Table of Content

15 June 2014, Volume 47 Issue 12

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Dissection of Genetic Components in the New High-Yielding Wheat Cultivar Chuanmai 104

LI Jun-1, 2 , WAN Hong-Shen-2, YANG Wu-Yun-2, WANG Qin-2, ZHU Xin-Guo-2, HU Xiao-Rong-2, WEI Hui-Ting-3, TANG Yong-Lu-2, LI Chao-Su-2, PENG Zheng-Song-4, ZHOU Yong-Hong-1

Scientia Agricultura Sinica. 2014, 47(12):  2281-2291.  doi:10.3864/j.issn.0578-1752.2014.12.001

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【Objective】The objective of the study is to dissect the genetic components of the new high-yielding wheat cultivar Chuanmai 104 developed by crossing wheat cultivars Chuanmai 42 with Chuannong 16 and detect the parental contribution to it. 【Method】Chuanmai 104 and its parents were genotyped using all the involved markers covering the whole genome to dissect the genetic components of Chuanmai 104. The parental contributions to the yield-related function of the genomic regions of Chuanmai 104 from each parent were also analyzed according to the authors’ previous-identified QTLs.【Result】Among the 859 polymorphic genetic loci (22 were missing in Chuanmai 104), the alleles at 522 loci in Chuanmai 104 were from the parent Chuanmai 42, while the alleles at the other 315 loci were from the other parent Chuannong 16. Therefore, Chuanmai 104 inherited more alleles from the parent Chuanmai 42 (60.8%). Parental contributions to Chuanmai 104 differed among A, B and D genomes and chromosomes. The frequencies of the alleles from Chuanmai 42 at the investigated loci of A, B and D genomes were 55.00%, 60.20% and 67.27%, respectively. These loci from Chuanmai 42 were broadly distributed on chromosomes 3A, 5A, 7A, 1B, 5B, 7B, 3D, 4D, 5D and 7D, and the other loci from Chuannong 16 were located on chromosomes 4A, 3B, 4B, 6B, 1D, 2D and 6D. A total of 68 genomic regions with the genetic distance larger than 5 cM were detected in Chuanmai 104, which were inherited from its parents and the total genetic length was 3,089.6 cM all over the whole map. Among these genomic regions, the parents Chuanmai 42 and Chuannong 16 contributed about 36 and 32 genomic regions to Chuanmai 104, respectively. The genomic regions inherited from Chuanmai 42 were distributed on chromosomes 3D, 5D, 7A, 7B and 7D, and the other regions from Chuannong 16 were located on chromosomes 3B, 4B and 6D. Chuanmai 104 inherited more genomic regions from Chuanmai 42 in A and D genomes and the genomic regions inherited from Chuannong 16 were more than those from Chuanmai 42 in B genome. In these detected genomic regions on chromosomes 1B, 1D, 2B, 4A, 4D, 5A, 5B, 5D and 7A, about nine genomic regions with Chuanmai 42-type haplotype and five genomic regions with Chuannong 16-type haplotype were significantly associated with yield-related traits by QTL mapping using the Chuanmai 42×Chuannong 16 RILs, respectively. In the yield-related genomic regions, the QTL alleles increasing the spike number per square meter on 1D, 2B and 4A chromosomes were provided by the parent Chuannong 16, while QTL alleles associated with more grain number per spike on 1BS chromosome arm and 4A chromosome were from the other parent Chuanmai 42. Moreover, Chuanmai 104 inherited the QTL alleles with higher thousand-kernel weight from both parents on 5B, 4A and 4D chromosomes, respectively. The pyramiding of these additive QTL alleles from each parent enhanced yield-related traits, which led directly to the character of high yield potential of Chuanmai 104. 【Conclusion】The parental contributions of Chuanmai 42 and Chuannong 16 to their offspring Chuanmai 104 in a whole genome scale was confirmed. Chuanmai 104 inherited the desirable properties of higher grain number per spike from Chuanmai 42, spike number per square meter from Chuannong 16, and thousand-kernel weight combined from both parents by phenotypic and QTL analysis. These characteristics are the genetic basis contributed to the high yield potential of Chuanmai 104.

