Long non-coding RNAs are major contributors to transcriptome changes in sunflower meiocytes with different recombination rates

Abstract

Background: Meiosis is a form of specialized cell division that marks the transition from diploid meiocyte to haploid gamete, and provides an opportunity for genetic reassortment through recombination. Experimental data indicates that, relative to their wild ancestors, cultivated sunflower varieties show a higher recombination rate during meiosis. To better understand the molecular basis for this difference, we compared gene expression in male sunflower meiocytes in prophase I isolated from a domesticated line, a wild relative, and a F1 hybrid of the two.

Results: Of the genes that showed differential expression between the wild and domesticated genotypes, 63.62 % could not be identified as protein-coding genes, and of these genes, 70.98 % passed stringent filters to be classified as long non-coding RNAs (lncRNAs). Compared to the sunflower somatic transcriptome, meiocytes express a higher proportion of lncRNAs, and the majority of genes with exclusive expression in meiocytes were lncRNAs. Around 40 % of the lncRNAs showed sequence similarity with small RNAs (sRNA), while 1.53 % were predicted to be sunflower natural antisense transcripts (NATs), and 9.18 % contained transposable elements (TE). We identified 6895 lncRNAs that are exclusively expressed in meiocytes, these lncRNAs appear to have higher conservation, a greater degree of differential expression, a higher proportion of sRNA similarity, and higher TE content relative to lncRNAs that are also expressed in the somatic transcriptome.

Conclusions: lncRNAs play important roles in plant meiosis and may participate in chromatin modification processes, although other regulatory functions cannot be excluded. lncRNAs could also be related to the different recombination rates seen for domesticated and wild sunflowers.

Keywords: Homologous recombination; Meiosis; Prophase I; RNA-seq; Transcriptomics; lncRNA; miRNA.

Fig. 1

Diagram showing the numbers and proportions of differentially expressed genes (DEG) between the domesticated and wild genotypes grouped by coding class. Global percentage of DEGs (first row) was calculated based on the total number of genes (59,058). Percentages of genes with and without protein coding potential were calculated with reference to number of DEGs (29,469). Percentages of lncRNA and unclassified genes were calculated with reference to non-protein coding genes (18,767). Last row in the diagram presents percentages in each category with reference to the total DEG number (29,469)

Fig. 2

Bioinformatic pipeline to determine the lncRNA nature of transcripts and Venn diagrams with number of lncRNA expressed by genotype. Protein coding was determined by comparing transcripts with peptide databases (TAIR 10, NCBI RefSeq and sunflower peptides); all transcript with one or more hits to peptides with a bitscore ≥ 90 and E value < 1e-6 were discarded as potential lncRNAs in (1). To determine if the transcripts had a blast hit with the draft of the sunflower genome, a threshold of bitscore ≥ 90 and E-value < 1e-6 was employed in (2). Only transcripts with a CPC score ≤ −1 and CPAT score ≤ 0.3 were considered as lncRNA by filters in (3). Venn diagrams: a) Expression of lncRNAs with significant similarity with the sunflower genome. b Expression of lncRNAs with no significant similarity with the sunflower genome. c Total number of lncRNAs detected

Fig. 3

Expression overview of genes grouped by coding class (Protein-coding, lncRNA or Unclassified) in somatic and meiocyte transcriptomes. a Bar chart showing the coding class proportions by transcriptome. b Bar chart comparing the number of genes exclusively expressed in somatic or meiocyte transcriptomes by coding class. c Box plots for the distributions of the decimal log expression of genes in meiocytes and somatic transcriptomes by coding class

Fig. 4

Bar charts for the number of reads per length (bp) in small RNA populations of sunflower meiocytes in two genotypes. Reads were classified as ‘Unique’ for sequences found only once, or ‘Redundant’ for sequences found more than one time within genotypes. a Results for the wild genotype Ac-8. b Results for the domesticated genotype HA89

Fig. 5

Bar charts for the number of reads per length (bp) in small RNA populations of sunflower meiocytes mapping to protein-coding, lncRNA or unclassified transcripts in two genotypes. a Results for the wild genotype Ac-8. b Results for the domesticated genotype HA89

Fig. 6

Transposable elements (TEs) in sunflower lncRNAs. a Relative percentages of DNA Transposons and Retrotransposons found within 2326 (9.18 %) sunflower lncRNA. b Bar chart for the number of lncRNA containing TEs per TEs family. DNA Transposons: DH = Helitron, DT = Unknown DNA transposon, DTA = hAT, DTC = CACTA, DTH = Harbinger, DTM = Mutator, DXX = MITE. Retrotransposons: RI = unknown non-LTR retrotransposons, RIL = LINEs, RLC = Copia retrotransposons, RLG = Gypsy retrotransposons, RLR = retroviruses, RLX = Unknown LTR retrotransposons, RX = Unknown retrotransposons