Characterization Of MiRNA And PhasiRNA In Litchi And The Relevant Regulation Pathway Of MiRNA During Fruitlet Abscission Induced By Carbohydrate Stress

Litchi(Litchi chinensis Sonn.)is an important tropical and subtropical fruit tree which originated in China.Massive physiological fruitlet drop during fruit development is one of the key factors affecting the heathy development of the litchi industry.To date,studies on the molecular mechanism related to fruit abscission mainly focused on m RNA level,rarely on the level of small RNAs(sRNAs),which have been emerging as a critical regulator in plant growth and development.miRNA is a class of sRNAs with a length of about 20-22 nt.It can not only regulate m RNA at post transcriptional level,but also induce the generation of secondary small RNA,namely phasi RNAs,which subsequently participate in all kinds of life activities.What are the regulatory roles of miRNAs and phasi RNAs in litchi? Which miRNA is involved in the process of fruitlet abscission in litchi? In order to answer these questions,in this study we firstly generated different deep-sequencing libraries from different tissues of litchi,including sRNA libraries,Parallel Analysis of RNA Ends(PARE)libraries and strandspecific RNA sequencing(ssRNA-seq)libraries.In combination with full-sequenced litchi genome information,we characterized main miRNA-and phasi RNA-involved regulatory pathways in litchi.We unexpectedly discovered a new mechanism of miRNA-induced phasi RNA generation coupled with alternative splicing/alternative polyadenylation.Secondly,in a treatment of "girdling plus defoliation" which promoted fruitlet abscission,we screened miRNAs and their target genes involved in this process.These result offered important reference value for litchi sRNA regulated mechanism,especially for molecular regulation of litchi fruitlet abscission at post transcriptional lever.Main results obtained in this study are listed as follows:(1)Six sRNA libraries and four PARE libraries data were constructed and used to identify miRNAs and their target genes at the genome-wide level,and 42 known and 40 new miRNA families were obtained,including 239 and 51 members respectively.Among them,32 known miRNA families(214 members)and 13 new miRNA families(20 members),have in total 395 target genes identified.Most of these target genes are transcription factors,disease resistance genes,and genes involved in plant hormone metabolism and signal transduction.Besides conserved target genes,new target genes,which had not been reported in other plants,were identified in litchi for known miRNAs,suggesting that known miRNAs can not only keep their basic function in evolution,but also gain new target genes to adapt the changing external world.(2)sRNA libraries were used to search for PHAS loci which generated phasi RNA in the litchi genome.A total of 515 PHAS loci were found,420 of them were likely coding genes with the other 95 non-coding.For the coding PHAS loci,R-genes account for the largest group,including 180 loci,followed by genes encoding Pentatricopeptide repeat-containing protein,Retrotransposon gag protein,Kinase,Auxin response factor,and MYB transcription factor.We identified miRNA triggers for 52 PHAS loci,including 6 plant typical miRNA-phasi RNA pathways,miR482/2118-NB-LRR,miR828-MYB,miR390-TAS3,miR393-TIR1/ARF,miR167-ARF and miR3954-NAC,and a litchi-specific pathway: miRN1-NB-LRR.The majority of PHAS loci(463)did not have miRNA triggers identified,suggesting that the biogenesis mechanism of phasi RNA would be more complex than what we thought before.(3)We found that miR482/2118 family shows diverse functions in litchi.In addition to target disease resistance genes,it can target a large number of non-coding genes,triggering phasi RNA production.Interestingly,the miR482/2118 mediated phasi RNA generation could be coupled with alternative splicing/alternative polyadenylation to generate sequence-distinct phasi RNA from different genomic regions,expanding the range of downstream target genes.There are 29 of these loci found in litchi,with 24 of them coupled with miR482/2118 and 4 coupled with miR3954.Hence,we proposed a new mechanism of phasi RNA generation coupled with alternative splicing/alternative polyadenylation,which can be potentially developed into a biotechnic approach to regulate many unrelated genes simultaneously.(4)The "girdling + defoliation" treatment could significantly promote fruitlet abscission 20 days after flowering,due to the carbohydrate stress induced.The fruitlet abscission rate increased significantly after 48 h and fruitlets almost fell completely at 96h(the abscission rate was 95%,while the control was only 8.2%).Assay of hormone contents,including ETH,ABA and IAA,found that carbohydrate stress promoted the release of ETH and induced the accumulation of ABA content,but IAA content decreased,giving rise to low IAA/ABA ratio in abscission zone and fruitlet.These three endogenous hormones showed greater difference in fruitlet than pedicel with abscission zone,suggesting that the balance of endogenous IAA,ABA and ethylene is one of the most important physiological factors causing litchi fruitlet abscission.(5)A total 36 sRNA libraries were constructed from pedicel with abscission zone and fruitlet samples collected at 0h,6h,12 h,24h and 48 h after the "girdling + defoliation" treatment.We found that 40 miRNA families(23 known miRNA families,including 77 family members,and 13 new miRNA families,including 21 family members)showed significant difference in expression after the treatment.These miRNAs are involved in a number of regulatory networks,including miR858-MYB,miR159-MYB,miR482/2118-R-gene,miR397-Laccase,miR160-ARF,miR393-TRI1,miR172-AP2,miR319-TCP,miR156-SPL,miR396-GRE,miR164-NAC,miRN1-Rgene,miR396-CP-RD21 a,miR396-IAA14 and miR396-Zn F and so on,suggesting that the regulation of miRNA is very enormous in the process of fruitlet abscission induced by carbohydrate stress(6)According to the visual evaluation method "ab initio" combined with Kendall ?-consecutive and ?-trend statistical method,In the correlation analysis of expression pattern of the miRNA and target genes during litchi fruitlet abscission,we found "Mix"(mixed correlation)miRNA/genes pairs are the largest group,with "FN"(negative correlation in the whole process)and "FP"(positive correlation in the whole process)miRNA/target gene pairs accounting for only a small part.Different targets genes of the same miRNA or target genes of different miRNA members from the same miRNA family showed different expression correlation patterns,suggesting that the regulation of target genes by miRNAs is very complicated during the fruitlet abscission caused by carbohydrate stress.(7)Expression of 18 miRNAs and their target genes were re-evaluated by q RT-PCR,confirming that ten miRNA-involved regulatory pathways are probably involved in the process of fruitlet abscission in litchi,and they are miR160-ARF,miR393-TIR1,miR396-IAA14,miR397-Laccase,miR159-CKX1,miR156-SPL,miR482/2118-PGK,miR396-CP-RD21 a,miR396-Zn F and miRN1-R-gene.