Supporting in situ conservation of the genetic diversity of crop wild relatives using genomic technologies.

The last decade has witnessed huge technological advances in genomics, particularly in DNA sequencing. Here, we review the actual and potential application of genomics in supporting in situ conservation of crop wild relatives (CWRs). In addition to helping in prioritization of protection of CWR taxa and in situ conservation sites, genome analysis is allowing the identification of novel alleles that need to be prioritized for conservation. Genomics is enabling the identification of potential sources of important adaptive traits that can guide the establishment or enrichment of in situ genetic reserves. Genomic tools also have the potential for developing a robust framework for monitoring and reporting genome-based indicators of genetic diversity changes associated with factors such as land use or climate change. These tools have been demonstrated to have an important role in managing the conservation of populations, supporting sustainable access and utilization of CWR diversity, enhancing accelerated domestication of new crops and forensic genomics thus preventing misappropriation of genetic resources. Despite this great potential, many policy makers and conservation managers have failed to recognize and appreciate the need to accelerate the application of genomics to support the conservation and management of biodiversity in CWRs to underpin global food security. Funding and inadequate genomic expertise among conservation practitioners also remain major hindrances to the widespread application of genomics in conservation.

Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species.

Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology ("DArTseq"). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies' diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.

Genetics and Genomics of African Rice (Oryza glaberrima Steud) Domestication.

African rice (Oryza glaberrima Steud) is one of the two independently domesticated rice species, the other one being Asian rice (Oryza sativa L.). Despite major progress being made in understanding the evolutionary and domestication history of African rice, key outstanding issues remain controversial. There appears to be an underlying difficulty in identifying the domestication centre and number of times the crop has been domesticated. Advances in genomics have provided unprecedented opportunities for understanding the genetic architecture of domestication related traits. For most of the domestication traits, the underlying genes and mutations have been identified. Comparative analysis of domestication genes between Asian and African rice has revealed that the two species went through an independent but convergent evolution process. The genetic and developmental basis of some of the domestic traits are conserved not only between Asian and African rice but also with other domesticated crop species. Analysis of genome data and its interpretation is emerging as a major challenge facing studies of domestication in African rice as key studies continue giving contradictory findings and conclusions. Insights obtained on the domestication of this species are vital for guiding crop improvement efforts.

Advances in Molecular Genetics and Genomics of African Rice (Oryza glaberrima Steud).

African rice (Oryza glaberrima) has a pool of genes for resistance to diverse biotic and abiotic stresses, making it an important genetic resource for rice improvement. African rice has potential for breeding for climate resilience and adapting rice cultivation to climate change. Over the last decade, there have been tremendous technological and analytical advances in genomics that have dramatically altered the landscape of rice research. Here we review the remarkable advances in knowledge that have been witnessed in the last few years in the area of genetics and genomics of African rice. Advances in cheap DNA sequencing technologies have fuelled development of numerous genomic and transcriptomic resources. Genomics has been pivotal in elucidating the genetic architecture of important traits thereby providing a basis for unlocking important trait variation. Whole genome re-sequencing studies have provided great insights on the domestication process, though key studies continue giving conflicting conclusions and theories. However, the genomic resources of African rice appear to be under-utilized as there seems to be little evidence that these vast resources are being productively exploited for example in practical rice improvement programmes. Challenges in deploying African rice genetic resources in rice improvement and the genomics efforts made in addressing them are highlighted.

Development of species diagnostic SNP markers for quality control genotyping in four rice (Oryza L.) species.

Species misclassification (misidentification) and handling errors have been frequently reported in various plant species conserved at diverse gene banks, which could restrict use of germplasm for correct purpose. The objectives of the present study were to (i) determine the extent of genotyping error (reproducibility) on DArTseq-based single-nucleotide polymorphisms (SNPs); (ii) determine the proportion of misclassified accessions across 3134 samples representing three African rice species complex (Oryza glaberrima, O. barthii, and O. longistaminata) and an Asian rice (O. sativa), which are conserved at the AfricaRice gene bank; and (iii) develop species- and sub-species (ecotype)-specific diagnostic SNP markers for rapid and low-cost quality control (QC) analysis. Genotyping error estimated from 15 accessions, each replicated from 2 to 16 times, varied from 0.2 to 3.1%, with an overall average of 0.8%. Using a total of 3134 accessions genotyped with 31,739 SNPs, the proportion of misclassified samples was 3.1% (97 of the 3134 accessions). Excluding the 97 misclassified accessions, we identified a total of 332 diagnostic SNPs that clearly discriminated the three indigenous African species complex from Asian rice (156 SNPs), O. longistaminata accessions from both O. barthii and O. glaberrima (131 SNPs), and O. sativa spp. indica from O. sativa spp. japonica (45 SNPs). Using chromosomal position, minor allele frequency, and polymorphic information content as selection criteria, we recommended a subset of 24 to 36 of the 332 diagnostic SNPs for routine QC genotyping, which would be highly useful in determining the genetic identity of each species and correct human errors during routine gene bank operations.

