A semi-dwarf gene BnaA6.rga-ds was previously obtained from Brassica napus mutant ds-1 through map-based cloning. It encodes a gain-of-function mutant DELLA protein that constitutively represses the signal transduction of gibberellin acid (GA). In this research, it was predicted that both BnaA6.rga-ds and its wild-type BnaA6.RGA were acidic proteins. Sequence alignment of amino acid showed that BnaA6.RGA and its homologues of other plant species had a C-termini which were more conservative than their N termini. Prediction of secondary and tertiary structures indicated that the protein mainly contained α-helixes and random coils. Phylogenetic analysis revealed that BnaA6.RGA had high homology to RGAs from other Brassicaceae plants, including Arabidopsis, Thellungiella halophila and Capsella rubella. BLAST in B. napus genome showed that BnaRGAs had 4 copies. Subcellular localization showed that both BnaA6.RGA and BnaA6.rga-ds were located in nucleus. It suggested their function as transcription regulators of downstream genes. BnaA6.rga-ds gene was transformed into B. napus cv Westar by Agrobacterium-mediated method, 13 positive transgenic plants were obtained and dwarf phenotype were found among them. These results suggested that BnaA6.rga-ds gene could regulate the height of Brassicaceae plants.
Soybean [Glycine max (L.) Merr.] is an important source of plant protein. Identification of quantitative trait loci (QTL) for protein content in soybean could provide a base for molecular marker-assisted selection breeding and elite genes-discovering in soybean. Advanced backcross lines were developed with variety Zhonghuang 13, an elite and popular variety in Yellow-Huai-Hai Valley as the recurrent parent, and a Japanese cultivar Dongshan 69 as the donor parents. Segregation of SSR markers in random and extremely selected BC2F2 populations of Zhonghuang 13 × Dongshan 69 were analyzed, and QTLs of protein content were detected with one-way ANOVA and Chi-square test. The results showed that: (1) A total of 42 QTLs associated with the protein content were detected in four populations by the two methods. 5 QTLs for protein content linked to Sat_202, Satt170, Satt282, Sat_074 and Satt146 were identified repeatedly in both random and extremely selected populations; (2) 5 main effective QTLs for protein content linked to Sat_077, Sat_202, Satt282, Satt146 and Satt712 were identified in both random and bilaterally selected BC2F2 populations by one-way ANOVA (P≤0.05). They were located in linkage groups C1, C2, D1b, F and J. Satt282 and Satt712 had the highest effective values, with F value 8.77, 7.80 and 9.01, 11.61 respectively; QTLs linked with these markers could explain 13.55%, 21.40% and 13.85%, 28.82% phenotypic varation in the two populations, respectively. (3) 8 QTLs detected by Chi-square test in the extremely selected population were the same as those identified by One-way ANOVA in random population. In total, 42 QTLs related to protein content were mapped. One-way ANOVA was an effective method for bilateral selected populations, Chi-square test was more effective in QTLs detection for directional selected populations. Detection of these QTL could facilitate the high protein breeding in soybean variety.
To breed cold tolerant Brassica rapa in Northern China, recurrent selection strategy were used with 4 winter rapes in 2 circumstances as Lanzhou and Jiuquan. Over-winter ratio and agronomic traits were investigated using 3 generations at the same location. Results showed that over-winter ratios were significantly increased in higher recurrent selected generations. A significant linear algebra was found between over-winter ratio and recurrent selection, but the selection effects were significant different among the materials. Plant height, silique density on main inflorescence, seed per silique, thousand seed weight and single plant yield were increased in higher generations, except silique per plant. The increase of over-winter ratio, the above traits and their corresponding genetic gains were relatively lower in cold tolerance varieties. Ecological environment obviously influenced recurrent selection effect. The over-winter ratios were lower in Jiuquan which was more frigid, but the genetic gains were higher. Correlation and stepwise regression analysis showed that plant height and silique density on main inflorescence were significantly correlated to single plant yield. The above traits had direct positive contributions to single plant yield in C0 population, but they changed in higher generations in C1 to C3. However, silique per plant had direct negative contribution to single plant yield in C1 and C2 from Longyou 6 (highly tolerant) and C1 and C3 from Tianyou 2 (tolerant). In conclusion, recurrent selection in much colder environments was effective to breed cold tolerance winter rape.
