De novo, high-quality assembly and annotation of the halophyte grass Aeluropus littoralis draft genome and identification of A20/AN1 zinc finger protein family

Abstract

Background: Aeluropus littoralis is considered a valuable natural forage plant for ruminant livestock and is highly tolerant to extreme abiotic stresses, especially salinity, drought, and heat. It is a monocotyledonous halophyte, has salt glands, performs C4-type photosynthesis and has a close genetic relationship with cereal crops. Moreover, previous studies have shown its huge potential as a reservoir of genes and promoters to understand and improve abiotic stress tolerance in crops.

Results: The sequencing and hybrid assembly of the A. littoralis genome (2n = 2X = 20) using short and long reads from the BGISeq-500 and PacBio high-fidelity (HiFi) sequencing platforms, respectively. Using the k-mer analysis method, the haploid genome size of A. littoralis was estimated to be 360 Mb (with a heterozygosity rate of 1.88%). The hybrid assembled genome included 4,078 contigs with a GC content of 44% and covered 348 Mb. The longest contig and the N50 values were 5.1 Mb and 133.77 kb, respectively. The Benchmarking Universal Single Copy Ortholog (BUSCO) value was 91.1%, indicating good integrity of the assembled genome. The discovered repetitive elements accounted for 90.6 Mb, representing 26.03% of the total genome, and included a significant component of transposable elements (11.48%, ~40 Mb). Using a homology-based approach, 35,147 genes were predicted from the genome assembly. We next focused our analysis on the zinc-finger A20/AN1 gene family, a member of which (AlSAP) was previously shown to confer increased tolerance to osmotic and salt stresses when it was over-expressed in tobacco, wheat, and rice. Here, we identified the complete set of members of this family in the Aeluropus littoralis genome, thereby laying the foundation for their future functional analysis in cereal crops. In addition, the expression patterns of four novel genes from this family were analyzed by qPCR.

Conclusion: This resource and our findings will contribute to improve the current understanding of salinity tolerance in halophytes while providing useful genes and allelic variation to improve salinity and drought tolerance in cereals through genetic engineering and gene editing, respectively.

Keywords: Aeluropus littoralis; Abiotic stress tolerance; Halophyte; Next generation sequencing; Zinc-finger A20/AN1 genes.

Fig. 1

Salinity-induced morphological and anatomical changes in A. littoralis. A Changes in the shoot and root systems of A. littoralis under different NaCl concentrations. B Excretion of salt crystals through the salt glands of A. littoralis. C Root cross-section anatomy of A. littoralis under control and 300 mM NaCl conditions (D). The cell layers are the epidermis (ep), endodermis (en), cortex (co), exodermis (ex), sclerenchyma (sc), the stele (st), central metaxylem (cmx), metaxylems (mx), and aerenchyma (Ae). Leaf cross-section autofluorescence of A. littoralis under control (E) and 300 mM NaCl conditions (F). The structures are as follows: salt gland (Sg), xylem (Xyl), phloem (Ph), sclerenchyma cell (Sc), mesophyll cell (mc), bundle sheath cell (bs), stroma (S), and epidermis cell (ec). Scale bars: 100 μm

Fig. 2

Chromosome karyotype analysis of Aeluropus littoralis, Oryza sativa, and Triticum turgidum

Fig. 3

A. littoralis genome assembly assessment and comparative synteny analysis. A Benchmarking universal single-copy orthologs (BUSCO) evaluation results for the assembled A. littoralis genome, indicating high completeness based on conserved gene content. B Syntenic genomic blocks between A. littoralis and selected monocot species. (Eleusine coracana, Zea mays, Setaria italica, Sorghum bicolor, Oryza sativa, and Triticum turgidum). Blue lines denote indicate conserved syntenic genomic regions

Fig. 4

Bar graph of enriched KEGG pathways identified in the genome of A. littoralis

Fig. 5

Computational analysis of A. littoralis SAP family members. Gene structures of the A. littoralis SAP genes. A The CDS, upstream/downstream UTR, and introns are illustrated by blue, orange, and black lines, respectively. B The conserved domains of the AlSAP proteins analyzed via the MEME v5.4.1 tool. C Tertiary structure analysis of A. littoralis SAP family members predicted via alphafold 3 server (https://alphafoldserver.com/welcome). D Heatmap of the predicted subcellular localization of A. littoralis SAP family members

Fig. 6

Phylogenetic relationships of stress-associated proteins (SAPs) from Aeluropus littoralis (Al), Eleusine coracana (Ec), Setaria italic (Si), Sorghum bicolor (Sb), Oryza sativa(Os), Zea mays(Zm), Hordeum vulgare (Hv), Arabidopsis thaliana (At), Cucumis sativus (Cs), Jatropha curcas (Jc), Medicago truncatula (Mt), and Solanum tuberosum (St). A total of 153 SAPs were divided into five groups. The accession numbers of the protein sequences used to conduct this phylogenetic analysis are listed in Table S6

Fig. 7

Expression profiles of selected AlSAP genes in the leaves and roots of A. littoralis subjected to salinity and osmotic stress. Transcript levels of AlSAP genes were quantified in leaf and root tissues following treatment with 300 mM NaCl (salt stress) and 10% PEG-6000 (osmotic stress). Data represent mean expression values ± SE (n = 3 replicates). Different lowercase letters indicate significant differences (p< 0.05) according to Duncan’s test)

Fig. 8

Analysis of the cis-acting elements in the promoter region of selected AlSAP genes. The 2000 bp upstream start codon of the selected AlSAP genes were analyzed with the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cis-acting elements were classified into four major classes: hormone responsive cis-elements, development-related cis-elements, light responsive cis-elements, and stress-responsive cis-elements