Assessment of Genetic Variation and Population Structure of Diverse Rice Genotypes Adapted to Lowland and Upland Ecologies in Africa Using SNPs.

Using interspecific crosses involving Oryza glaberrima Steud. as donor and O. sativa L. as recurrent parents, rice breeders at the Africa Rice Center developed several 'New Rice for Africa (NERICA)' improved varieties. A smaller number of interspecific and intraspecific varieties have also been released as 'Advanced Rice for Africa (ARICA)'. The objective of the present study was to investigate the genetic variation, relatedness, and population structure of 330 widely used rice genotypes in Africa using DArTseq-based single nucleotide polymorphisms (SNPs). A sample of 11 ARICAs, 85 NERICAs, 62 O. sativa spp. japonica, and 172 O. sativa spp. indica genotypes were genotyped with 27,560 SNPs using diversity array technology (DArT)-based sequencing (DArTseq) platform. Nearly 66% of the SNPs were polymorphic, of which 15,020 SNPs were mapped to the 12 rice chromosomes. Genetic distance between pairs of genotypes that belong to indica, japonica, ARICA, and NERICA varied from 0.016 to 0.623, from 0.020 to 0.692, from 0.075 to 0.763, and from 0.014 to 0.644, respectively. The proportion of pairs of genotypes with genetic distance > 0.400 was the largest within NERICAs (35.1% of the pairs) followed by ARICAs (18.2%), japonica (17.4%), and indica (5.6%). We found one pair of japonica, 11 pairs of indica, and 35 pairs of NERICA genotypes differing by <2% of the total scored alleles, which was due to 26 pairs of genotypes with identical pedigrees. Cluster analysis, principal component analysis, and the model-based population structure analysis all revealed two distinct groups corresponding to the lowland (primarily indica and lowland NERICAs) and upland (japonica and upland NERICAs) growing ecologies. Most of the interspecific lowland NERICAs formed a sub-group, likely caused by differences in the O. glaberrima genome as compared with the indica genotypes. Analysis of molecular variance revealed very great genetic differentiation (FST = 0.688) between the lowland and upland ecologies, and 31.2% of variation attributable to differences within cluster groups. About 8% (1,197 of 15,020) of the 15,020 SNPs were significantly (P < 0.05) different between the lowland and upland ecologies and formed contrasting haplotypes that could clearly discriminate lowland from upland genotypes. This is the first study using high density markers that characterized NERICA and ARICA varieties in comparison with indica and japonica varieties widely used in Africa, which could aid rice breeders on parent selection for developing new improved rice germplasm.

Role of genomics in promoting the utilization of plant genetic resources in genebanks.

Global efforts have seen the world's plant genetic resources (PGRs) conserved in about 1625 germ plasm repositories. Utility of these resources is important in increasing the resilience and productivity of agricultural production systems. However, despite their importance, utility of these resources has been poor. This article reviews the real and potential application of the current advances in genomic technologies in improving the utilization of these resources. The actual and potential application of these genomic approaches in plant identification, phylogenetic analysis, analysing the genetic value of germ plasm, facilitating germ plasm selection in genebanks as well as instilling confidence in international germ plasm exchange system is discussed. We note that if genebanks are to benefit from this genomic revolution, there is need for fundamental changes in the way genebanks are managed, perceived, organized and funded. Increased collaboration between genebank managers and the user community is also recommended.

Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice.

Amylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT-HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the approach of sequencing bulks was able to identify genes on different chromosomes associated with this complex trait.

Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences.

