) is a microarray-based method allowing for detection of DNA polymorphism at several thousand loci in a single assay without relying on DNA sequence information. The objective of this study was the development and application of Diversity Arrays technology for rye.ResultsUsing the Pst I/Taq I method of complexity reduction we created a rye diversity panel from DNA of 16 rye varieties and 15 rye inbred lines, including parents of a mapping population consisting of 82 recombinant inbred lines. The usefulness of a wheat diversity panel for identification of DArT markers for rye was also demonstrated. We identified 1022 clones that were polymorphic in the genotyped ILs and varieties and 1965 clones that differentiated the parental lines L318 and L9 and segregated in the mapping population. Hierarchical clustering and ordination analysis were performed based on the 1022 DArT markers to reveal genetic relationships between the rye varieties and inbred lines included in the study. Chromosomal location of 1872 DArT markers was determined using wheat-rye addition lines and 1818 DArT markers (among them 1181 unique, non-cosegregating) were placed on a genetic linkage map of the cross L318 × L9, providing an average density of one unique marker every 2.68 cM. This is the most saturated rye linkage map based solely on transferable markers available at the moment, providing rye breeders and researches with a better choice of markers and a higher probability of finding polymorphic markers in the region of interest.ConclusionThe Diversity Arrays Technology can be efficiently and effectively used for rye genome analyses - assessment of genetic similarity and linkage mapping. The 11520-clone rye genotyping panel with several thousand markers with determined chromosomal location and accessible through an inexpensive genotyping service is a valuable resource for studies on rye genome organization and in molecular breeding of the species.

Genetic structure, linkage disequilibrium and signature of selection in sorghum: Lessons from physically anchored dart markers

Reference populations are valuable resources in genetics studies for determining marker order, marker selection, trait mapping, construction of large-insert libraries, cross-referencing marker platforms, and genome sequencing. Reference populations can be propagated indefinitely, they are polymorphic and have normal segregation. Described are two new reference populations who share the same parents of the original wheat reference population Synthetic W7984 (Altar84/ Aegilops tauschii (219) CIGM86.940) x Opata M85, an F(1)-derived doubled haploid population (SynOpDH) of 215 inbred lines and a recombinant inbred population (SynOpRIL) of 2039 F(6) lines derived by single-plant self-pollinations. A linkage map was constructed for the SynOpDH population using 1446 markers. In addition, a core set of 42 SSR markers was genotyped on SynOpRIL. A new approach to identifying a core set of markers used a step-wise selection protocol based on polymorphism, uniform chromosome distribution, and reliability to create nested sets starting with one marker per chromosome, followed by two, four, and six. It is suggested that researchers use these markers as anchors for all future mapping projects to facilitate cross-referencing markers and chromosome locations. To enhance this public resource, researchers are strongly urged to validate line identities and deposit their data in GrainGenes so that others can benefit from the accumulated information.

DArT markers: diversity analyses, genomes comparison, mapping and integration with SSR markers in Triticum monococcum

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r2 decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod.

A study of allelic diversity underlying flowering-time adaptation in maize landraces

BackgroundTriticum monococcum (2n = 2x = 14) is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. DArT (D iversity Ar rays T echnology) employs a hybridisation-based approach to type thousands of genomic loci in parallel. DArT markers were developed for T. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating DArT and microsatellite markers.ResultsA DArT array, consisting of 2304 hexaploid wheat, 1536 tetraploid wheat, 1536 T. monococcum as well as 1536 T. boeoticum representative genomic clones, was used to fingerprint 16 T. monococcum accessions of diverse geographical origins. In total, 846 polymorphic DArT markers were identified, of which 317 were of T. monococcum origin, 246 of hexaploid, 157 of tetraploid, and 126 of T. boeoticum genomes. The fingerprinting data indicated that the geographic origin of T. monococcum accessions was partially correlated with their genetic variation. DArT markers could also well distinguish the genetic differences amongst a panel of 23 hexaploid wheat and nine T. monococcum genomes. For the first time, 274 DArT markers were integrated with 82 simple sequence repeat (SSR) and two morphological trait loci in a genetic map spanning 1062.72 cM in T. monococcum. Six chromosomes were represented by single linkage groups, and chromosome 4Am was formed by three linkage groups. The DArT and SSR genetic loci tended to form independent clusters along the chromosomes. Segregation distortion was observed for one third of the DArT loci. The Ba (black awn) locus was refined to a 23.2 cM region between the DArT marker locus wPt-2584 and the microsatellite locus Xgwmd33 on 1Am; and the Hl (hairy leaf) locus to a 4.0 cM region between DArT loci 376589 and 469591 on 5Am.ConclusionDArT is a rapid and efficient approach to develop many new molecular markers for genetic studies in T. monococcum. The constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.

Aluminum stress stimulates the accumulation of organic acids in root apices of Brachiaria species

Landraces (traditional varieties) of domesticated species preserve useful genetic variation, yet they remain untapped due to the genetic linkage between the few useful alleles and hundreds of undesirable alleles. We integrated two approaches to characterize the diversity of 4,471 maize landraces. First, we mapped genomic regions controlling latitudinal and altitudinal adaptation and identified 1,498 genes. Second, we used F-one association mapping (FOAM) to map the genes that control flowering time, across 22 environments, and identified 1,005 genes. In total, we found that 61.4% of the single-nucleotide polymorphisms (SNPs) associated with altitude were also associated with flowering time. More than half of the SNPs associated with altitude were within large structural variants (inversions, centromeres and pericentromeric regions). The combined mapping results indicate that although floral regulatory network genes contribute substantially to field variation, over 90% of the contributing genes probably have indirect effects. Our dual strategy can be used to harness the landrace diversity of plants and animals.