SNP genotyping by High Resolution Melting Curve (HRM) is a simple and effective, higher sensitivity and stronger specificity method. Optimization and establishment of soybean HRM-based SNP genotyping system is the precondition and basis for soybean SNP research in future. In this study, small amplicon after adding the internal calibration was used to optimize and establish SNP genotyping system. The results showed that it was better to make PCR product length 50-80 bp, the melting temperature at 72-82 °C and Tm value between 55-62 °C when designing a SNP primer. A total of 10 μL PCR reaction system should contain 25 ng DNA template, 0.6 pmol SNP primers and 1 μL LC Green dye. After PCR reaction, 3 pmol high and low temperature internal calibration should be added to each wells, and then denatured for HRM analysis. Moreover, the optimized SNP genotyping system was used to genotyping a recombinant inbred lines (RILs) population, and the results demonstrated that the RILs could be completely divided into 2 genotypes of their parents by the optimized system, with improved accuracy and efficiency. The optimized SNP genotyping system established in this study will provide a useful foundation for developing SNP markers, constructing high density genetic maps, QTL identification in the future.
For sesame breeding and production, spontaneous outcrossing rates (SOR) were investigated using 2 pairs of morphology markers under 2 planting patterns in 5 areas during 2010 to 2012. In one pair, cultivars with determinated growth habit (D) and closed capsule (C) were used. Both traits were controlled by recessive single genes. Corresponding normal (N) trait materials were used as allogeneic pollen sources. In the other pair, cultivars with monofloral (M) and trifloral (T) traits were used. The monofloral was controlled by a dominant single gene and was used as allogeneic pollen source. The 2 patterns were mixed and drilling. Results showed that, under mixed planting patterns, SOR was 5.13% - 23.35% with an average of 12.69%. There were great differences among different planting region, years and cultivars. SOR was directly related to distances among allogeneic pollen sources, SOR gradually decreased with the increase of planting rows. There were differences among planting regions. The SOR were not detected beyond allogeneic pollen sources of about 20-30 lines (8~12m). This study suggested an effective isolation distance of 400m and a buffer zone of 8-12m, which was sufficient to meet the demand of sesame seed purity of 99.9%.
Root exudates of big peanut (Arachis hypogaea L.) variety with strong continuous cropping obstacle resistance Luhua 11 and small peanut variety with weak continuous cropping obstacle resistance Luhua 12 secreted during seedling and flowering stages after continuous planting for different years were collected by continuous collection method, and their main components were identified by the method of gas chromatography-mass spectrometry. Contents of phenolic acid (aldehyde) in peanut continuous cropping soil were determined by using liquid chromatography. Results showed that 11 and 8 kinds of components were identified from the root exudates of Luhua 11 at seedling and flowering stage respectively. Compared with Luhua 11, cinnamic acid was not found in the root exudates of Luhua 12 at seedling stage, and hexanehexol were found at flowering stage additionally. Root secretion of p-hydroxybenzene phenylmethyloctanoic acid of Luhua 11 increased obviously after continuous cropping for 5 years. Luhua 12 root secretion of long chain fatty acid substance palmitic acid and stearic acid and alcohol substance mannite increased significantly after continuous cropping for 5 years. After these 2 varieties of peanut continuous cropping for 5 years, contents of phenolic acids (aldehyde) allelochemicals p-Hydroxybenzoic acid, phthalic acid, cinnamic acid, 2,5-Dimethylbenzaldehyde and 2,6-Di-tert-butylphenol in soil presented a cumulative trend. And the total accumulation of Luhua 11 was greater than that of Luhua 12. The responses of root exudates of different peanut variety with different resistance to continuous cropping were inconsistent. Continuous cropping can affect the secretion and accumulation of peanut root exudates, which affect the strength of the allelopathy.
