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Genome survey and genetic characterization of Acacia pachyceras O. Schwartz - PubMed

  • ️Sun Jan 01 2023

Genome survey and genetic characterization of Acacia pachyceras O. Schwartz

Nazima Habibi et al. Front Plant Sci. 2023.

Abstract

Acacia pachyceras O. Schwartz (Leguminoseae), a woody tree growing in Kuwait is critically endangered. High throughput genomic research is immediately needed to formulate effective conservation strategies for its rehabilitation. We therefore, performed a genome survey analysis of the species. Whole genome sequencing generated ~97 Gb of raw reads (92x coverage) with a per base quality score above Q30. The k-mer analysis (17 mer) revealed its genome to be 720Mb in size with an average guanine-cytosine (GC) ratio of 35%. The assembled genome was analyzed for repeat regions (45.4%-interspersed repeats; 9%-retroelements; 2%-DNA transposons). BUSCO assessment of completeness of genome identified 93% of assembly to be complete. Gene alignments in BRAKER2 yielded 34,374 transcripts corresponding to 33,650 genes. Average length of coding sequences and protein sequences were recorded as 1,027nts and 342aa, respectively. GMATA software filtered a total of 901,755 simple sequence repeats (SSRs) regions against which 11,181 unique primers were designed. A subset of 110 SSR primers were PCR validated and demonstrated for its application in genetic diversity analysis of Acacia. The SSR primers successfully amplified A. gerrardii seedlings DNA depicting cross transferability among species. The principal coordinate analysis and the split decomposition tree (bootstrapping runs of 1000 replicates) distributed the Acacia genotypes into two clusters. The flow cytometry analysis revealed the A. pachyceras genome to be polyploid (6x). The DNA content was predicted as 2.46 pg, 1.23 pg, and 0.41 pg corresponding to 2C DNA, 1C DNA and 1Cx DNA, respectively. The results provide a base for further high throughput genomic studies and molecular breeding for its conservation.

Keywords: SSR primers; flow cytometry; gene annotation; microsatellite; transposable element; whole genome sequencing; woody tree.

Copyright © 2023 Habibi, Al Salameen, Vyas, Rahman, Kumar, Shajan, Zakir, Razzack and Al Doaij.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1

Map of Kuwait (A) showing the location of Acacia tree in Sabah Al-Nature Reserve and KISR greenhouse. Map was drawn using the ArcGIS v10.4.1. (B) The single mature tree of Acacia pachyceras growing in the Sabah Al-Nature Reserve area of Kuwait.

Figure 2
Figure 2

K-mer analysis for genome size prediction of Acacia pachyceras. The x-axis represents the depth and the corresponding frequencies are plotted on the y-axis. The values on the right-hand side panel shows the k-mer analysis parameters and the genome size (k-mer number/peak depth). Peaks at 25x and 50x are due to the repetitive and heterozygous genome sequences respectively.

Figure 3
Figure 3

De novo Assembly of Acacia pachyceras. (A) Assembly statistics. Genome was assembled using MaSuRCA-4.0.3. (B) GC content of Acacia pachyceras genome. The x-axis represents the GC% and sequencing depth is plotted on y-axis. Sliding window at 10kb non-overlapping bases was used for GC% estimation. (C) BUSCO assessment of completeness of genome assembly. BUSCO was run in genome mode using metaeuk as the gene predictor. The x-axis presents the BUSCO % and the Y-axis denotes the genome name.

Figure 4
Figure 4

(A) Distributions of di- to hexanucleotide (SSR) motif types with repeat numbers ranging from 4 to 14 in the assembled genomic sequences of Acacia pachyceras (B) Primers designed against the filtered SSR motifs employing Primer 3.

Figure 5
Figure 5

(A) Principal Coordinate Analysis (PCoA) plot of the pairwise genetic distances between 17 genotypes of A. pachyceras based on 189 polymorphic SSR loci. (B) A split decomposition network tree of seventeen Acacia genotypes. The splits tree v5 algorithm was used to construct the tree with 999 permutations and bootstrapping.

Figure 6
Figure 6

(A) Flow cytometric histograms for genome size estimation and ploidy assessment of A. pachyceras. The pink peaks correspond to the A. pachyceras nuclie whereas the blue peak represents the standard Panicum miliaceum (2C=2.09 pg; 2n=4x=36). The nuclei of reference and sample species were extracted and stained with propidium iodide and analyzed simultaneously. Relative fluorescence is plotted on the x-axis and the nuclei count are shown on the y-axis. (B) Bar plots of minimum fluorescence intensities of A. pachyceras and P.miliaceum nuclei. (C) Flow cytometric histograms for genome size estimation and ploidy assessment of A. gerrardii. (D) Bar plots of minimum fluorescence intensities of A. gerrardii and P. miliaceum nuclei.

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Grants and funding

We thank the Kuwait Foundation for Advancement of Sciences (KFAS Grant no. PR18-12SL-12) and the Kuwait Institute for Scientific Research (KISR Grant no. FB134C) to fund this research.