RNA-Seq Transcriptome Profiling of Ethiopian Lowland Bamboo (Oxytenanthera Abyssinica (A.Rich) Munro Under Drought and Salt Stresses and SSR Based Genetic Diversity Analysis o f Ethiopian Highland Bamboo (Arundinaria Alpina K. Schum).
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Date
2019-07-02
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Addis Ababa University
Abstract
Bamboos are perennial grasses classified under family Poaceae and subfamily Bambusoideae and are
among the fastest growing plants on earth. Although bamboos have ecological and economic importance
in Ethiopia, scientific inquiry particularly on genomics, transcriptomics and genetic diversity and
structure is lacking. Ethiopian lowland bamboo (O. abyssinica) adapted in hot areas of Ethiopia hence
inquiry on global transcriptome profiling is very important to elucidate the most important genes,
transcript factors and metabolic pathways associated to abiotic stress. To do so, plastic pot germinated
seedlings of lowland bamboo were subjected to 200 mM NaCl and 25% PEG-6000 (Poly Ethylene
glycol) to induce salt and drought stress, respectively. Using the Illumina sequencing platform, fifteen
cDNA libraries were constructed and sequenced to generate the first drought and salt stress global
transcriptome profiling of the species. Following quality control, 754,444,646 clean paired-ends reads
were generated, and then de novo assembled into 406,181 unigenes. Functional annotation against the
public databases presented annotation of 217,067 (53.4%) unigenes, where NCBI-Nr 203,777, Swissport
115,741, COG 81,632 and KEGG 80,587. Prediction of Transcripts Factors (TFs) has generated 4,332
TFs organized into 64 TF families. Analysis of Differentially Expressed Genes (DEGs) provided 65,471
genes where 569 genes belongs to all stresses. Transcript factors (TFs) with a higher number of
differentially expressed genes include bZIP (49), WRKY (43), MYB (38), AP2/ERF (30), HD-ZIP (25)
and MYB related (21).
Such 569 genes could serve for engineering plants for multiple abiotic tolerances.
Despite the important role of bZIPs in plants, particularly in abiotic tolerances, bZIP family members and
their corresponding functions remain elusive in the lowland bamboo. Through genome-wide analysis, a
total of 162 bZIP lowland bamboo TFs having the bZIP binding domain (PF00170) were identified. For
the identified bZIPs, functional annotation, phylogenetic relationship and their expression under drought and salt stress were investigated. The expression profile of the bZIP TFs revealed that majority of the
bZIPs were highly responsive to drought and salt stress as 99 of the 162 TFs were up-regulated.
Metabolic pathway analysis revealed that environmental information processing and genetic information
processing categories were the only represented pathway which implies that the bZIPs are associated to
drought and salt stress.
Understanding on the genetic diversity and structure of highland bamboo is important particularly for
conservation. For genetic diversity analysis 150 samples, representing 15 populations, 10 samples from
each population were collected. The sources of SSR primers were; first, primers developed through
reduced-representation genome sequencing of lowland bamboo were checked for their polymorphism and
transferability, then 7 primers were selected and used for highland bamboo. Second, primers were
directly taken from literature from Chinese Moso bamboo, after checking their transferability 9 primers
were used. The genetic diversity indices (H= 0.398 and I= 0.639) for the 16 SSR markers used were
moderate in capturing genetic variability. Both individual phylogeny and population structure merged the
15 populations in to nine clusters, with less geographic origin or collection based pattern. UPGMA
clustered the 15 populations in 2, while NJ clustered in to 3, however pattern of their grouping is not in
line with their pattern of origin of collection. The principal component analysis revealed that individuals
are highly dispersed and did not group the populations into stridently distinct clusters
which tells the
presences of high genetic admixture possibly attributed to gene flow or the reproductive biology of the
species. In conclusion, the RNA-Seq based global transcriptome profiling has generated transcriptomics
resources for lowland bamboo which can be employed for itself and related species for further research.
Most importantly the study uncovered key stress responsive genes, transcription factors, metabolic
pathways
and even genes differentially expressed to all stress groups which could be used as the basis
for further studies aiming to confer plants for multiple abiotic stress tolerances. The study on bZIPs
genome wide analysis also provided valuable information on how bZIPs are highly associated to abiotic
stress tolerance since only pathways related to stress tolerances were represented in metabolic pathway analysis. The genetic diversity and population structure analysis of highland bamboo uncovered the
presences of moderate genetic variability with high genetic admixture. The information could be used as a
basis particularly for conservation interventions and for further investigations.
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Keywords
Arundinaria Alpina, Abiotic Stress, Differentially Expressed Genes, Genetic Diversity, Oxytenanthera, Abyssinica, Rna-Seq, Ssr, Transcriptomics