Isolation and Functional Analysis of Salt-Responsive Gene TaSRP in Wheat

HU Di-1, 2 , XU Zhao-Shi-2, CUI Xiao-Yu-2, CHEN Ming-2, LI Lian-Cheng-2, MA You-Zhi-2, ZHANG Xiao-Hong-1

Scientia Agricultura Sinica. 2014, 47(12):  2292-2299.  doi:10.3864/j.issn.0578-1752.2014.12.002

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【Objective】 Salt is one of the major constraints to yield and quality of wheat in China. How to improve salt tolerance of crops has become an important objective of breeding. The study provided experimental data for further study of the function and the mechanism of TaSRP gene. 【Method】 TaSRP was obtained by analysis of salt treatment wheat de novo transcriptome assembly. Homologous sequences of TaSRP in O. sativa, Arabidopsis and Glycine max were selected using the NCBI BLAST analysis program. Homology analysis and multiple alignments were performed with DNAMAN and MEGA5.05. Real-time PCR of TaSRP specific primers was used to analyze the expression patterns under various abiotic stresses. The coding region of TaSRP was fused to the N-terminal end of GFP under control of the CaMV 35S promoter as 16318hGFP-TaSRP. The 16318hGFP- TaSRP and control vectors were transformed into Arabidopsis protoplasts for subcellular localization. Results were visualized by a confocal microscopy after 16h at room temperature under dark conditions. The promoter region of TaSRP was amplified by PCR using specific primers. Cis-elements responding to abiotic stresses were analyzed by referring to the plant cis-element database PLACE （http: //www.dna.affrc.go.jp/PLACE）. Full length TaSRP cDNA was ligated into the vector pBI121 under control of the CaMV 35S promoter to construct an expression vectors for wild-type Arabidopsis plants. The pBI121-TaSRP construct was introduced into Agrobacterium tumefaciens C58C1 strain cells. Transgenic plants were used for salt resistant experiment. 【Result】 TaSRP, a ricin B-like lectin gene, was obtained by searching the results of salt treatment wheat de novo transcriptome assembly. TaSRP, containing a EUL domain, belongs to EUL family. Homology analysis and multiple alignments showed that there was a high degree of sequence similarity between TaSRP and OSR40 (OSR40g3, OSR40g2 and OSR40c1), but a low degree of sequence similarity between TaSRP and Arabidopsis and G. max. The subcellular localization assay indicated that TaSRP located in cytoplasm. RT-PCR analysis revealed that the expression of TaSRP was upregulated by ABA and salt, but not affected by drought. Isolation of the TaSRP promoter revealed a core promoter element and some cis-acting elements responding to abiotic stresses. Analysis of real-time quantitative PCR showed that TaSRP was up-regulated by high-salt and ABA stresses. The total root lengths of three TaSRP lines were significantly longer than those of wild-type plants in salt treatments (125 mmol•L-1 and 150 mmol•L-1 NaCl) in Arabidopsis plants. These results indicated that TaSRP lines improved salt tolerance of Arabidopsis. 【Conclusion】 TaSRP increased tolerance to salt stress of Arabidopsis plants.

Research on Heredity and Gene Expression Differences for Nitrogen Metabolism-Related Indices of Wheat Parents and Their Hybrids at Seedling Stage

WANG Lin-Lin-1, WANG Ping-1, 2 , WANG Zhen-Lin-1, SUN Ai-Qing-1, YANG Min-1, WANG Chun-Wei-1, YI Xiao-Mei-1, HAN Xiao-Yu-1, YIN Yan-Ping-11

Scientia Agricultura Sinica. 2014, 47(12):  2300-2312.  doi:10.3864/j.issn.0578-1752.2014.12.003

