Identification Of Genes Associated With Taproot Heterosis Formation And CMS Occurrence In Radish(Raphanus Sativus L.)

Heterosis is a kind of phenomenon that F1 hybrids are superior to parents in terms of growth rate,viability,reproduction rate,disease resistance,stress resistance,biomass and yield.Radish(Raphanus sativus L.)is an annual or biennial herb of the root vegetable crop with high nutritional and medicinal value in the world.Radish is a typical cross-pollinated crop and the heterosis is very obvious in radish-At present,many studies on the heterosis in radish are focused on the molecular marker prediction,general combining ability and physiological index determination,which greatly limits the research on the formation mechanism of radish heterosis from molecular genetic aspect.In this study,the radish inbred lines ’LU127’ and ’YH’ were used as the parents to generate advantage hybrids Fi,and systematically identified the differentially expressed genes and miRNAs of ’LU127’,’YH’ and ’F1’ at the taproot thickening stage for studying the relationship with heterosis.The taproot heterosis formation-related gene RsSUT1 of radish was cloned and isolated,overexpression and suppression vector of RsSUT1 was constructed,which might lay a foundation for the transgenic study of radish.The miRNAs and target genes involved in cytoplasmic male sterility(CMS)occurrence during anther development were identified.The main findings are as follows:1.The radish reciprocal Fi hybrids were generated by using ’LU127’ and ’YH’ as the parents,and the heterosis analysis of the traits were observed.The traits of plant height,leaf divergence,leaf length and taproot length in reciprocal Fi hybrids have obvious mid-parent heterosis(MPH),the values of taproot length and plant weight in reciprocal Fi hybrids were even higher than the parents,reaching the better-parent heterosis(BPH).The differentially expressed genes(DEGs)and differentially expressed miRNAs(DEMs)between the Fi(LU127 as the maternal parent)and its parents were identified by RNA-seq and sRNA-seq analysis.According to the expression patterns of DEGs and DEMs,the results showed that 32.4%of the total DEGs(3888)between Fi hybrid and its parents were non-additively expressed genes(1259),and the number of transgressive genes(842)was two times than the number of parental expression level dominance(ELD)genes(417).15 additively expressed miRNAs,five transgressive miRNAs and 3 parental ELD miRNAs were identified in DEMs.The genetic regulatory network model of the radish taproot heterosis formation was proposed,auxin signal transduction-related genes such as IAAs,TIR1,GH3 and SAURs,Cytokinine signal transduction-related genes AHK2,AHK4,AHP4,ARR4 and ARR6,brassinosteroid signal transduction-related genes TCH4 and CYCD3 were involved in plant hormone signal transduction pathway to regulate cell enlargement,cell elongation and cell division to promote the thickening of taproots in F1 hybrid.The sucrose transporter 1(SUT1)involved in sucrose transport and translocation pathways,sucrose synthase 3 and 4(SUS3,SUS4),sucrose phosphate synthase 1F(SPS1F),β-glucosidase genes(BGLUs)and UDP glucose-dehydrogenase gene(UDG3,UDG4)involved in starch and sucrose metabolism played roles in promoting the accumulation of sucrose and formation of taproot heterosis F1 hybrid by regulating sucrose synthesis and metabolism,glucose metabolism and sucrose transport and translocation.The results will provide a theoretical basis for dissecting the molecular mechanism of heterosis formation and provide a valuable platform for further genetic improvement of yield traits in radish breeding programs.2.The cDNA and gDNA sequences of the RsSUT1(sucrose transporter 1)gene were isolated from the radish.The RsSUTl gene contained two introns and three exons,the full-length of gDNA and cDNA was 1,724 and 1,654 bp,respectively,which contains 1,545 bp open reading frame(ORF).The sequence can be deduced to encode 514 amino acids,The protein SUT1 has 12 transmembrane domains,which belonged to a classic hydrophobic protein.Phylogenetic analysis showed that RsSUTl was closely related to BrSUT1 protein of Brassica rapa.RsSUTl gene was expressed in leaves and taproots of three different radish materials.The expression level of F1 in leaves and taproots were higher than that in its parents.The artificial miRNA sequence was designed to silence the RsSUT1 gene,the pri-amiR-RsSUTl was obtained by overlapping PCR.The engineering bacteria(EHA105)containing overexpression vector of pCAMBIA2301-RsSUTl and suppression vector of pCAMBIA2301-pri-amiR-RsSUT1 were constructed.These results might provide a foundation for the further study of RsSUTl gene function.3.A comparative small RNA-seq was conducted in floral buds of CMS line ’WA’ and its maintainer line ’WB’.A total of 162 known miRNAs belonging to 25 conserved and 24 non-conserved miRNA families were isolated and 27 potential novel miRNA families were identified in floral buds of radish.Of these miRNAs,28 known and 14 potential novel miRNAs were differentially expressed during anther development.The regulatory network of CMS occurrence-related ARF16 targeted by miR160a,TIR1 targeted by miR393a PPR targeted by miR158b-3p,AG05 targeted by miR396b,SPL3 targeted by miR156a and MYB101 targeted by miR159a were involved in auxin signal transduction pathway,regulation of organellar gene expression,miRNA-mediated target silencing and regulation of floral organ development was constructed.These findings could contribute new insights into complex miRNA-mediated regulatory network of CMS occurrence and facilitate further dissection of molecular mechanism underlying heterosis in radish.