Maize-soybean relay strip intercropping is one of the widespread practices in south western and southern China soybean production area. Soybean grows under shade and subsequent lighting recovery in maize-soybean relay strip intercropping. To select soybean variety with better adaptation to intercropping, 2 soybean varieties (Gongxuan 1 and Guixia 3) were planted in intercropping , and their growth responses to shade and lighting recovery, their yields were compared in this study. During shade period, aboveground dry mass and leaf area of both varieties reduced, mass allocation increased in stem but decreased in leaf. Gongxuan 1 had higher leaf mass allocation ratio than Guixia 3, and response index (RI) to intercropping for aboveground dry mass and leaf area were lower than those of Guixia 3. During light recovery period, aboveground dry mass and leaf area increased rapidly, and the preference of leaf mass allocation ratio declined. Gongxuan 1 showed higher aboveground dry mass, leaf mass allocation ratio and leaf area than Guixia 3. No difference on yields between 2 varieties in intercropping were observed, but only Guixia 3 showed significant reduction on yield in intercropping compared with monoculture. Yield of soybean in intercropping were positively correlated to aboveground dry mass and leaf area during recovery period. RI of yield showed significant correlation with RIs of aboveground dry mass, and leaf area during both shade and recovery periods. In conclusion, soybean varieties better adapted to relay strip intercropping could accumulate more aboveground dry mass and form larger leaf area during light recovery period, and were less affected during shade period.
To understand light-temperature characteristics for introduced spring-sowing rapeseeds in Tibet, factor analysis was applied on 18 semi-winter rapeseed (Brassica napus L.) cultivars from the southern and eastern China. Results showed that the accumulated variance contribution rate of 3 common factors (light-temperature factors at vegetative stage, budding and flowering periods, silique stage) accounted for 100%, reflecting total 9 light-temperature indicators and their relationships. During the whole growth period, the most influenced period by light-temperature was nutritional period, followed by budding and flowering stages. Cultivars from regions adjacent to Tibet were light-temperature insensitive, the others were light-temperature sensitive. t test showed stronger vegetative growth and longer growth period of sensitive cultivars. But no significant yield difference was found. It could be concluded that in Tibet spring-sowing area, the semi-winter rapeseed insensitive to light-temperature could be used for breeding the high-yield and early-mature variety.
To optimized nitrogen management on oil flax/soybean intercropping system, field experiment was carried out to investigate N effect on dry matter accumulation and yield of 2 crops. 3 different N rates (0, 75 and 150 kg/hm2) were applied on monocropping and intercropping respectively. Results showed that fitted logistic equations of oil flax dry matter accumulation were significant at P<0.01. The maximum rate of dry matter accumulation of monocropping oil flax occurred at flowering stage. It moved to fruiting stage under 150kg/hm2 N on intercropping oil flax. Under monocropping, total dry matter accumulation and grain dry matter accumulation were increased with N application, and reached the highest under 150kg/hm2 treatment (T3). Under intercropping in contrast, 75kg/hm2 N application (T5) led to the top dry matter accumulation. Compared to monocropping, the maximum intercropping oil flax yield (as 3 204.80kg/hm2) was obtained due to higher translocation of dry matter (before anthesis), higher dry matter accumulation (after anthesis), and higher contribution rate of dry matter assimilation to grains. Additionally, mutually beneficial effect was greater than competition effect in oil flax/soybean intercropping system. In conclusion, half N application (75kg/hm2 N) under flax/soybean intercropping obtained high yield of 3 204.80kg/hm2 flax seeds
To regulate safflower growth and seeds quality, hormone effects were investigated by using GA (30, 100, 170mg/L) and its biosynthesis inhibitor PP333 (30, 90, 150mg/L). Hormones were sprayed at elongation stage. Several agronomic traits were measured and fatty acid in vegetative organs and seeds were analyzed by gas chromatography/mass spectrometry (GC-MS). Results showed that GA increased plant height and branch length significantly, and decreased primary branches and total capitulums per plant. Initial flowering date was delayed for 1-4 days. GA (30mg/L) increased seed linoleic acid to 67.83%, which was significantly higher than that of water control (62.58%). PP333 significantly decreased branch length, delayed the initial flowering date for 1-2 days with little effects on other traits. PP333 (150mg/L) increased seed oleic acid to 20.05%, which was significantly higher than that of control (15.24%). It also remarkably increased the seed O/L at 20d after flowering. These results indicated that 30mg/L GA could be used to improve seed linoleic acid, and 150mg/L PP333 to improve oleic acid.