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【Objective】This study aims to analyze the heredity and gene expression differences of wheat nitrogen metabolism- related indices, which will provide a theoretical basis for selecting wheat cultivars of high nitrogen utilization.【Method】Six wheat varieties with different nitrogen use efficiency and their three hybrid progenies were used as materials which were grown in Hoagland’s nutrient solution with 3 nitrate levels at seedling stage, that are low nitrogen (LN), normal nitrogen (CK) and high nitrogen (HN) at 0.2, 4 and 8 mmol•L-1, respectively. Heredity of morphology, nitrogen metabolism-related enzymes, nitrogen content and nitrogen accumulation in wheat seedling stage and gene expression differences of root and leaf under three nitrogen levels were explored. 【Result】Root length and fresh weight under low nitrogen level were significantly higher than those under normal nitrogen level and their negative heterosis was observed. Seedling height and fresh weight indicating mid-parent heterosis and even super-parent heterosis showed a parabolic trend with the nitrogen level increasing. Root length declined quickly originally and then leveled off with increasing level of nitrogen, while seedling height increased quickly at first and then trended to be stable with the rate of nitrogen increasing. The variation of fresh weight in root and seedling were consistent with root length and seedling height. Nitrogen content of root increased, while that of leaf increased initially and then declined slightly with the rising nitrogen level, nitrogen accumulation as well. Heterosis of nitrogen content in root and leaf was significant under the high nitrogen level. The activity of nitrate reductase and glutamine synthetase was increased with increasing level of nitrogen. Heterosis of nitrate reductase activity was negative, while that of glutamine synthetase activity was positive. Heteroses of nitrate reductase activity and root traits were basically identical, while that of glutamine synthetase activity and seedling traits were mainly consistent. The expression levels of three nitrate transporter genes that performed positive heterosis declined sharply at the beginning and subsequently kept stable with the rising nitrogen level. The expression levels of NRT1.1 and GS1c in leaf all increased at first and then declined with the increasing rate of nitrogen. Their performance of heterosis was positive. The expression levels of NRT1.1 in root showed a substantial increase, while that in leaf decreased significantly under nitrogen stress. Most indices of high nitrogen utilization variety at low N level (YM35) under low nitrogen stress and high nitrogen utilization variety at high N level (DK138) under high nitrogen stress were relatively high. Ten indices of the combination of YM35×DK138 were higher among 15 indices under low nitrogen level, and then 6 and 5 indices were higher under normal and high nitrogen levels, respectively. 【Conclusion】The changing trends of root indices closely related with nitrogen absorption declined quickly at first and then leveled off with the increasing level of nitrogen. The regularity and gene expression levels of these indices related to nitrogen utilization were coincident with which increased quickly originally and then trended to be stable with the rate of nitrogen increasing. Most indices of three crosses showed positive heterosis under three different nitrogen levels. Combination of high nitrogen utilization variety at low N level × high nitrogen utilization variety at high N level was beneficial for breeding of good progeny under the low nitrogen environment. High nitrogen utilization variety for parent mating was beneficial to breed good progeny under the normal nitrogen environment.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Difference of Yield and Its Formation Mechanism of Indica-Japanica Inter-Subspecific Hybrid Rice with Different Canopy Leaf Types

JIANG Yuan-Hua, ZHANG Hong-Cheng, WEI Hai-He, ZHAO Ke, XU Jun-Wei, DAI Qi-Gen, HUO Zhong-Yang, XU Ke, WEI Hai-Yan, GUO Bao-Wei

Scientia Agricultura Sinica. 2014, 47(12):  2313-2325.  doi:10.3864/j.issn.0578-1752.2014.12.004

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【Objective】The objective of this study was to reveal the difference of grain yield and its formation mechanism between clown leaf moderate type(CLMT) and clown leaf long type(CLLT) of Yongyou series of indica-japonica-hybrid-rice under the super high yield cultivation conditions. 【Method】 A field experiment was conducted with CLMT combination such as A26/F9250, A16/F9250, Yongyou 12, Yongyou 13 and CLLT combination such as A43/F8585, A41/ F8585, Yongyou 15, Yongyou 11 as materials in rice-wheat cropping areas (Yangzhou, Changshu of Jiangsu) in the lower reaches of the Yangtze River in 2011-2012. Difference in grain yield, the sink structure, stem and sheath material accumulation and transfer, photosynthetic characteristics and superior and inferior grain-filling characteristics between CLMT and CLLT of indica-japonica hybrid rice were analyzed systematically. 【Result】 Under the super high yield cultivation conditions, the grain yield of CLMT was significantly higher than that of CLLT, the yields of CLMT were 11.62 t•hm-2(Changshu, 2011), 11.98 t•hm-2(Yangzhou, 2011), 12.51 t•hm-2(Changshu, 2011), and 12.30 t•hm-2(Yangzhou, 2011), and were 8.85%, 9.75%, 9.60%, and 10.26% higher than those of CLLT, respectively. Effective panicles, effective spikelets per panicle, total spikelets of the CLMT were higher than those of CLLT. Total sink weight of CLMT was equal with CLLT, while 1000- grain weight, filling index of the CLMT were lower than those of CLLT. It was indicated that the increasement of filling index was the key point of higher yield of CLMT. Stem and sheath weight at heading and milky stages, stem and sheath apparent output, apparent output rate, apparent transfer rate from heading to maturity of CLMT were lower than that of CLLT, but stem and sheath weight in maturity, the maximum output, the maximum output rate, maximum transport rate from heading to milk stage of CLMT was higher than those of CLLT. It was found that translocation mechanism of stem and sheath of CLMT was more reasonable than those of CLLT. Leaf area index of CLMT was lower than that of CLLT at heading stage, and equal to that of CLLT at milk stage, and higher than that of CLLT at maturity . Photosynthetic rate of flag leaf of CLMT was equal to that of CLLT at heading and milk stages, and was higher than that of CLLT at maturity stage. Leaf area duration of CLMT was equal to that of CLLT from heading stage to milk stage and was higher than that of CLLT from milky stage to maturity stage. The decreasing rate of leaf area and photosynthetic rate of CLMT were lower than those of CLLT both from heading to milk stage and from milk stage to maturity. It was indicated that CLMT had photosynthetic production advantages during the late solid phase. For superior grain, initial growth power (Ro), time of the relative maximum grain filling rate (Tmax), active grain filling period (D) and effective filling time (T99) of CLMT were higher or longer than those of CLLT, while the relative maximum grain filling rate (RGRmax), the relative average grain filling rate (RGRmean) in superior grain of CLMT were lower slightly than those of CLLT. For inferior grain, RO of CLMT was lower than that of CLLT, while Tmax, D, T99, RGRmax, RGRmean of CLMT were longer or higher than those of CLLT. Both for superior grain and for inferior grain, the grain filling rate of CLMT was higher than those of CLLT. 【Conclusion】Under the mechanized and simplified super high cultivation conditions, there was no big difference in total sink weight between CLMT and CLLT, but matter translocation of stem and sheath at early grain filling stage, photosynthetic characteristics at later grain filling stage of CLMT were higher than that of CLLT, therefore，the grain filling rate and yield of CLMT were higher than that of CLLT. Reasonable stem sheath matter transport mechanism, strong photosynthetic production performance and gentle lasting filling characteristics were the mainly physiological basis of the formation of the yield advantage.

