Combination of linkage and association mapping with genomic prediction to infer QTL regions associated with gray leaf spot and northern corn leaf blight resistance in tropical maize.

Among the diseases threatening maize production in Africa are gray leaf spot (GLS) caused by Cercospora zeina and northern corn leaf blight (NCLB) caused by Exserohilum turcicum. The two pathogens, which have high genetic diversity, reduce the photosynthesizing ability of susceptible genotypes and, hence, reduce the grain yield. To identify population-based quantitative trait loci (QTLs) for GLS and NCLB resistance, a biparental population of 230 lines derived from the tropical maize parents CML511 and CML546 and an association mapping panel of 239 tropical and sub-tropical inbred lines were phenotyped across multi-environments in western Kenya. Based on 1,264 high-quality polymorphic single-nucleotide polymorphisms (SNPs) in the biparental population, we identified 10 and 18 QTLs, which explained 64.2% and 64.9% of the total phenotypic variance for GLS and NCLB resistance, respectively. A major QTL for GLS, qGLS1_186 accounted for 15.2% of the phenotypic variance, while qNCLB3_50 explained the most phenotypic variance at 8.8% for NCLB resistance. Association mapping with 230,743 markers revealed 11 and 16 SNPs significantly associated with GLS and NCLB resistance, respectively. Several of the SNPs detected in the association panel were co-localized with QTLs identified in the biparental population, suggesting some consistent genomic regions across genetic backgrounds. These would be more relevant to use in field breeding to improve resistance to both diseases. Genomic prediction models trained on the biparental population data yielded average prediction accuracies of 0.66-0.75 for the disease traits when validated in the same population. Applying these prediction models to the association panel produced accuracies of 0.49 and 0.75 for GLS and NCLB, respectively. This research conducted in maize fields relevant to farmers in western Kenya has combined linkage and association mapping to identify new QTLs and confirm previous QTLs for GLS and NCLB resistance. Overall, our findings imply that genetic gain can be improved in maize breeding for resistance to multiple diseases including GLS and NCLB by using genomic selection.

Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet.

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a 'poor man's crop', finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

A high-density linkage map of finger millet provides QTL for blast resistance and other agronomic traits.

Finger millet [Eleusine coracana (L.) Gaertn.] is a critical subsistence crop in eastern Africa and southern Asia but has few genomic resources and modern breeding programs. To aid in the understanding of finger millet genomic organization and genes underlying disease resistance and agronomically important traits, we generated a F2:3 population from a cross between E. coracana (L.) Gaertn. subsp. coracana accession ACC 100007 and E. coracana (L.) Gaertn. subsp. africana , accession GBK 030647. Phenotypic data on morphology, yield, and blast (Magnaporthe oryzae) resistance traits were taken on a subset of the F2:3 population in a Kenyan field trial. The F2:3 population was genotyped via genotyping-by-sequencing (GBS) and the UGbS-Flex pipeline was used for sequence alignment, nucleotide polymorphism calling, and genetic map construction. An 18-linkage-group genetic map consisting of 5,422 markers was generated that enabled comparative genomic analyses with rice (Oryza sativa L.), foxtail millet [Setaria italica (L.) P. Beauv.], and sorghum [Sorghum bicolor (L.) Moench]. Notably, we identified conserved acrocentric homoeologous chromosomes (4A and 4B in finger millet) across all species. Significant quantitative trait loci (QTL) were discovered for flowering date, plant height, panicle number, and blast incidence and severity. Sixteen putative candidate genes that may underlie trait variation were identified. Seven LEUCINE-RICH REPEAT-CONTAINING PROTEIN genes, with homology to nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance proteins, were found on three chromosomes under blast resistance QTL. This high-marker-density genetic map provides an important tool for plant breeding programs and identifies genomic regions and genes of critical interest for agronomic traits and blast resistance.

Population genetic structure and migration patterns of the maize pathogenic fungus, Cercospora zeina in East and Southern Africa.

Cercospora zeina is a causal pathogen of gray leaf spot (GLS) disease of maize in Africa. This fungal pathogen exhibits a high genetic diversity in South Africa. However, little is known about the pathogen's population structure in the rest of Africa. In this study, we aimed to assess the diversity and gene flow of the pathogen between major maize producing countries in East and Southern Africa (Kenya, Uganda, Zambia, Zimbabwe, and South Africa). A total of 964 single-spore isolates were made from GLS lesions and confirmed as C.zeina using PCR diagnostics. The other causal agent of GLS, Cercospora zeae-maydis, was absent. Genotyping all the C.zeina isolates with 11 microsatellite markers and a mating-type gene diagnostic revealed (i) high genetic diversity with some population structure between the five African countries, (ii) cryptic sexual recombination, (iii) that South Africa and Kenya were the greatest donors of migrants, and (iv) that Zambia had a distinct population. We noted evidence of human-mediated long-distance dispersal, since four haplotypes from one South African site were also present at five sites in Kenya and Uganda. There was no evidence for a single-entry point of the pathogen into Africa. South Africa was the most probable origin of the populations in Kenya, Uganda, and Zimbabwe. Continuous annual maize production in the tropics (Kenya and Uganda) did not result in greater genetic diversity than a single maize season (Southern Africa). Our results will underpin future management of GLS in Africa through effective monitoring of virulent C.zeina strains.

