Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis

Abstract

MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide RNAs that regulate gene expression at the transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress, and pathogen responses. miRNAs with their targets have been widely studied in model plants, but limited knowledge is available on the small RNA population of cotton (Gossypium hirsutum)-an important economic crop, and global identification of related targets through degradome sequencing has not been developed previously. In this study, small RNAs and their targets were identified during cotton somatic embryogenesis (SE) through high-throughput small RNA and degradome sequencing, comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1. A total of 36 known miRNA families were found to be differentially expressed, of which 19 miRNA families were represented by 29 precursors. Twenty-five novel miRNAs were identified. A total of 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families, respectively, and 16 transcripts were targeted by eight novel miRNAs. Interestingly, four trans-acting small interfering RNAs (tas3-siRNAs) were also found in degradome libraries, three of which perfectly matched their precursors. Several targets were further validated via RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). The profiling of the miRNAs and their target genes provides new information on the miRNAs network during cotton SE.

Fig. 1.

The length distribution of small RNAs in CK and EC libraries. (This figure is available in colour at JXB online.)

Fig. 2.

The length distribution of known miRNAs during cotton SE. (This figure is available in colour at JXB online.)

Fig. 3.

Differentially expressed known miRNA families during cotton SE. (This figure is available in colour at JXB online.)

Fig. 4.

Differentially expressed novel miRNAs during cotton SE. (This figure is available in colour at JXB online.)

Fig. 5.

TAS3 transcripts identified during cotton SE. (A) Target sites of miR390 and tas3-siRNA genes on zhu2_CL11974Contig1. (B) Target sites of miR390 and tas3-siRNA genes on zhu2_gi|164318918|gb|ES828745.1|ES828745. (C) Target sites of miR390 and tas3-siRNA genes on zhu2_gi|164333334|gb|ES843623.1|ES843623. (D) Target sites of miR390 and tas3-siRNA genes on zhu2_CL11405Contig1. Alignment of miR390 with a portion of its target sequence. Two target sites of ghr-miR390 are indicated which were upstream and downstream of the of the tas3-siRNA-producing site respectively. The solid line indicates matched RNA base pairs, ‘.’ Indicates G:U pairs, ‘:’ represents mismatches. ghr-TAS3a D6 (+), ghr-TAS3b D7 (+), and ghr-TAS3c D8 (+) are indicated by underlining (This figure is available in colour at JXB online.)

Fig. 6.

Relative expression levels of five known miRNAs during cotton SE. (A) miR156, (B) miR167, (C) miR3476, (D) miR390, (E) miR164. The equation ratio=2-ΔCt was applied to calculate the relative expression level using GhUBQ7 as the reference gene; three biological replicates and three technical replicates were performed. 0h, hypocotyls; 6h, 12h, 24h, 48h, 5d: hypocotyls induced for 6, 12, 24, and 48h, and 5 d, respectively; NEC, non-embryonic callus; EC, embryonic callus; GE, globular-stage somatic embryo; TE, torpedo-stage somatic embryo; CE. cotyledon-stage somatic embryo.

Fig. 7.

Relative expression levels of six target genes (five ARF genes and one SPL gene) during cotton SE. (A) zhu2_CL4794Contig1 (ARF8) targeted by miR167. (B) zhu2_CL3970Contig2 (ARF6) targeted by miR167. (C) zhu2_CL11135Contig1 (ARF6) targeted by miR167. (D) zhu2_CL3970Contig1 (ARF6) targeted by miR167. (E) zhu2_CL3001Contig1 (ARF6) targeted by miR167. (F) zhu2_gi|164260183|gb|ES799263.1|ES799263 (SPL9) targeted by miR156. The equation ratio=2-ΔCt was applied to calculate the relative expression level using GhUBQ7 as the reference gene; three biological replicates and three technical replicates were performed. 0h, hypocotyls; 6h, 12h, 24h, 48h, 5d, hypocotyls induced for 6, 12, 24, and 48h, and 5 d, respectively; NEC, non-embryonic callus; EC, embryonic callus; GE, globular-stage somatic embryo; TE, torpedo-stage somatic embryo; CE, cotyledon-stage somatic embryo.

Fig. 8.

Validation of miRNA target genes by RLM 5’-RACE. Cleavage site for SPL9 (zhu2_gi|164260183|gb|ES799263.1|ES799263) targeted by miR156 (A); ARF8 (zhu2_CL4794Contig1) and ARF6 (zhu2_CL3970Contig2, zhu2_CL15585Contig1) targeted by miR167 (B–D); TIR1 (zhu2_CL14972Contig1) targeted by miR393 (E); ARF16 (zhu2_gi|164303952|gb|ES831580.1|ES831580) targeted by miR160 (F); NF-YA3 (zhu2_CL17360Contig1) targeted by miR169 (G); CZSOD (zhu2_gi|164324123|gb|ES849495.1|ES849495) targeted by miR398 (H); TAS3 (zhu2_CL11974Contig1) targeted by miR390 (I); CZSOD2 (zhu2_CL2800Contig1) targeted by miR159 (J); SurE-like phosphatase transcript (zhu2_CL3163Contig2) targeted by miR3476* (K); GRF8 (zhu2_CL3508Contig2) transcript targeted by miR396 (L); and ARF3 (zhu2_CL14460Contig1) transcript targeted by TAS3a D6 (+) (M). The solid lines indicate matched RNA base pairs, ‘.’ indicates G:U pairs, ‘:’ represent other types of mismatch. The black arrow indicates a cleavage site verified by RLM 5’-RACE, with the frequency of cloned RACE products shown above the alignment.