To investigate the effects of molybdenum (Mo) fertilizer on different rapeseed (Brassica napus) genotype, field trials were conducted using 30 varieties. Results indicated that SPAD value of rapeseed seedlings, dry matter weight and yield of matured plants were increased under Mo application (+Mo, ammonium molybdate 0.75kg/hm2) treatment. The maximum increase rates were 20%, 29.53% and 37.68% respectively. The 1 000-seed weight was increased and pod numbers were decreased. No significant difference was observed in seed number per pod between Mo application and control treatments (-Mo, zero ammonium molybdate). Mo application increased Mo content in root, straw, shell and seed. It also increased plant Mo accumulation. Among them, plant Mo content and accumulation were increased by 6.1% - 88.2% and 70.5% - 87.5% respectively. Mo content differences among genotypes reached 5 folds. It suggested that higher Mo content genotypes could be selected, and Mo fertilizer could be used to improve the rapeseed Mo content for human nutrition.
To understand the anti-virus function of SGS3 gene in Brassica napus, gene BnSGS3 was cloned from rapeseed cultivar Zhongshuang 6 by 5'-RACE and nested PCR. Sequence analysis showed that BnSGS3 includes 2 638bp, 4 introns and a 1 824bp complete ORF. DNAMAN comparison result showed that the similarities between BnSGS3 and other SGS3 genes were 54.9% to 97.5%. Over-expressing vector BnSGS3-Ov and interfering vector BnSGS3-Si were constructed and transformed into Arabidopsis thaliana Columbia ecotype by floral-dip method. 30 transgenic plants of each vector were obtained verified by herbicide spray and PCR identification. After cucumber mosaic virus (CMV) inoculation, BnSGS3-Ov transgenic Arabidopsis plants showed moderate resistance to CMV with slighter symptoms than those from BnSGS3-Si transgenic plants. These BnSGS3-Ov plants could grow and flower normally. But BnSGS3-Si plants were seriously infected by CMV with abnormal plants and no flower. Youcai mosaic virus (YoMV) inoculation result showed that both transgenic and wild type plants were susceptive. It suggested that BnSGS3 gene conferred moderate resistance to CMV, but no obvious resistance to YoMV.
The pathogens of scorch spot disease were isolated from peanut leaves collected in Linshu, Shandong Province and other places. They were identified as Leptosphaerulina arachidicola by sequence analysis of genomic ITS region. 3 of 12 Linshu isolates were resistant to carbendazim on PSA medium containing 33.3mg/L carbendazim, while other isolates were susceptible. Carbendazim resistant isolates could grow on PSA medium containing 3333.3mg/L carbendazim and were also resistant to 30% azoxystobin, 20% tebuconazole+hey oxime amine, and 70% polyram, but susceptible to 30% tebuconazole, 10% difenoconazole, propiconazole, 25% mancozeb +8% triazolene and 70% propineb. There was no significant difference in colony diameters between carbendazim resistant and susceptible isolates on PSA medium after 12 days.
Nowadays, peanut web blotch caused by Phoma arachidicola is a serious leaf disease, which is distributed widely in the production field of peanut. In order to evaluate the new fungicides on peanut web blotch disease, the indoor toxicity, field control effect agronomic traits and pod yield of 13 types of fungicides for pathogenic fungus of peanut web blotch were tested. The results indicated that EC50 value of propiconazole of 1.9344mg/L was the most effective, and EC50 values of boscalid, kresoxim-methyl-boscalid, carbendazim and difenoconazole were less than 6 mg/L. In field test, 100.00 mg/L propiconazole was the best and could control 67.90% disease, and further increased the yield of 43.86%, compared to control; followed by pyraclostrobin-metiram, boscalid with controlling effect of 64.93% and 62.84% respectively. According to the indoor toxicity and field control tests, propiconazole, boscalid, pyraclostrobin-metiram, Kresoxim-methyl-boscalid and difenoconazole were effective in controlling peanut web blotch disease. These fungicides were used in rotation in different period of disease, to reduce the resistance and control field peanut web blotch disease effectively.