Morphological and Molecular Characterization of Toxigenic Aspergillus flavus from Groundnut Kernels in Kenya.

Pathogenesis of Aspergillus flavus on important agricultural products is a key concern on human health due to the synthesis and secretion of the hazardous secondary metabolite, aflatoxin. This study identified and further characterized aflatoxigenic A. flavus from groundnuts sampled from sundry shops in Kenya using integrated morphological and molecular approaches. The groundnuts were plated on potato dextrose agar for isolation and morphological observation of A. flavus based on macroscopic and microscopic features. Molecular characterization was done through amplification and comparison of the partial sequence of the ITS1-5.8S-ITS2 region. The expression analysis of aflR, aflS, aflD, aflP, and aflQ genes in the aflatoxin biosynthesis pathways was conducted to confirm the positive identification of A. flavus. The gene expression also aided to delineate toxigenic isolates of A. flavus from atoxigenic ones. Morphologically, 18 isolates suspected to be A. flavus were identified. Out of these, 14 isolates successfully amplified the 500 bp ITS region of A. flavus or Aspergillus oryzae, while 4 isolates were not amplified. All the remaining 14 isolates expressed at least one of the aflatoxigenic genes but only 5 had all the genes expressed. Partial sequencing revealed that isolates 5, 11, 12, 13, and 15 had 99.2%, 97.6%, 98.4%, 97.5%, and 100% homology, respectively, to the A. flavus isolate LUOHE, ITS-5.8S-ITS2, obtained from the NCBI database. The five isolates were accurate identification of atoxigenic A. flavus. Precise identification of toxigenic strains of A. flavus will be useful in establishing control strategies of the fungus in food products.

UGbS-Flex, a novel bioinformatics pipeline for imputation-free SNP discovery in polyploids without a reference genome: finger millet as a case study.

BACKGROUND: Research on orphan crops is often hindered by a lack of genomic resources. With the advent of affordable sequencing technologies, genotyping an entire genome or, for large-genome species, a representative fraction of the genome has become feasible for any crop. Nevertheless, most genotyping-by-sequencing (GBS) methods are geared towards obtaining large numbers of markers at low sequence depth, which excludes their application in heterozygous individuals. Furthermore, bioinformatics pipelines often lack the flexibility to deal with paired-end reads or to be applied in polyploid species. RESULTS: UGbS-Flex combines publicly available software with in-house python and perl scripts to efficiently call SNPs from genotyping-by-sequencing reads irrespective of the species' ploidy level, breeding system and availability of a reference genome. Noteworthy features of the UGbS-Flex pipeline are an ability to use paired-end reads as input, an effective approach to cluster reads across samples with enhanced outputs, and maximization of SNP calling. We demonstrate use of the pipeline for the identification of several thousand high-confidence SNPs with high representation across samples in an F3-derived F2 population in the allotetraploid finger millet. Robust high-density genetic maps were constructed using the time-tested mapping program MAPMAKER which we upgraded to run efficiently and in a semi-automated manner in a Windows Command Prompt Environment. We exploited comparative GBS with one of the diploid ancestors of finger millet to assign linkage groups to subgenomes and demonstrate the presence of chromosomal rearrangements. CONCLUSIONS: The paper combines GBS protocol modifications, a novel flexible GBS analysis pipeline, UGbS-Flex, recommendations to maximize SNP identification, updated genetic mapping software, and the first high-density maps of finger millet. The modules used in the UGbS-Flex pipeline and for genetic mapping were applied to finger millet, an allotetraploid selfing species without a reference genome, as a case study. The UGbS-Flex modules, which can be run independently, are easily transferable to species with other breeding systems or ploidy levels.

The role of Kenya in the trans-African spread of maize streak virus strain A.

