| Canola(Brassica napus) is one of the most important oil crops in the world. In China, more than 85% of the canola production is from Yangtze River basin. However, drought occurrs frequently in Autumun when canola is seeded in this area with the climate change. In order to achieve high seedling establishment under drought conditions, selecting high seed vigor accessions and/or seed type and genetic improvement of drought tolerance at early seedling stages is of great importance for stabilizing and expanding the planting area of winter canola and edible oil production.This project studied seed characteristics, temperature and drought stress on seed germination and seedling growth, evaluated drought tolerance of canola at early seedling stage and flowering stage, and elucidated the gene expression mechanism responding to drought. The main results are as follows:(1) Accessions with different genetic bases varied in germination characteristics. Based on germination speed(T50), 137 canola accessions were grouped into three categories: fast, medium and slow, with 9(7%) in the fast category, 12(9%) in the slow category and the rest in the medium category of germination. Based on T50 and seedling vigor index, four accessions with high seed vigour and four accessions with low seed vigor were identified. Seed germination was not affected by seed weight and seed dormancy, which may be controlled by genetic difference among accessions.(2) Seed germination chacteristics differed due to different seed coat colour and seed size within accession. Canola seeds of Huashuang 5 were sorted based on seed coat color into black, dark brown to light brown, and black seeds and were also sorted based on seed size into large(2.0 mm~2.5 mm), medium(1.7 mm~2.0 mm and small(1.5 mm~1.7 mm). From dark to light seed coat colors, germination percentage and germination index declined and mean germination time increased; emergence percentage, seedling vigor index, shoot and root length decreased; seed oil content decreased but protein and soluble sugar content increased. However, seed size only affect germination speed, thousand seed weight, fresh weight, dry weight, oil content and soluble sugar content. Medium seed showed fastest germination and highest oil content. Therefore, medium and black seed could have the highest early vigor among all seed types.(3) Relatively higher temperature, 35/30 °C, caused physiological damage during seed germination. Investigations were carried out to study the responses of canola(Brassica napus) under contrasting temperature regimes(day/night °C): 35/30(high temperature) and 15/10(low temperature) in comparison with 25/20(optimal temperature) at early seedling stage. The results indicated that high temperature inhibited seedling establishment, while low temperature restrained seed germination. Both high and low temperatures showed detrimental effects on seedling growth as revealed by reduced establishment percentage, seedling vigor index and fresh weight due to accumulated reactive oxygens. High temperature resulted in decreased chlorophyll content, superoxide dismutase(SOD) and peroxidase(POD) activities, while catalase activities(CAT) increased; while low temperature resulted in delayed growth metabolism revealed by increased SOD activities, reduced POD activities, unchanged CAT activities; much higher increment of proline content was observed under high temperature than low temperature.(4) Drought stress occurred at seeding stage inhibited emergence; germination could not occur when osmotic potential was lower than-0.5 MPa; the germinated seeds and seedlings were hard to recover from drought injury. Drought stress was simulated by PEG6000. When Huashuang 5 was exposed to-0.1 MPa ~-1.0 MPa drought stress at seeding stage, germination percentage decreased when osmotic potentials were lower than-0.5 MPa; emergence percentage reduced when the osmotic potential was lower than-0.1 MPa, seedlings could not emerge when the osmotic potential was lower than-0.6 MPa, the lower the osmotic potential, the more reduced the germination and emergence percentage. Huaza 9 and Zhongshuang 11 were exposed to drought stress at 0 d(T0), 1 d(T1), 3 d(T3) and 5 d(T5) after seeding. The earlier the drought stress occurred the more severe the inhibition of germination and emergence. For T0 and T1, the emergence percentages were 0%~12% under-0.5 MPa. Canola couldn’t emerge under-1.0 MPa and-1.5 MPa. For T3 and T5, the emergence percentage was 29%~79% and 5%~40% for the two cultivars, respectively. Canola couldn’t emerge under-1.5 MPa. When recovered from drought stress, the difference between final seedling establishment percentage of T0(-0.5 MPa and-1.5 MPa) and control was not significant; compared with control, the final establishment percentages for T1, T3 and T5 after seeding were significantly reduced, relatively higher reduction was observed under T1 and T5. The final establishment percentages may depended on the germinating status, T1 and T5 were germinated seeds and freshly emerged seedlings, respectively.