To unravel the effects of cAMP on biological characteristics and pathogenicity of sunflower pathogen Verticillium dahlia, growth rate, conidia production, germination rate of conidia spores, microsclerotia production, crude toxin and pathogenicity of V. dahliae were measured after V. dahlia cultured in medium with 10 mmol/L cAMP. Results showed that pathogen conidia was increased by 55.45% in the medium, but conidia germination rate, microsclerotia yield, crude toxin production and disease index were reduced by 69%, 46.8%, 35.27% and 7.28% respectively. In conclusion, exogenously cAMP promoted V. dahliae conidia yield, inhibited conidia germination rate, and reduced microsclerotia yield, crude toxin production and pathogenicity. Among them, cAMP inhibition effect on microsclerotia formation was greater.
To biocontrol the sunflower Sclerotinia rot, 135 endophytic bacteria strains were isolated from Orobanche cumana, a kind of root-holoparasitic weed of sunflower. The bacteria were dual cultured to investigate the inhibition rate of Sclerotinia mycelia growth. Then antagonistic effects were tested on leaves in vitro, and sclerotia germination were screened under bacteria against Sclerotinia sclerotiorum of sunflower. Strains LIEH 92 and LIEB 54 were found efficiently suppressing S. sclerotiorum with efficacy of 68.48% and 62.33% on detached leaves. Sclerotia did not germinate under sterile fermentation filtrate treatment. Thus the 2 endophytic bacteria had great potentials in biocontrol on sunflower sclerotinia rot. Except ositive xylose fermentation and negative Vopes-prokauer test, strain LIEH 92 had the same morphological and physio-biochemical characteristics as Serratia marcescens. 16S rDNA phylogenetic analysis showed that the majority of strains belonging to Serratia of enterobacteriaceae had over 99% similarity to LIEH 92. Thus the strain LIEH 92 was preliminarily identified as a new subspecies of S. marcescens.
For a long time, determination of relative percentage content of individual fatty acid was used as the main determination method for soybean fatty acid, which was hard to reflect the absolute concentration of fatty acid in soybean seeds. Therefore, it is important to develop an absolute quantitative method for fatty acid determination. In this study, a quantitative method for fatty acid determination was established through heated-methylation extraction method and gas chromatography (GC) analysis. Five individual fatty acid methyl esters (i.e. methyl palmitate, methyl stearate, methyl oleate, methyl linoleate and methyl linolenate) were used as the standard samples. According to the standard curves (R2 > 0.99) and the regression equations of five kinds of fatty acid methyl esters, the absolute concentration of fatty acid of soybean seeds could be determined accurately. Through determination of fatty acids in four soybean varieties with different oil content and comparison of the total fatty acid contents with the total fat contents of them, we suggested the extraction efficiency and detection reproducibility of fatty acid could be significantly improved. In addition, the total concentration of five fatty acid detected by this method accounted for more than 94% of total fat content in soybean seeds. It concluded that this method could be used to detect not only the relative percentage content but also the absolute concentration of individual fatty acids in soybean seeds, which could play an important role in soybean fatty acid determination and breeding.
