Identification of MicroRNAs and their Targets Associated with Embryo Abortion during Chrysanthemum Cross Breeding via High-Throughput Sequencing

Abstract

BackgroundMicroRNAs (miRNAs) are important regulators in plant development. They post-transcriptionally regulate gene expression during various biological and metabolic processes by binding to the 3’-untranslated region of target mRNAs to facilitate mRNA degradation or inhibit translation. Chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental flowers with increasing demand each year. However, embryo abortion is the main reason for chrysanthemum cross breeding failure. To date, there have been no experiments examining the expression of miRNAs associated with chrysanthemum embryo development. Therefore, we sequenced three small RNA libraries to identify miRNAs and their functions. Our results will provide molecular insights into chrysanthemum embryo abortion.ResultsThree small RNA libraries were built from normal chrysanthemum ovules at 12 days after pollination (DAP), and normal and abnormal chrysanthemum ovules at 18 DAP. We validated 228 miRNAs with significant changes in expression frequency during embryonic development. Comparative profiling revealed that 69 miRNAs exhibited significant differential expression between normal and abnormal embryos at 18 DAP. In addition, a total of 1037 miRNA target genes were predicted, and their annotations were defined by transcriptome data. Target genes associated with metabolic pathways were most highly represented according to the annotation. Moreover, 52 predicted target genes were identified to be associated with embryonic development, including 31 transcription factors and 21 additional genes. Gene ontology (GO) annotation also revealed that high-ranking miRNA target genes related to cellular processes and metabolic processes were involved in transcription regulation and the embryo developmental process.ConclusionsThe present study generated three miRNA libraries and gained information on miRNAs and their targets in the chrysanthemum embryo. These results enrich the growing database of new miRNAs and lay the foundation for the further understanding of miRNA biological function in the regulation of chrysanthemum embryo abortion.

Identification and comparative profiling of miRNAs in herbaceous peony (Paeonia lactiflora Pall.) with red/yellow bicoloured flowers

“…The number of sRNAs was far less than the report of miRNAs responsive to Botrytis cinerea in P. lactiflora 21, which demonstrated that the mechanism of colour formation was less complicated compared with the stress response in P. lactiflora . With respect to the length distribution of sRNAs, 21–24 nt sRNAs dominated the sRNA transcriptome in red outer-petal and yellow inner-petal with the 21-nt sRNAs being the most abundant in length, which was in accordance with the reports in P. lactiflora 21, P. persica 17 and C. indica 22, while not in agreement with 24-nt sRNAs being the most abundant in C. tinctorius 16 and D. morifolium 20. This was mainly because of the plant species and the specific enzymes that were used to process the different length of sRNAs40.…”

MicroRNA and Putative Target Discoveries in Chrysanthemum Polyploidy Breeding

“…In chrysanthemum cross breeding, embryonic development is a crucial stage for seed formation, but embryo abortion is prevalent in chrysanthemum distant hybridization [ 31 , 32 ]. Previous studies explored this barrier at the level of cell structure, gene expression, and miRNA regulation [ 19 , 24 ], in which 227 miRNAs were identified in hybrid embryos from cross C. morifolium and diploid C. nankingense . Because of the importance of chromosome doubling in cross breeding, in the present study, we performed the hybridization using C. morifolium and tetraploid C. nankingense ; 179 miRNAs were identified in three embryo samples (Table S2 ).…”

“…RNA samples used for sequencing were reverse transcribed using PrimeScript miRNA qPCR starter kit ver 2.0 (Takara Bio, Dalian, China). qRT-PCR was performed using the SYBR Premix EX Taq Kit (Takara, Dalian, China), and the PCR amplification was done as described by Zhang et al [ 24 ]. Three biological replicates were performed for each sample, and relative expression levels were calculated by the 2 −△△CT method.…”

“…Although previous studies analyzed many different reasons leading to plant embryo abortion, including maternal genotypes [ 20 ], parent ploidy [ 21 , 22 ], and gene regulation [ 6 , 23 ], the molecular mechanisms regulating embryo development are poorly understood. Zhang et al revealed the gene, protein, and miRNA change in the stage of chrysanthemum embryo development [ 19 , 24 ] and provided numerous information of embryo abortion in chrysanthemum hybridization breeding. Evidence supporting a major role for chromosome doubling in overcoming chrysanthemum embryo abortion has been obtained from some studies [ 22 , 25 ], but the functions of miRNA remain largely uncharacterized.…”

Current status and prospects of chrysanthemum genomics

“…특히 miRNA의 target mRNA가 대부분이 전사조절인 자로 하위유전자의 발현에도 영향을 주기 때문에 그 영향 력이 매우 커서 국화에서도 NGS를 활용하여 miRNA가 전 게놈 수준에서 분석되었다 (Xia et al 2015;Zhang et al 2015). 원연교잡 후에 확보된 3종류의 배에서 228개의 miRNA가 동정되었으며, 각각의 배에서 특이적으로 발현되는 miRNA 도 규명되었다 .…”