| Rapeseed(Brassica napus), one of the most important oil crops worldwide, is also the main fuel, feed and energy crops, which has the largest planting area and the highest annual production in the world. With the improvement of rapeseed yield and quality, and the growing of seed set rate, lodging has become a common problem that should not be neglected. Ideotype can improve utilization efficiency of light energy. So it would be necessary for breeding of the high-yield, high-quality and high-efficiency of B.napus if we can discover and use the variety traits of B.napus. Stem bending as a specific trait of plant type has potential values in rapeseed breeding. In addition, identification and characterization of candidate genes responsible for stem bending trait also provide novel insights into understanding of the genetic mechanisms of stem growth and development in plant.In this study, F1 and F2 hybridization populations derived from cross between B.napus mutant(stem bending1, stb1) and high-yield B.napus cultivar Zhongshuang11(ZS11) were used for the inheritance analysis and QTL mapping based on the rapeseed 60 K SNP chips array and whole genome resequencing-based QTL-Seq method. Then differentially expressed genes(DEGs) between ZS11 and stb1 in the main stem were identified by RNA-Seq and those DEGs with non-synonymous mutation existed between the ZS11 and stb1 were regarded as candidate genes. The main results are as follows:1. The phenotypes of stb1 mutant which derived from a natural mutation of high generation RIL population GH06 × ZY821 and control accession ZS11 were compared in this study. The results showed that the key period of the stem bending occurrs is approximately 10 days before to 5 days after the early flowering stage, which means the period from the end of the stem elongation to the early flowering. We observed that the number of catheter, development degree of stem xylem, sclerenchyma and parenchyma cells in stb1 mutant is far less than the control ZS11 according to the frozen section and electron microscope scanning in the early flowering stage. The ratio of S/G lignin in stb1 was significantly lower than that of ZS11 according to the lignin monomer detection by GC-MS. The phenotype observation confirmed that compared with controls, the development of vascular system was abnormal and lignin components also changed greatly in stb1. After treatwent with topping or auxin spraying on the shoot apica meristem at the beginning of bolting stage and before stem bending occurred, the main stem of stb1 mutants recovered upright, while the lateral branch became more bending than those mutants without treatment. The results suggests that mutation phenotype of stb1 may caused by abnormal metabolism of auxin or polar transport of auxin(PAT).2. The F1 population plants from reciprocal crosses of stb1 and ZS11 all showed upright stem phenotype just as same as the ZS11, implying that the stb1 mutants were controlled by recessive gene. To analyze the genetic law, we examined the 273 individuals in the F2 population, and the results found that there were differences in the degree of bending among different individuals, and the bending initial altitude were not always the same. A total of 215 and 58 plants showed upright and bending stem in the F2 population, respectively. The phenotypic separation ratio between straight and curved stem was about 3:1, and Chi square test value in the Chi-square test was χ2=2.05<χ2(0.05)= 3.84, suggesting that the genetic laws of stb1 mutant might be in accordance with Johann Gregor Mendel autosomal recessive single gene inheritance.3. Among the F2 segregation population, 30 bend-stem plants, 64 straight-stem plants and two parents were choosen for extraction of genomic DNA, which were used for genotyping based on the Brassica 60 K chip array. Of the 52157 SNPs(single nucleotide polymorphisms), a total of 47139 SNP markers could be detected within more than 90 samples, which means that the SNP chip hybridization in our study was successfully and suitable for subsequent mapping analysis. There were 16159 SNP markers showed differences between parent materials ZS11 and stb1 mutant, and 11721 of SNP markers without obvious separation were found to be suitable for genetic linkage map construction. With the SNP genotype data of 94 F2 plants, a genetic linkage map contains 19 linkage groups, including 1840 SNPs, covering 1821.72 c M with an average distance of 0.99 c M were constructed using Joinmap 4.0. The single marker SNP loci analysis made by Win QTL Cartographer 2.5 showed that STB1 gene was mapped on A01 chromosome, and between Bn-A01-p2421445 and Bn-A01-p3939508, positioning interval genetic distance of 1.802 c M. BLASTN analysis of all SNP markers within the intervals against the B. napus genome sequences revealed that the distance between Bn-A01-p2421445 and Bn-A01- p4230829 on the A01 chromosome is 1.995 Mb.4. In total, 3073 DEGs were identified between the upright stem of ZS11 and the bending stem of stb1 based on RNA-seq analysis. Of those DEGs, 1424 and 1649 genes were up-regulated and down-regulated, respectively. GO enrichment analysis analyzed by Bin GO showed that down-regulated in the stb1 mutant mainly enriched in the biosynthesis of phosphoinositide, long sunshine light cycle, polyol biosynthesis, auxin polar transport, ultraviolet(UV) light response, etc. A total of 29 down-regulated genes involved in PAT were also identified. In addition, SAUR41 which associated with cell differentiation and formation of root meristem, ACL5 that related to the stem internode elongation and the development of vascular bundle, and AVP1 that adjust p H value outside the body such as affect ghrelin output protein localization in stb1 mutant were also significantly down-regulated. The expression levels of PAL1, PAL2, PAL4, C4 H, 4CL5 and COMT1 genes involved in lignin biosynthesis in stb1 mutant were also significantly down-regulated. In the interval regions between Bn-A01-p2421445 and Bn-A01-p4230829 and 200 kb of flanking sequence, five down-regulated genes which associated with auxin bio-synthesis, transport and signal transduction were likely to be the candidate genes of STB1. And among them, APM1 and CCD8 participate PAT, ARF16 and IAA11 participate in the signal transduction of auxin, while PRHA is regulated by the auxin positively and closely associated with microtubule organization development.5. Based on the whole genome resequencing data of stb1 and ZS11, the In Del(Insertion/Deletion) mutation loci within the scope of Bn-A01-p2421445 and Bn-A01-p4230829 and the distribution characteristics of on the reference genome sequences were analyzed. According to the results of the sequence alignment, the In Dels that aligned within the range of QTL mapping uniquely were choosen for development of Indel primers using the Geneious 4.8.5. Of the 27 pair of primers, 8 of them could be successfully amplified and showed length polymorphism between stb1 and ZS11. These markers will also be used for fine mapping of STB1 gene in the larger F2 population. |