To optimize the extraction technology of peanut flavonoids, the effects of extraction solvent, solid-liquid ratio, extraction time and temperature on flavonoids extraction rate were determined by using peanut leaves and roots. Results showed that the optimum conditions for extracting flavonoids in dry leaves and roots were to shake extraction under dark conditions at 25℃ for 18h using 70% ethanol as extraction solvent with 1:233 of solid-liquid ratio. Alternatively, peanut flavonoids were extracted using 70% ethanol at 60℃ for 1h. For fresh leaves of peanut, the optimum extracting conditions were to shake extraction under dark conditions at 25℃ for 18h using 100% ethanol with 1:40 of solid-liquid ratio. Alternatively, peanut flavonoids were extracted using 100% ethanol with 1:40 of solid-liquid ratio at 80℃ for 1h. Finally, the extract solution was filtrated and used for measurement of flavonoids by Al(NO3)3 colorimetry. Total flavonoids contents in leaves of different peanut cultivars showed significant differences, ranged from 2.97 to 11.1mg/gDW. Much higher flavonoids contents were detected in Nongda818, Dabaiyu and Huayu20.
A method was developed for determination of daidzein in vegetable oil by solid phase extraction-high performance liquid chromatography (HPLC) tandem mass spectrometry. The extraction procedure and liquid chromatography tandem mass spectrometry conditions were optimized. The linear regress equation was Y=8 544.77×X+58 269.4, with correlation coefficient of 0.998 5. The limit of detection and limit of quantification were 0.02μg/kg and 0.06μg/kg respectively. The recoveries of daidzein in oil samples were in the range of 97.6-117.2% with RSD less than 8.3%. The results showed that only soybean oil contained daidzein. The proposed method could be used in detecting daidzein and adulteration in vegetable oil, which further provided critical technical supports to ensure the vegetable oil quality and consumer security.
Branch and pod angles are important plant architecture traits, which are closely related to ideal plant type, yield, high-density cultivation and mechanical harvesting. For simple and accurate collection of these angles, digital image method was used to set up an angle measurement method for rapeseed. According to their branch angles, 22 materials were measured and grouped into 3 typical types: compact (<30º, 3), loose (>40º, 11) and intermediate (30º-40º, 8). According to pod angles, the materials were grouped into 2 types: horizontal (close to 90º, 3) and inclined (<80º, 19). To test the accuracy of this method, 5 materials were studied by comparing with the traditional manual measurements. T test and ANOVA analysis showed a high conformity on angle distinguishing. The high consistency of standard curve was y=1.019 2x-1.363 2 with corresponding coefficient of determination as 0.991 9. It indicated that the above image processing was simple, accurate and easy to apply to rapeseed.
Based on ASP.net and VB.net technology, a network database of Chinese peanut varieties and their genealogy were constructed by Microsoft SQL Server 2008. In this database, a total of 2 498 varieties, including the improved varieties, native varieties and introduced varieties were recorded. Among which, 565 were examined and approved. Among them, 254 were widely used by farmers. The database can provide an excellent tool for peanut variety retrieval, genealogy tracing and key parent evaluation.
To build an intelligent image diagnose technique, an intelligent diagnose of nutrients deficiency in rapeseed (Brassica napus L.) were proposed according to color variation characteristics of leaves from nitrogen, phosphorus and potassium deficiency. Using the Kawasaki soilless formula, rapeseed seedlings were cultivated and leaf images were collected. An image library was created with 4 types (normal, N deficiency, P deficiency and K deficiency). Template image with 4 gradients was set up using the GrabCut algorithms on significant color feature images. Others were subdivided into training and testing images. Matching indexes were calculated between with the training and template images using color histogram backprojection, which were utilized to train the Bayesian classifier for classification characteristics. Matching indexes of the testing images were computed into the classifier for obtaining the nutrients deficiency diagnostic of rapeseed. All algorithms were implemented with VC++ and Open CV. Results showed that the proposed method was able to accurately identify the common nutrients deficiency. Thus the above image diagnose could provide a constructive example for other nutrient deficiency diagnose.
Phenolic acids are natural antioxidant ingredients. Esterification improves the lipophilicity of phenolic compounds, and also improves their biological activities. This paper reviewed the research progress of chemical and enzymatic synthesis methods of lipophilic phenolic acid esters. Their antioxidant activity was also included. Problems on synthesis method and antioxidant activity evaluation method were discussed with future application. It was to provide supports for wider use of lipophilic phenolic acid esters in food, pharmaceuticals, cosmetics and other fields as novel lipophilic antioxidants.