Maize streak virus (MSV), the causal agent of maize streak disease (MSD), is the most important viral pathogen of Africa's staple food crop, maize. Previous phylogeographic analyses have revealed that the most widely-distributed and common MSV variant, MSV-A1, has been repeatedly traversing Africa over the past fifty years with long-range movements departing from either the Lake Victoria region of East Africa, or the region around the convergence of Zimbabwe, South Africa and Mozambique in southern Africa. Despite Kenya being the second most important maize producing country in East Africa, little is known about the Kenyan MSV population and its contribution to the ongoing diversification and trans-continental dissemination of MSV-A1. We therefore undertook a sampling survey in this country between 2008 and 2011, collecting MSD prevalence data in 119 farmers' fields, symptom severity data for 170 maize plants and complete MSV genome sequence data for 159 MSV isolates. We then used phylogenetic and phylogeographic analyses to show that whereas the Kenyan MSV population is likely primarily derived from the MSV population in neighbouring Uganda, it displays considerably more geographical structure than the Ugandan population. Further, this geographical structure likely confounds apparent associations between virus genotypes and both symptom severity and MSD prevalence in Kenya. Finally, we find that Kenya is probably a sink rather than a source of MSV diversification and movement, and therefore, unlike Uganda, Kenya probably does not play a major role in the trans-continental dissemination of MSV-A1.

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies.

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity.

Symbiotic Efficiency of Native Rhizobia Nodulating Common Bean (Phaseolus vulgaris L.) in Soils of Western Kenya.

This study was conducted to determine the abundance and symbiotic efficiency of native rhizobia nodulating common bean in Kisumu and Kakamega, Kenya. Soil sampling was carried out in three farms that had been used for growing common bean for at least two seasons and one fallow land with no known history of growing common bean or inoculation. Abundance of soil rhizobia and symbiotic efficiency (SE) were determined in a greenhouse experiment. Native rhizobia populations ranged from 3.2 × 10(1) to 3.5 × 10(4) cells per gram of soil. Pure bacterial cultures isolated from fresh and healthy root nodules exhibited typical characteristics of Rhizobium sp. on yeast extract mannitol agar media supplemented with Congo red. Bean inoculation with the isolates significantly (p < 0.05) increased the shoot dry weight and nitrogen (N) concentration and content. The SE of all the native rhizobia were higher when compared to a reference strain, CIAT 899 (67%), and ranged from 74% to 170%. Four isolates had SE above a second reference strain, Strain 446 (110%). Our results demonstrate the presence of native rhizobia that are potentially superior to the commercial inoculants. These can be exploited to enhance bean inoculation programmes in the region.

Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes.

Finger millet is an allotetraploid (2n = 4x = 36) grass that belongs to the Chloridoideae subfamily. A comparative analysis has been carried out to determine the relationship of the finger millet genome with that of rice. Six of the nine finger millet homoeologous groups corresponded to a single rice chromosome each. Each of the remaining three finger millet groups were orthologous to two rice chromosomes, and in all the three cases one rice chromosome was inserted into the centromeric region of a second rice chromosome to give the finger millet chromosomal configuration. All observed rearrangements were, among the grasses, unique to finger millet and, possibly, the Chloridoideae subfamily. Gene orders between rice and finger millet were highly conserved, with rearrangements being limited largely to single marker transpositions and small putative inversions encompassing at most three markers. Only some 10% of markers mapped to non-syntenic positions in rice and finger millet and the majority of these were located in the distal 14% of chromosome arms, supporting a possible correlation between recombination and sequence evolution as has previously been observed in wheat. A comparison of the organization of finger millet, Panicoideae and Pooideae genomes relative to rice allowed us to infer putative ancestral chromosome configurations in the grasses.

The genetic map of finger millet, Eleusine coracana.

Restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), expressed-sequenced tag (EST), and simple sequence repeat (SSR) markers were used to generate a genetic map of the tetraploid finger millet (Eleusine coracana subsp. coracana) genome (2n = 4x = 36). Because levels of variation in finger millet are low, the map was generated in an inter-subspecific F(2) population from a cross between E. coracana subsp. coracana cv. Okhale-1 and its wild progenitor E. coracana subsp. africana acc. MD-20. Duplicated loci were used to identify homoeologous groups. Assignment of linkage groups to the A and B genome was done by comparing the hybridization patterns of probes in Okhale-1, MD-20, and Eleusine indica acc. MD-36. E. indica is the A genome donor to E. coracana. The maps span 721 cM on the A genome and 787 cM on the B genome and cover all 18 finger millet chromosomes, at least partially. To facilitate the use of marker-assisted selection in finger millet, a first set of 82 SSR markers was developed. The SSRs were identified in small-insert genomic libraries generated using methylation-sensitive restriction enzymes. Thirty-one of the SSRs were mapped. Application of the maps and markers in hybridization-based breeding programs will expedite the improvement of finger millet.