(5) Shoot length significantly reduced, while root to shoot length ratio increased. Under-0.1 MPa~-0.5 MPa, the seedling growth of Huashuang 5 decreased with the decreased osmotic potential, shoot length decreased by 22.4%~49.5%, root length decreased by 1.1%~15.8%, while root to shoot length ratio increased by 27.9% ~ 67.1%. The earlier the drought stress occurred within 5 days after seeding, the more damage the seedling growth. During drought stress, the highest root length, shoot length, fresh weight and dry weight were observed for T5 and the lowest for T0. When recovered from drought stress, seedling growth was significantly lower than that under control conditions. While relatively better seedlings were observed for T0, and worst seedlings were observed for T1.(6) Root to shoot length could be used as an indicator of drought tolerance. 140 canola accessions were used to evaluate drought tolerance at early seedling stage. Under drought stress(-0.5 MPa), root length and root to shoot length ratio significantly increased by 14.3% and 91.0%, but shoot length and fresh weight significantly decreased by 40.1% and 60.1% compared to the control. Meanwhile, the level of genetic variation detected among all accessions was affected by drought stress. Variation in root length was slightly increased, but variation in root to shoot length ratio greatly increased under drought conditions, which may be of great importance in drought tolerance screening. Base on drought stress index, 12 drought tolerant and 15 drought sensitive accessions were identified. Apart from shoot length, the differences of seedling traits between drought tolerant and sensitive accessions were all significant under drought conditions(-0.5 MPa and-1.0 MPa). Root to shoot length ratios of drought tolerant accessions were 1.1 cm and 1.2 cm longer than those of drought sensitive accessions under-0.5 MPa and-1.0 MPa, respectively.(7) Accessions which were consistently drought tolerant at seedling and flowering stage were identified. Nine canola accessions with diverse drought tolerance at early seedling stage were used to eveluate drought tolerance at flowering stage. Drought stress occurred at flowering stage resuted in 3.4%~37.9% yield decrease. Based on yield decrease, four accessions were identified including two accessions, L129(Zhongshuang 6) and L134(CHINAB), which were consistently drought tolerant and two accessions, L86(Kubanskij) and L130(CR2264/85), which were consistently drought sensitive。(8) Canola drought tolerant candidate genes were identified by combing genome-wide association study and RNA sequencing. 66 accessions representing drought tolerant, medium and drought sensitive accessions were genotyped using 25,495 60 K Brassica SNPs. Through a genome-wide association approach, 16 significantly associated loci were identified. Two drought sensitive B. napus accessions were used for RNA-seq, and differentially expressed genes between normal and drought stress conditions were identified. Altogether 79 candidate genes differentially expressed in both accessions were identified, of which 8 genes were putatively associated with drought tolerance.This study indicated that there were large genetic variations among natural canola populations. For canola production, accessions with fast germination and emergence and high drought tolerance were identified, and dark seed coat color and medium seed size associated with the above selected accessions should be seeded at 15 °C ~25 °C to achieve full seedling establishment by direct seeding. Drought stress caused more damage in germinated seeds and freshly emerged seedlings than ungerminated seeds. The critical point for germination of canola was-0.5 MPa, based on which suitable water management could be conducted to avoid drought damage. Root to shoot length was a reliable indicator for drought tolerance; it was involved in 10 out of 16 identified marker-trait associations. Therefore, the morphology, physiology and genetic mechanism of drought tolerance could be further studied based on root to shoot length ratio. The SNP markers associated with drought tolerance could be used to explore the drought tolerant candidate genes. Water use efficiency and drought tolerance mechanism varied among growth stages i.e. germination, seedling growth, flowering and podding. Based on our results, accessions witch are consistently drought tolerance at seedling and flowering stage were identified and the mechanism needs to be studied at phenotypic, physiological and molecular levels. |
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