Effect of Different Diammonium Phosphate Levels on Nutrient Uptake of Super-High Yielding Soybean

ZHAO Yu-Kun-1, ZHANG Hui-Jun-1, AO Xue-1, WANG Hai-Ying-1, WANG Wen-Bin-2, SONG Shu-Hong-2, XIE Fu-绨1

Scientia Agricultura Sinica. 2014, 47(12):  2326-2334.  doi:10.3864/j.issn.0578-1752.2014.12.005

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【Objective】A pot experiment was conducted to find out the nutrient uptakes of nitrogen (N), phosphorus (P), potassium (K) of super-high yielding soybean cultivars and their difference with a common cultivar. 【Method】In 2011 and 2012, two super-high yielding cvs. Liaodou 14, Zhonghuang 35 and one common cv. Liaodou 11 were used in the pot experiment to investigate the effects of different diammonium phosphate (DAP) application levels on soybean N, P, K accumulation and distribution. The experiment was laid out in a complete randomized experiment design with three replications. Diammonium phosphate (DAP) was applied at soybean seedling stage. The treatments consisted of two cultivers and five levels of DAP (0, 50, 100, 150 and 200 mg per kg dry soil). Three pots were selected at random at the stages of V3, R2, R6, R7 and R8. Grains, soybean-roots, and aboveground vegetative samples were collected from all the treatments. The plant samples were dried in the oven at 105℃ for 30 min and 80℃ at least for 72 h to achieve constant weight. The plant samples were weighed, ground and digested with H2SO4-H2O2 acid mixture method. Nitrogen content was estimated by Kjeldahl (KN520) method. Phosphorus and potassium were respectively determined by ammonium molybdate colorimetric assay (UV-2450) and flame photometer (PEAA800). N, P, K accumulation was calculated by multipling dry mass with N, P, K percentage content. The nutrient harvest index was calculated by dividing the accumulative nutrient amounts of grains with total nutrient amounts produced by the plants. Required nutrients of producing weight grain per se were calculated by the accumulation of nutrients and grain yield.【Result】The absorption, transportation and distribution of N, P, K varied with different cultivars and DAP application levels. Super-high yielding cultivars needed to accumulate more nutrients. Their N and P accumulations were significantly higher than common cultivar in addition to seedling. K accumulation of roots was significantly higher than common cultivar in medium pod filling. Super high-yielding cultivars had a stronger capacity of nutrient transportation. Their harvest indexs of N, P and K were significantly higher than common cultivar. However, less nutrients for super cultivars were required to produce weight grain per se. Super-high yielding cultivars had a higher nutrient use efficiency. The application of DAP significantly increased the uptake of nutrients. The results showed that the accumulative amounts and harvest index of N, P, K showed an increasing tendency with the increment of DAP application levels, so as the required nutrient amounts of producing weight grain per se. Among them, the amounts of N and P accumulation, K harvest index and required N and P amounts of producing weight grain per se were maximized by the application of 200 mg•kg-1 of both super-high yielding cultivars and common cultivar. The required N and P amounts of producing weight grain per se of super-high yielding soybean cvs. Liaodou 14, and Zhonghuang 35 increased less than that of common soybean cv. Liaodou 11. The accumulative amounts of K, N and P harvest index, K requirement of producing weight grain per se decreased as the DAP application level was above 150 mg•kg-1. Super-high yielding soybean cvs. Liaodou 14, Zhonghuang 35 decreased less than that of common soybean cv. Liaodou 11. 【Conclusion】Super-high yielding cultivars had a stronger capacity of nutrient absorption and higher transferring efficiency of nutrients. Fertilization significantly enhanced the capacity of N, P, K absorption and transportation, but it reduced the efficiency of nutrient utilization.

