Cloning And Functional Analysis Of A Major QTL,qSLWA9 For The Silique Length And Seed Weight In Brassica Napus L.

Rapeseed?Brassica napus L.?is the second-leading source of vegetable oil worldwide and improving its seed yield is always the significant object in B.napus breeding program.Silique is an important reproductive and photosynthetic organ and three major factors?siliques per plant,seeds per silique,and seed weight?influencing grain yield are closely related with silique development.Silique length and seed weight are both two important yield-influencing traits controlled by quantitative trait loci?QTLs?in oilseed rape.Many QTLs have been identified,however,the number of cloned genes underlying these QTLs is quite few at present.In our previous study,we detected a major QTL,qSLWA9,for silique length and seed weight using recombinant inbred lines populations derived from the cross between S1 and S2,two inbred lines that showed significant differences in silique length and seed weight.We developed a pair of near-isogenic lines?NILs?,designated NIL?S1?and NIL?S2?,from a residual heterozygous line containing a heterozygous chromosome segment encompassing the qSLWA9 locus.In this study,we conducted fine mapping for qSLWA9using NIL-F₂ population developed from crossing the NIL?S1?and NIL?S2?.We identified BnaA9.CYP78A9 that underlies the major QTL by map-based cloning,and analyzed the expression pattern and origion of BnaA9.CYP78A9.Besides,we primarily studied the function of BnaA9.CYP78A9 in regulating silique development in rapeseed.The main results were described as follows:1.Fine mapping of qSLWA9.We developed a NIL-F₂ population by crossing the NIL?S1?and NIL?S2?to do fine mapping of qSLWA9 and an F₂ population with 9,737individuals was used for genotype and phenotype analysis.We ultimately narrowed down qSLWA9 to a genomic region flanked by makers SL5 and SL15.This genomic region corresponds to 89-kb and 86-kb regions in the Chiifu-401-42?B.rapa?and ZS11?B.napus?reference genomes,respectively,and SL5 is on unassigned A9_random scaffold 1826 and SL15 is on a chromosome A9 pseudomolecule in the Darmor-bzh?B.napus?reference genome.The region contains 13 predicted protein-coding genes.2.Candidate genes prediction of qSLWA9.According to the annotation of B.napus and B.rapa,we analyzed the expression and sequence of genes in the candidate region and found that the level of BnaA09g55530D transcription in S1 silique valves was much higher than that in S2 and BnaA09g55520D was deleted in S1 genome,which suggested that BnaA09g55520D and BnaA09g55530D were the candidate genes.3.Comparative sequencing and cloning of qSLWA9.We compared the upstream regulatory sequence and coding sequence of BnaA09g55530D between S1 and S2.We did not find any sequence variation in the coding regions but found four single nucleotide polymorphisms in the 3.9-kb upstream regulatory region from the translation start site of BnaA09g55530D,and we identified a 12.3-kb deletion and the deleted sequence was replaced by a 3.7-kb insertion in the distal end of the 3.9-kb upstream regulatory region of the translation start site of BnaA09g55530D in S1.The rapeseed genetic transformation assay confirmed that the insertion of 3.7-kb enhanced the expression of BnaA09g55530D,resulting in long siliques and large seeds.BnaA09g55530D is the Arabidopsis orthologous gene of AT3G61880?CYP78A9?and was designated as BnaA9.CYP78A9.4.Expression pattern and protein subcellular localization of BnaA9.CYP78A9.BnaA9.CYP78A9 was ubiquitously expressed in cotyledons,roots,stems,leaves,flower buds,silique valves,and seeds but preferentially expressed in silique valves.During all stages of fruit development,BnaA9.CYP78A9 was expressed at significantly higher levels in S1 than in S2.The subcellular localization of BnaA9.CYP78A9 was mainly localized to the plasma membrane.5.Analysis of the 3.7-kb inserted in the upstream region of BnaA9.CYP78A9.The 3.7-kb insertion was a partial CACTA-like transposable element?TE?that included one?TGTTTCTTGTAGTG?of the terminal inverted repeats on the right side and a gene encoding a transposase.A series of 5'truncations of the 3.7-kb TE were fused with the minimal 35S promoter to drive GUS reporter gene expression in transgenic Arabidopsis and quantitative GUS assays using transgenic Arabidopsis plants indicateded that the 3.7-kb TE acts as an enhancer and the 3.7-kb TE sequence in fact contain two independent enhancer domains:one located between-4.58 and-4.12 kb and the other located between-4.12 and-3.92 kb from the translation start site,and the latter is weaker than the former.6.The origin of the 3.7-kb inserted in the upstream region of BnaA9.CYP78A9.We developed a presence/absence?PAV?PCR marker to specifically detect the presence or absence of the 3.7-kb CACTA-like TE according to the sequence comparation between S1and S2.We genotyped a set of 245 rapeseed accessions using the PAV marker and the TE insertion was detected in 25 Chinese accessions.In addition,the presence of the TE in oilseed rape was consistently associated with long siliques and large seeds.We also genotyped a collection of 174 Chinese B.rapa lines using the PAV marker and identified three lines containing the TE whose average silique length was much greater than that of lines without the TE insertion.The above results indicated that TE insertion had existed within the B.rapa gene pool in China and was presented in B.rapa predated the formation of the allopolyploidization of B.napus.7.The function study of BnaA9.CYP78A9.The kinetic growth of siliques and transcript levels of BnaA9.CYP78A9 suggested that the long siliques are mainly due to their higher elongation rate and longer elongation phase after fertilization by the increased BnaA9.CYP78A9 transcript levels.The cell size compared in silique valves between S1 and S2 under a scanning electron microscope indicated that the long siliques are mainly due to their greatly increased cell length.The long-silique variety S1 and BnaA9.CYP78A9 highly expressing transgenic plants had higher IAA concentration than the regular-silique variety S2 and transgene-negative plants indicated that BnaA9.CYP78A9 may influence silique elongation development by increasing auxin concentration.