Rice is the most important crop in the world, acting as the staple food for over half of the world's population. The evolutionary relationships of cultivated rice and its wild relatives have remained contentious and inconclusive. Here we report on the use of whole chloroplast sequences to elucidate the evolutionary and phylogenetic relationships in the AA genome Oryza species, representing the primary gene pool of rice. This is the first study that has produced a well resolved and strongly supported phylogeny of the AA genome species. The pan tropical distribution of these rice relatives was found to be explained by long distance dispersal within the last million years. The analysis resulted in a clustering pattern that showed strong geographical differentiation. The species were defined in two primary clades with a South American/African clade with two species, O glumaepatula and O longistaminata, distinguished from all other species. The largest clade was comprised of an Australian clade including newly identified taxa and the African and Asian clades. This refined knowledge of the relationships between cultivated rice and the related wild species provides a strong foundation for more targeted use of wild genetic resources in rice improvement and efforts to ensure their conservation.

The biosynthesis, structure and gelatinization properties of starches from wild and cultivated African rice species (Oryza barthii and Oryza glaberrima).

The molecular structure and gelatinization properties of starches from domesticated African rice (Oryza glaberrima) and its wild progenitor (Oryza barthii) are determined and comparison made with Asian domesticated rice (Oryza sativa), the commonest commercial rice. This suggests possible enzymatic processes contributing to the unique traits of the African varieties. These have similar starch structures, including smaller amylose molecules, but larger amounts of amylose chains across the whole amylose chain-length distribution, and higher amylose contents, than O. sativa. They also show a higher proportion of two- and three-lamellae spanning amylopectin branch chains (degree of polymerization 34-100) than O. sativa, which contributes to their higher gelatinization temperatures. Fitting amylopectin chain-length distribution with a biosynthesis-based mathematical model suggests that the reason for this difference might be because O. glaberrima and O. barthii have more active SSIIIa and/or less active SBEIIb enzymes.

Influence of ethnolinguistic diversity on the sorghum genetic patterns in subsistence farming systems in eastern Kenya.

Understanding the effects of actions undertaken by human societies on crop evolution processes is a major challenge for the conservation of genetic resources. This study investigated the mechanisms whereby social boundaries associated with patterns of ethnolinguistic diversity have influenced the on-farm distribution of sorghum diversity. Social boundaries limit the diffusion of planting material, practices and knowledge, thus shaping crop diversity in situ. To assess the effect of social boundaries, this study was conducted in the contact zone between the Chuka, Mbeere and Tharaka ethnolinguistic groups in eastern Kenya. Sorghum varieties were inventoried and samples collected in 130 households. In all, 297 individual plants derived from seeds collected under sixteen variety names were characterized using a set of 18 SSR molecular markers and 15 morphological descriptors. The genetic structure was investigated using both a Bayesian assignment method and distance-based clustering. Principal Coordinates Analysis was used to describe the structure of the morphological diversity of the panicles. The distribution of the varieties and the main genetic clusters across ethnolinguistic groups was described using a non-parametric MANOVA and pairwise Fisher tests. The spatial distribution of landrace names and the overall genetic spatial patterns were significantly correlated with ethnolinguistic partition. However, the genetic structure inferred from molecular makers did not discriminate the short-cycle landraces despite their morphological distinctness. The cases of two improved varieties highlighted possible fates of improved materials. The most recent one was often given the name of local landraces. The second one, that was introduced a dozen years ago, displays traces of admixture with local landraces with differential intensity among ethnic groups. The patterns of congruence or discordance between the nomenclature of farmers' varieties and the structure of both genetic and morphological diversity highlight the effects of the social organization of communities on the diffusion of seed, practices, and variety nomenclature.

Conservation and utilization of African Oryza genetic resources.

Africa contains a huge diversity of both cultivated and wild rice species. The region has eight species representing six of the ten known genome types. Genetic resources of these species are conserved in various global germplasm repositories but they remain under collected and hence underrepresented in germplasm collections. Moreover, they are under characterized and therefore grossly underutilized. The lack of in situ conservation programs further exposes them to possible genetic erosion or extinction. In order to obtain maximum benefits from these resources, it is imperative that they are collected, efficiently conserved and optimally utilized. High throughput molecular approaches such as genome sequencing could be employed to more precisely study their genetic diversity and value and thereby enhance their use in rice improvement. Oryza sativa was the first crop plant to have its reference genome sequence released marking a major milestone that opened numerous opportunities for functional characterization of the entire rice genome. Studies have however demonstrated that one reference genome sequence is not enough to fully explore the genetic variation in the Oryza genus, hence the need to have reference sequences for other species in the genus. An overview of the state of conservation and utilization of African Oryza is hereby presented. Progress in the release of reference genome sequences for these species is also highlighted.