PLANT PROTECTION

Genetic Diversity of Botrytis cinerea Isolates from Broad Bean

HUANG Yan-1, 2 , ZHU Zhen-Dong-1, DUAN Can-Xing-1, WU Xiao-Fei-1, DONG Fang-Yang-2

Scientia Agricultura Sinica. 2014, 47(12):  2335-2347.  doi:10.3864/j.issn.0578-1752.2014.12.006

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【Objective】Botrytis cinerea is one of the most important pathogens causing chocolate spot, blossom blight and pod rot in broad bean. B. cinerea is a species complex that comprises two cryptic species, Group I and Group II, and exhibits a great morphological and genetic diversity. However, the status of B. cinerea species complex from broad bean as well as their genetic characteristics have been poorly understood. Therefore, this study aims to determine the status and investigate the genetic diversity and genetic differentiation of isolates of B. cinerea complex from broad bean obtained from six different geographic regions. 【Method】The B. cinerea isolates were observed for cultural characteristics on potato dextrose agar. Transposons types of the isolates were detected using Flipper- and Boty-specific primers. Group determination of the isolates was analyzed by PCR-RFLP of Bc-hch gene, and their genetic characteristics was analyzed using SSR markers. 【Result】 All the 100 isolates were identified as B. cinerea group II. Three colony types, including mycelial type, sclerotial type and conidial type, were produced, and 76 isolates belonged to sclerotial type. Three transposon genotypes were identified in 92 isolates, which contained Boty-only, Fliper-only and Boty + Fliper genotypes. The most prevalent transposon genotype was Boty-only which was detected in isolates from Chongqing, Sichuan, Gansu and Hebei, while genotype Fliper-only was only found in isolates from Jiangsu. SSR analysis detected 92 multilocus genotypes (MLGs) among the 100 isolates with averaged gene and genotypic diversity of 0.5140 and 0.9982, respectively. Analysis of AMOVA revealed that genetic variation within populations accounted for 86.70% of the total genetic variation. The standardized index of association (rD) was estimated to range from 0.0312 to 0.1261 with the average value of 0.0491. In all comparsions, the values of fixation index (FST) ranged from 0.0085-0.2650 with the average value of 0.1330. All 100 isolates were divided into 10 genetic groups by UPGMA clustering analysis, and most of genetic groups included isolates from different geographic regions. In addition, two genetic groups were inferred in 92 MLGs by Bayesian analysis, and both populations from Chongqing and Sichuan belonged to one group, the population from Qinghai belonged to the other group, while popultions from Jiangsu, Hebei and Gansu were admixture of two genetic groups.【Conclusion】The populations of B. cinerea complex from broad bean made up of only B. cinerea group II. The sclerotial type was main colony morphology. Most of the isolates belonged to transposon genotypes whose distribution existed distinct geographic difference. Great genetic diversity was found in all six populations, and genetic variation mainly presented within populations. Sexual reproduction was the main mode of reproduction. Moderate to large genetic differentiation was detected among most populations.

Comparison of Secretome Extraction Methods in Verticillium dahliae

XIAO Hong-Li, GUI Yue-Jing, QI Wei-Yan, CHEN Jie-Yin, LI Lei, XU Ming, DAI Xiao-Feng

Scientia Agricultura Sinica. 2014, 47(12):  2348-2356.  doi:10.3864/j.issn.0578-1752.2014.12.007

