| Sunflowers(Helianthus annuus) are annual herbaceous plants belonging to the family of Compositae. They are valuable in economy, in food industry and as one of the important oil crops in China. It is rich in genetic resources and has many varieties. The wide range of varieties created through artificial breeding in different habitats, are generally categorized into three types, i.e., the edible, the oil and the intermediate types. Sunflower black stem disease(Leptosphaeria lindquistii) is an important quarantine sunflower fungal diseases. As reported, this disease has occurred occasionally in some limited regions of Xinjiang. In recent years, studies on the morphological identification, characterization, disease symptom analyses, specific molecular detection of the black stem disease has been abundant, and the molecular mechanism studies for sunflower resistance breeding to this pathogen has been sporadic.In this research, the edible and oil type sunflower seeds, each 20 varieties, were used as materials(of which, 24 varieties was from the United States and gifted from Tianjin Entry-exit Inspection and Quarantine Bureau and 16, domestically purchased). the sizes of seeds and kernels, the contents of protein, crude fat and relative fatty acid composition, carbohydrate of the kernels were respectively measured and comparatively analyzed; Genomic DNAs of the 40 varieties of sunflower plants were extracted and used as templates to PCR amplify their ITS sequences by the universal primers ITS1/4 and the products sequenced bi-directionally; these ITS sequences were aligned and compared using Clustalx1.81 and Bio Edit7.0.9 in reference to the registered sunflower ITS sequence in Gen Bank. A phylogenetic tree was constructed with MEGA 5.10; DNASP5.0 were used for diversity analysis; two previously reported susceptible and resistant sunflower varieties, respectively Kangdi KWS303 and MT792 G, was used as the plant materials and the BXC1 isolate we isolated and identified by ITS molecular detection was used as the pathogen. Under controlled laboratory conditions, the stems of seedlings of both varieties were challenged with the black stem pathogen BXC1 isolate. And 10 days after, when disease symptom became evident, fluorescence quantitative PCR was used to detect the expression differences of the selected 16 genes at the symptomized stem tissues of the susceptible and resistant sunflower varieties.The main results are as follows: 1. Results of morphological and main components comparison of the seeds and kernels showed that the edible sunflower seeds and kernels are larger(long / short diameter for seeds: 1.75 ± 0.21 / 0.79 ± 0.08 cm; for kernels: 1.18 ± 0.06 / 0.54 ± 0.05 cm) than the oil seeds and kernels(long / short diameter for seeds: 1.04 ± 0.10 / 0.52 ± 0.06 cm; for seed kernel: 0.88 ± 0.07 / 0.45 ± 0.04 cm); and the seed coats of the former was mostly white striped in gray background; and the seed coats of the latter was black and seeds was full enriched; the average kernel protein content of edible sunflower kernel was larger(19%) than that of the edible sunflower kernels(15%); the oil contents of the edible and oil sunflower kernels were in the range of 30.03%~42.37% and 30.00%~47.24%, respectively; the main fatty acid components of sunflower seed oil were palmitic, stearic, and linoleic acid, with a total relative content of oleic and linoleic acid of about 90%; 70% of the edible sunflower seeds had a relative linoleic acid content more than 60%, and 58% of the oil sunflower seeds had a relative oleic acid content more than 80%; the contents of total sugar, reducing sugar and non-reducing sugar between the edible and oil sunflower seed kernels had no significant difference. The differences in the contents of some major components(such as kernel protein, fatty acid components) between the edible and oil type sunflower seed kernels could be used as targets in breeding and selecting of novel sunflower varieties. Sunflowers are divided into edible and oil types, mainly based on its seed coat morphology, seed and kernel size and convenience in hand-hulling and eating.2. ITS sequence analysis and clustering results show that sunflower ITS sequences are 650 bp in total length, of which, 267 bp ITS1, 159 bp 5.8S and 224 bp ITS2; sunflower ITS sequences have 9 single nucleotide polymorphic sites and the GC content is 52.4%; there are three haplotypes with a haplotype diversity(Hd) of 0.532. Results from the haplotyping and phylogenetic analysis of the sunflower ITS sequences are in good accordance and, for the first time, indicate that the edible and oil sunflower varieties are basically clustered separately and it is quite likely that the edible sunflower varieties were descended from the oil type varieties. This novel result provides a basis for further phylogenetic studies among sunflower varieties. 3. Gene expression analysis results showed that the expression levels of the 6 genes, PP2 A, Mn SOD1, HDZTF, BCCP, ELT and PDE in the control of the resistant plant were all lower than those in the susceptible plant. Ten days after the inoculation, the expression levels of these genes increased significantly(up-regulated), while their expression in the susceptible plant was significantly reduced(down regulated), indicating that they are likely related to resistance; the expression levels of the 7 genes, TST, CPC, CAT, Cu Zn SOD1, Cu Zn SOD2, MYBR and MYBF in the control of the resistant plant were higher than those of the susceptible plant, and 10 days after inoculation, their expressions were significantly up-regulated in the susceptible plant, while the their expressions in the resistant plant were significantly down-regulated, suggesting that these are most likely susceptible genes to the pathogen; the expression levels of the remaining 3 genes, PAL2, POD and WRKY were higher in the control of the susceptible plant than those in the resistant plant; 10 days after inoculation, the expressions of the 3 genes were all down-regulated in both the susceptible and the resistant plant, indicating that they are probably generally responded genes in sunflowers to this pathogen. |
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