Distribution, Diversity and Salinity Induced Transcriptomics in Doum palm (Hyphaene compressa)

Abstract

Doum palm (Hyphaene compressa) is one of the few perennial evergreen plants that grow in Arid and Semi-Arid Lands (ASALs) of Africa. However, ethnobotanical knowledge about the plant is scanty and limited genetic data has been documented. Despite the doum palm's widespread presence in ASALs characterized by high saline soils, little is known about its salinity tolerance mechanisms. This study aimed to determine the ethnobotanical knowledge, the morphological and genetic diversity, develop molecular markers for diversity studies and to characterize the genes involved in salinity tolerance in H. compressa accessions from Kenya. Sampling of ethnobotanical data was done in four ASAL regions of Kenya; Turkana, Tharaka Nithi, Kwale and Tana River using the snowball technique. Interview schedules were administered to informants to determine the domestication status, utilization and the biotic and abiotic stresses impacting its growth: Responses from 79 respondents were evaluated. To ascertain the morphological variability of the vegetative and fruit features of H. compressa and to define its morphotypes, 90 H. compressa accessions were further analyzed. For morphological diversity, a total of 19 morphological characters, including seven quantitative and twelve qualitative aspects of fruit and vegetative attributes were used. Genotyping by sequencing (GBS) was done to determine the population structure and genetic diversity. Greenhouse experiments were conducted on accessions from Tharaka, Tana River and Turkana to evaluate salinity tolerance. Varying salinity levels of control, 100mM, 200mM and 300mM were imposed on the accessions for eight weeks. Morphological, physiological, proline content and ion content were determined. The RNA of the most tolerant accession was sequenced to identify differentially expressed genes (DEGs). Simple Sequence Repeats (SSR) markers were developed from unigenes obtained through RNA sequencing. Twenty of these primers were validated using 20 accessions from the four sampled regions. The results of this study showed that the sampled regions' levels of domestication varied, with the majority of the respondents exhibiting little interest in domesticating H. compressa. The study documented fourteen uses of H. compressa with food use (fruit) and soil erosion prevention (roots) scoring the greatest and least fidelity levels. Human intervention and pest infestation were the most prevalent biotic stresses, while salt and drought were the most prevalent abiotic stresses. All seven quantitative traits were highly effective at distinguishing doum palm phenotypes (p < 0.001). The 90 accessions belonged to five morphotypes, numbered 1, 2, 3, 4 and 5. Kwale accessions were heterogeneous. Genotyping by sequencing analysis on the other hand revealed two populations with high within-population diversity. Accessions from Turkana were grouped in one cluster while accessions from Tharaka, Tana River and Kwale accessions were grouped in another cluster. Moderate FST of 0.074 was obtained indicating moderate genetic differentiation in H. compressa. Tana River samples proved to be most tolerant to salinity stress followed by Turkana accessions. Tana River accessions also accumulated more proline and more biomass. Tana River control and salinity stressed samples at 300mM were sequenced. A total of 92,135 unigenes were obtained from de_novo assembly of the RNA data. In the current study, a total of 8611 DEGs were obtained with 3722 being up regulated and 4889 down regulated. A total of 25 gene ontology terms and 36 KEGG pathways were enriched. A total of 16,632 SSR markers were mined from the 92,135 unigenes at the rate of 1SSR per 5.5kb of unigenes. The AG/CT SSR motifs were the most frequent trinucleotide motifs. The validated SSR markers amplified 55 alleles at the rate of 2.75 alleles per locus. The SSR markers revealed higher genetic diversity indices compared to Single Nucleotide Polymorphism (SNPs) data obtained through GBS approach. Further, the SSR markers clustered the accessions into three populations based on STRUCTURE analysis. The use of H. compressa as food can build resilience of pastoralist communities who are susceptible to famine during drought. Overall, the study designated H. compressa into five morphotypes and two genetic clusters. This study also demonstrated that H. compressa is moderately to highly tolerant to salinity. The most salinity tolerant accessions are those from Tana River. This study has described numerous salinity induced DEGs, enriched GO terms and KEGG pathways in H. compressa which have shed light on its tolerance mechanisms. The first ever functional SSR markers have been developed for this plant which are crucial for diversity studies, marker assisted breeding and should be validated in other members of the genus Hyphaene and related taxa.