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【Objective】The objective of this study is to identify the high efficient method for secreted protein extraction in Verticillium dahliae, provide a technique basis for further functional characterization of V. dahliae secretome, and to construct a platform for functional verification of virulence factors and the mechanism research of V. dahliae. 【Method】Secreted proteins of the high virulent V. dahliae strain VdG1 were extracted by three methods, including ultra-filtration (UF), TCA precipitation and ion-exchange (IEX), followed by LC-MS/MS analysis for protein identification. The bioinformatics softwares WoLFPSORT, SignalP, TMHMM and PHOBIUS were used to predict classical secreted proteins. The classical secreted proteins were annotated by using CAZy (carbohydrate-active enZYmes) and PHI (pathogen-host interaction) databases. In addition, motif searchs was conducted via BLASTp, such as RxLx, LysM and Cys Pattern. The extracted proteins were used to detect the biological activity and pathogenicity on susceptible cotton. 【Result】By the three methods of TCA precipitation, IEX and UF, the extracted efficiency of V. dahliae secretome was 1.18, 0.96 and 0.56 mg?L-1, respectively. The SDS-PAGE result showed that TCA and IEX extracted samples had more efficiency and purity than that of UF. The numbers of classical secreted proteins were 124, 112 and 75, respectively, of which 98, 85 and 57 were possible pathogenic factors. In addition, ion-exchange had a high extraction efficiency for small molecular weight proteins and the extracted protein could induce wilt and necrosis symptom to cotton. 【Conclusion】To extract secreted proteins of V. dahliae by three methods, the lowest efficiency and purity, the minimum amount of classical secreted proteins, the fewest number of potential pathogenic factors were the UF extracted samples, and such method was quite time-consuming for large volume processing. TCA and IEX methods were fit to extract secreted proteins of V. dahliae, but TCA method couldn’t keep the sample’s biological activities and the extracted sample was just for 2-D electrophoresis detection. IEX method was firstly applied in the preparation of pathogenic fungi, and the operation was simple, convenient, all processes were completed by the instrument system. The extracted proteins had biological activity, and could cause cotton leaf wilt and necrotic symptom. In addition, the database and software comparative analysis found that the identified secreted proteins of V. dahliae contain 61 CAZy enzymes, 38 PHI proteins, 13 RxLx motif proteins, 1 LysM motif protein, 15 Cys Pattern motif proteins, and 2 VdNEP family proteins. The results will be used for screening the pathogenic factors and providing an important basis for further functional verification.

FoPLC4, Encoding Phospholipase C4, Is Involved in Sporulation and Pathogenicity in Fusarium oxysporum

SUN Ling, CHU Xiao-Jing, HAO Yu, ZHANG Hong-Bin, LIANG Yuan-Cun

Scientia Agricultura Sinica. 2014, 47(12):  2357-2364.  doi:10.3864/j.issn.0578-1752.2014.12.008

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【Objective】Cucumber Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum, is an important disease in cucumber (Cucumis sativus L.) and can result in serious economic loss. The objective of this study is to investigate the functions of phospholipase C4 (PLC4) in F. oxysporum.【Method】Chromosomal location of the FoPLC4 and structure of FoPLC4 were analyzed by bioinformatics methods. Based on the F. oxysporum f. sp. lycopersici genome database, the FoPLC4 was cloned using specific primers. The gene replacement vector carrying hygromycin phosphotransferase (HPH) gene was constructed based on the gene homologous recombination, and then transformed into protoplasts with PEG-mediated gene transformation. The FoPLC4 deletion mutant (?FoPLC4) was generated through hygromycin B selection and molecular assay. Biological characters of the mutant on PDA (potato dextrose agar) medium and its pathogenicity on cucumber seedlings were determined after inoculation of radicles with conidia suspensions.【Result】The FoPLC4 located on chromosome 4, contains 3 282 bp with no intron and encodes a long polypeptide of 1 093 amino acids (aa). The protein encoded by FoPLC4 resembled mammalian PLCδ and possessed five domains including an EF-hand domain, a pleckstrin homology domain, a C2 domain, and two catalytic domains. Transformants selected on hygromycin B plates were screened by PCR and RT-PCR. Compared with the wild type (WT) strain, the ?FoPLC4 mutant showed a similar colony growth rate, but exhibited sparse and fluffy aerial mycelium on PDA medium. The conidiation of the ?FoPLC4 mutant reduced by 82.2%, the ?FoPLC4 simultaneously produced micro- and macro-conidia while the WT strain did only produce micro-conidia, and macro-conidia accounted for 12.1% of all spores in the ?FoPLC4. Pathogenicity assay of the ?FoPLC4 showed that wilting symptoms of cucumber seedlings significantly reduced. After 10 days of inoculation, disease index decreased by 53.3% on inoculated seedlings. Furthermore, the functional complementation of ?FoPLC4 restored the characteristics of the WT strain.【Conclusion】FoPLC4 contains 3 282 bp with no intron and encodes a protein of 1 093 aa. Compared with WT, ?FoPLC4 significantly exhibited the change of spore types, decreased the number of spores and pathogenicity in cucumber seedlings. The results indicate that FoPLC4 plays multiple roles in the regulation of sporulation and pathogenicity in F. oxysporum.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Spatial Heterogeneity of Loess Tilled Slope Surface Roughness

