Damaris Achieng Odeny

Genomics-assisted breeding for boosting crop improvement in pigeonpea (Cajanus cajan)

Pigeonpea is an important pulse crop grown predominantly in the tropical and sub-tropical regions of the world. Although pigeonpea growing area has considerably increased, yield has remained stagnant for the last six decades mainly due to the exposure of the crop to various biotic and abiotic constraints. In addition, low level of genetic variability and limited genomic resources have been serious impediments to pigeonpea crop improvement through modern breeding approaches. In recent years, however, due to the availability of next generation sequencing and high-throughput genotyping technologies, the scenario has changed tremendously. The reduced sequencing costs resulting in the decoding of the pigeonpea genome has led to the development of various genomic resources including molecular markers, transcript sequences and comprehensive genetic maps. Mapping of some important traits including resistance to Fusarium wilt and sterility mosaic disease, fertility restoration, determinacy with other agronomically important traits have paved the way for applying genomics-assisted breeding (GAB) through marker assisted selection as well as genomic selection (GS). This would accelerate the development and improvement of both varieties and hybrids in pigeonpea. Particularly for hybrid breeding programme, mitochondrial genomes of cytoplasmic male sterile (CMS) lines, maintainers and hybrids have been sequenced to identify genes responsible for cytoplasmic male sterility. Furthermore, several diagnostic molecular markers have been developed to assess the purity of commercial hybrids. In summary, pigeonpea has become a genomic resources-rich crop and efforts have already been initiated to integrate these resources in pigeonpea breeding.

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.

Novel Sources of Witchweed (Striga) Resistance from Wild Sorghum Accessions

Sorghum is a major food staple in sub-Saharan Africa (SSA), but its production is constrained by the parasitic plant Striga that attaches to the roots of many cereals crops and causes severe stunting and loss of yield. Away from cultivated farmland, wild sorghum accessions grow as weedy plants and have shown remarkable immunity to Striga. We sought to determine the extent of the resistance to Striga in wild sorghum plants. Our screening strategy involved controlled laboratory assays of rhizotrons, where we artificially infected sorghum with Striga, as well as field experiments at three sites, where we grew sorghum with a natural Striga infestation. We tested the resistance response of seven accessions of wild sorghum of the aethiopicum, drummondii, and arundinaceum races against N13, which is a cultivated Striga resistant landrace. The susceptible control was farmer-preferred variety, Ochuti. From the laboratory experiments, we found three wild sorghum accessions (WSA-1, WSE-1, and WSA-2) that had significantly higher resistance than N13. These accessions had the lowest Striga biomass and the fewest and smallest Striga attached to them. Further microscopic and histological analysis of attached Striga haustorium showed that wild sorghum accessions hindered the ingression of Striga haustorium into the host endodermis. In one of the resistant accessions (WSE-1), host and parasite interaction led to the accumulation of large amounts of secondary metabolites that formed a dark coloration at the interphase. Field experiments confirmed the laboratory screening experiments in that these same accessions were found to have resistance against Striga. In the field, wild sorghum had low Area under the Striga Number Progressive curve (AUSNPC), which measures emergence of Striga from a host over time. We concluded that wild sorghum accessions are an important reservoir for Striga resistance that could be used to expand the genetic basis of cultivated sorghum for resistance to the parasite.

The early transcriptome response of cassava (Manihot esculenta Crantz) to mealybug (Phenacoccus manihoti) feeding

The mealybug, Phenacoccus manihoti, is a leading pest of cassava (Manihot esculenta Crantz), damaging this crop globally. Although the biological control of this mealybug using natural predators has been established, resistance breeding remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The transcriptome response of the green mite resistant cassava genotype AR23.1 was compared to P40/1 with no known resistance at 24 and 72 hours of mealybug infestation compared to non-infested mock. A total of 301 and 206 genes were differentially expressed at 24 and 72 of mealybug infestation for AR23.1 and P40/1 genotypes respectively, using a log2 fold change and P-value ≤ 0.05. Gene ontology functional classification revealed an enrichment of genes in the secondary metabolic process category in AR23.1 in comparison with P40/1, while genes in the regulation of molecular function, cellular component biogenesis and electron carrier categories were more significantly enriched in P40/1 than in AR23.1. Biological pathway analysis, based on KEGG, revealed a significant enrichment of plant-pathogen interaction and plant hormonal signal transduction pathways for a cohort of up-regulated and down-regulated DEGs in both genotypes. Defence-related genes such as 2-oxogluterate, gibberellin oxidase and terpene synthase proteins were only induced in genotype AR23.1 and not in P40/1, and subsequently validated by RT-qPCR. The study revealed a difference in response to mealybug infestation in the two genotypes studied, with AR23.1 showing a higher number of differentially expressed transcripts post mealybug infestation at 24 and 72 hours. Candidate defence-related genes that were overexpressed in the AR23.1 genotype post mealybug infestation will be useful in future functional studies towards the control of mealybugs.

Heritability Analysis and Phenotypic Characterization of Spider Plant (Cleome gynandra L.) for Yield

Knowledge on phenotypic diversity among existing spider plant accessions is a milestone in the improvement of spider plant, which is a highly nutritious indigenous vegetable in Kenya. A study involving agronomic and morphological characterization of 49 spider plant accessions assembled from East and South Africa was carried out at the University of Nairobi Field Station for two seasons in a randomized complete block design with three replications. Phenotypic data was collected on growth habit, flower, petiole, leaf and stem colour, petiole, leaf and stem hairiness, number of leaves per plant, plant height, number of primary branches, leaf length and width, single leaf area, and chlorophyll content according to FAO descriptors with modifications. Data was analyzed using both DARwin software V6 and Genstat Version 14. We observed significant differences among the traits implying great genetic variability among the evaluated spider plant accessions. The high genetic variation was further validated using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering method with stem and flower colour as key traits. The 49-spider plant accessions were clustered into 2 major groups, each consisting of Kenyan and South African accessions. Stepwise regression revealed that plant height had the most influence on yield in terms of number of leaves per plant. We also observed high heritability for several traits including days to flowering (91%), number of leaves per plant (99%), plant height (99%), number of primary branches (94%), chlorophyll content (94%), and single leaf area (87%). Our results reveal the high genetic variation between different spider plant accessions, especially from different regions of Africa that could be further exploited to improve productivity in the plant. The high heritability of most of the yield related traits is promising for improving yield in the crop through direct selection.