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. 2016 Apr 12:16:80.
doi: 10.1186/s12870-016-0770-z.

Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis

Jingli Chen  1 , Yi Zheng  2 , Li Qin  1 , Yan Wang  1 , Lifei Chen  1 , Yanjun He  1 , Zhangjun Fei  2   3 , Gang Lu  4
Affiliations

Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis

Jingli Chen et al. BMC Plant Biol. .

Abstract

Background: MicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination.

Results: Two independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes.

Conclusions: We identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in asparagus.

Keywords: Asparagus officinalis; Degradome analysis; High-throughput sequencing; Sex determination; miRNAs.

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Figures

Fig. 1
Fig. 1
Length distribution of unique sRNAs in male and female libraries of A. officinalis. Most of the generated reads were 24 (>30 %) and 21(>19 %) nucleotides long
Fig. 2
Fig. 2
Number of identified miRNAs in each conserved miRNA family in A. officinalis
Fig. 3
Fig. 3
Relative nucleotide bias at each miRNA position compared with the total RNA. Uridine (U) was the most common nucleotide at the 5′ end (>78 %), and the 10th nucleotides, which match to the cleavage site of targets, were mainly adenine (A) (~40 %)
Fig. 4
Fig. 4
Comparison of miRNA expression levels between asparagus male and female individuals through qRT-PCR. a Expression levels of 13 selected miRNAs between male and female plants. b Expression profiles of aof-miR159a, aof-miR167g, aof-miR172a and aof-miR172b during male and female flower development. F-0.5, 0.5 mm female flower buds; M-0.5, 0.5 mm male flower buds; F-4, 4.0 mm female flower; M-4, 4.0 mm male flower. *or **indicates a statistically significant difference between male and female flowers at the same stage at P < 0.05 or 0.01, respectively
Fig. 5
Fig. 5
Target plot (t-plots) of representative validated asparagus miRNA targets in different categories as confirmed by degradome sequencing
Fig. 6
Fig. 6
Detection of predicted miRNA target genes in asparagus by 5′ RLM-RACE. Arrows point to the cleavage sites of targeted mRNAs for four asparagus miRNAs. The Watson-Crick pairing (vertical dashes) and mismatch pairing (circles) are shown in the complementary pairing area of miRNA and its target. The denominator and numerator of the fraction indicate the number of sequenced monoclonal sequences and the number of monoclonal sequences with the cleavage site at the arrow, respectively. Only the monoclonal sequences with the cleavage sites in the complementary pairing area of miRNA/target or nearby 10 nucleotides were counted
Fig. 7
Fig. 7
Comparison of the expression levels of miRNA target genes between male and female individuals through qRT-PCR. a The expression level of EIF-2 (UN00815), ARF6 (UN12573) and AP2 (UN21982), targeted by aof-miR159, aof-miR167 and aof-miR172 family, respectively, in male and female plants. *or **indicates a statistically significant difference between male and female plants at P < 0.05 or 0.01, respectively. b The expression level of target genes in 0.5 mm and 4 mm female and male flower buds. F-0.5 and F-4 represent 0.5 mm and 4 mm female flower respectively; M-0.5 and M-4 represent 0.5 mm and 4 mm male flower, respectively. *or **indicates a statistically significant difference between male and female flowers at the same stage at P < 0.05 or 0.01, respectively
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