ZHANG Li-1, 2 , ZHANG Qing-Feng-1, 2 , ZHAO Long-Shan-1, 2 , WANG Jian-1, WU Fa-Qi-1

Scientia Agricultura Sinica. 2014, 47(12):  2365-2373.  doi:10.3864/j.issn.0578-1752.2014.12.009

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【Objective】The objective of this paper is to reveal the spatial heterogeneity of surface roughness of 4 typical loess tilled slopes during different erosive stages under a micro-scale (2 cm×2 cm) condition.【Method】When slopes with 4 different gradients and tillage measures were prepared (artificial backhoe, artificial digging, contour tillage and straight cultivated slope (check), an erosive rainfall with a rainfall intensity of 60 mm/h was carried out. Soil surface roughness relative elevation data points were taken by a laser scanner and analyzed with both the semivariogram function and fractal dimension models.【Result】The result of classical statistical analysis showed that the loess tillage slope surface roughness has a weak spatial variability. The result of the semivariogram analysis indicated that the loess tilled slope surface roughness had a higher spatial autocorrelation, their spatial autocorrelation scale of surface roughness ranged from 2.02 m to 3.82 m. The spatial heterogeneity caused by the spatial structure characteristic accounted for the greater proportion of the total heterogeneity. The fractal dimension analysis showed that the surface roughness had good fractal features, and it ranged from 1.59 to 1.91. With the increase of gradient, the spatial distribution of slope surface roughness tended to complex, its spatial heterogeneity was stronger. The spatial heterogeneities of the artificial backhoe (AB), artificial digging (AD) and contour tillage (CT) increase in turn within the scope of the small scale, and had a good effect on soil and water conservation. 【Conclusion】The main reason for the differences of the spatial heterogeneity of surface roughness is the spatial structural characteristics formed by the integrated interaction of human farming and slope and the artificial cultivation. The space configuration pattern of surface roughness is mainly controlled by slope gradient factor in a smaller scale range, and by the rainfall with its erosion process in a larger scale range. Results of the study will lay a foundation for understanding and quantitative description of the coupling relationship between the surface roughness and erosion, and will also provide a theoretical basis for guiding future tillage practices in the loess sloped regions.

Study on Soil Total N Estimation by Vis-NIR Spectra with Variable Selection

YANG Mei-Hua-1, 2 , ZHAO Xiao-Min-1, 2 , 3 , FANG Qian-1, 2 , XIE Bi-Yu-1, 2

Scientia Agricultura Sinica. 2014, 47(12):  2374-2383.  doi:10.3864/j.issn.0578-1752.2014.12.010

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【Objective】 Variable selection or feature selection is a critical step in data analysis of visible-near infrared (Vis-NIR) spectrum research. The aim of this study was to determine the soil total nitrogen (TN) contents through building models based on absorption features of soil TN using variable selection methods combined with Vis-NIR spectroscopy, and to provide a basis for the fast estimation of the content of soil TN.【Method】Representative 120 soil samples were collected from the typical red soil area of Ji’an County, Jiangxi Province. The TN contents and the Vis-NIR were measured in the laboratory. Several variable selection methods including principal component analysis (PCA), uninformative variable elimination (UVE) and UVE coupled with successive projections algorithm (SPA) were employed for Vis/NIR data, the models of partial least squares regression (PLSR) with leave-one-out cross-validation, least squares-support vector machine (LS-SVM), the back-propagation neural network (BPNN) and BPNN with optimized threshold and weight using genetic algorithm (GA-BPNN) combined different variable selection methods were calibrated and validated using independent data sets. 【Result】 The results showed that the application of UVE to the wavelengths reduced wavelengths from original 200 to 59 of which located in visible range and the rest located in the region of overtones and combinations in near infrared range. The application of SPA to the wavelengths preselected by UVE further reduced the wavelengths to only 5 for TN, including 820, 940, 1 040, 1 060 and 1 990 nm. LS-SVM models achieved competitive prediction performance compared with PLSR, BPNN and GA-PBNN based on 59 wavelengths with coef?cient of determination (R2) of 0.7492 and root mean square error (RMSEp) of 0.2921 and residual prediction deviation (RPD) of 1.8904 for soil TN. Furthermore, LS-SVM models achieved excellent prediction performance with PLSR, BPNN and GA-PBNN based on 5 wavelengths using variable selection UVE-SPA, with coef?cient of determination (R2) of 0.7945 and root mean square error (RMSEp) of 0.2499 and residual prediction deviation (RPD) of 2.0009 for soil total N. Nevertheless, LS-SVM, BPNN and GA-PBNN models based on 7 principal components was invalid.【Conclusion】 The overall results indicated that SPA was a powerful way for the variable selection, and Vis-NIR spectroscopy incorporated to SPA-LS-SVM was successful for the accurate determination of soil TN.

