QTL Mapping For Yield-related Blade Length And Width In Saccharina Japonica And Functional Validation Of Tic20 Gene

Saccharina japonica is one of the most important economic brown algae which distributed among the coastal areas of east Asia.In this study,the QTL mapping of S.japonica blade length and width traits carried out by constructing high-density linkage map.The identified candidate Tic20 gene’s functional research was carried out in gene cloning,subcellular localization,transmembrane topology analysis and protein interaction.The results are of great significance for analysing the molecular mechanism of S.japonica blade length and width traits’formation and the structure and function of Tic20 in S.japonica.On the basis of SLAF-seq,7627 SNP markers were selected to construct a linkage map with 31 linkage groups and an average genetic distance of 0.69 cM,and QTLs related to blade length and width phenotypes of S.japonica were located and analyzed.In total,12 QTLs contributing to blade length and 10 to width were detected.Some QTL intervals were detected for both blade length and width.Additive alleles for increasing blade length and width in S.japonica came from both parents.After the QTL interval regions were comparatively mapped to the current reference genome of S.japonica（MEHQ00000000）,14 Tic20（translocon on the inner envelope membrane of chloroplast）genes and three peptidase genes were identified.The expression levels of four Tic20 genes were different not only in the two parent sporophytes but also at different cultivation times within one parent.Besides,the SNP markers closely associated with blade length and width could be used to improve the selecting efficiency of S.japonica breeding.Tic20 is an important translocon protein that plays a role in protein transporting into the chloroplast.In this study,we cloned the S.japonica Tic20-14 gene,structural analysis of SjTic20-14 protein revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain and a C-terminal cyanobacterium-originated Tic20 domain.Using Phaeodactylum tricornutum as the transgenic verification model,indicated the innermost membrane localization of SjTic20-14 in the P.tricornutum chloroplast,N_in-C_in-terminal orientation with“M”-type transmembrane topology and the EF-hand domain entirely extruded into the chloroplast stroma.In addition,the trangenosis of SjTic20-14 promoted the growth of P.tricornutum.However,the actual localization and topology and further functional analysis of SjTic20-14 in S.japonica is needed.The high quality S.japonica membrane protein yeast two-hybrid libraries was constructed with light treated high quality S.japonica RNA.The library capacity was1.44×10⁷ cfu/m L with high library integrity and coverage;recombination rate of random amplification was 100%and the length of inserted fragments ranged from 0.4to 2.6 Kb,exhibiting a high abundant of inserted fragments.Using SjTic20-14 as bait to screen the interaction proteins with SjTic20-14,the total number of clones co-transformed into sieve libraries were 5.84×10⁶cfu.Total 99 cDNA sequences were obtained from 272 selected colonies,among which 74 had known functions while 25with unknown functions.Functional interaction analysis revealed that SjTic20-14 is widely involved in various life course like metabolic processes（phosphate-containing compound metabolism,nitrogen compound metabolism,carbohydrate metabolism and DNA metabolism,etc.）,cellular composition（biofilm and dynein）,transcriptional regulation,protein transport and photosynthesis.All the random chose 24 cDNAs for the rotary validation showed positive interaction（100%positive rate）.Quantitative detection of protein interacting strength showed significant differences in 3 types of interactions of the 17 in the 24 chose cDNA sequences of full-length SjTic20-14,EF-hand domain and Tic20 domain,respectively;suggesting that the structural integrity of SjTic20-14 had a certain impact on its function.With BiFC validation indicated the interactions Tic20-14,EF-hand and Tic20 domain with the light harvesting complex subtype 6（LHC6）respectively in P.tricornutum.This also indirectly reflects that SjTic20-14 may affect the photosynthesis of S.japonica by interacting with LHC6,thereby affecting the formation of the phenotype of S.japonica blade length and width,thus affecting the growth of S.japonica.