Accumulation Characteristics of Cadmium by Rice Cultivars in Soils and Its Species Sensitivity Distribution

SUN Cong, CHEN Shi-Bao, SONG Wen-恩, LI Ning

Scientia Agricultura Sinica. 2014, 47(12):  2384-2394.  doi:10.3864/j.issn.0578-1752.2014.12.011

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【Objective】Understanding of the accumulation characteristics of Cadmium by different rice cultivars in soils and its species sensitivity distribution is necessary for determination of the hazardous concentration of Cd to rice cultivars of 5% of the tested species (HC5), the threshold values (HC5) of Cd to rice species will provide vital information for protection of Cd pollution to different rice cultivars and thereafter the food chains in China.【Method】Two typical paddy soils were collected from southern China, and the soils were polluted by Cd with different Cd addition levels of 0, 1.2, 4.8, 10, 40, and 120 mg•kg-1 in soils. Eighteen different rice cultivars from the main rice producing areas around China were selected for test plant species in this study. The biomass of the rice cultivars, the Cd concentrations in plants and thereafter the bioconcentration factor (BCFs) were investigated using pot experiments in green houses. The log-logistic distribution model was used to determinate the dose-response relationship of Cd toxicity to rice cultivars based on different endpoints. The response relationships of Cd toxicity to18 different rice species were fitted by cumulative probability distribution functions Log-logistic distribution model. The species sensitivity distributions (SSD) of Cd to rice was performed with the Burr-III type distribution model, and the different species sensitivity distribution of Cd toxicity frequency and 95% of rice varieties based on the protection of Cd toxicity thresholds HC5 were obtained and meanwhile, the quantitative relationship of BCFs with the bioavailability of Cd in soils was also determined in this study.【Result】 The biomass of different rice species decreased with the increasing concentrations of Cd in acidic soil from Qiyang, biomass of rice X-42 which was the most sensitive species to Cd stress decreased by 86%, but the biomass of rice Z-120 showed stronger tolerance to Cd stress decreased by 51%. When added at a concentration of 4.8 mg•kg-1, the biomasses of JY-253, J-463, Z-611, J-899, T-15, X-6, T-167 reached the maximum, and the others were at the concentration of 1.2 mg•kg-1. Compared with Cd0, the biomass of different varieties of rice increased by 4%-56%. This showed the stimulating effect of relatively lower level of Cd on rice growth. In alkaline soils from Guangzhou, there was no significant difference in the biomass of different rice species with increasing concentrations of Cd. The biologic concentration factor (BCFs) of Cd to rice species decreased with the increased spiked levels of Cd in both soils. In the low-Cd concentration (1.2 mg•kg-1), BCF in the red soil ranged between 0.0056 (S-974)-0.0133 (T-15), 2.38-fold difference; but in the paddy soil, the range of variation between 0.0018 (L-42, LY-28) -0.0034 (J-899), 1.89-fold difference. However, the BCFs of rice in Qiyang soil was higher than in Guangzhou soil under the same Cd level 2-14 times difference. This may be related to the nature of the soil. Qiyang red soil is an acidic soil, organic matter scarce, highly effective state of the measured Cd contention, and strong poisoned rice, reduced the multi-biomass. On the contrary, Guangzhou paddy soil which is high in pH and more organic matter, increased Cd absorption capacity and holding power, weakening poison rice biomass did not change significantly. Rice X-45 had the highest Cd toxicity threshold concentration (EC50, mg•kg-1), while rice X-42 had the lowest EC50. The EC50 of 18 rice species ranged from 4.30-61.611 mg•kg-1, with the variations differed from 1.0 to 14.32 folds.【Conclusion】Significant differences of response of rice cultivars to Cd stress were observed among the tested rice plant species, and hormesis effect of Cd stress to rice cultivars was also observed in this study although Cd was not included in the nutrients for rice plants. The results indicated that distinct difference existed among the tested rice species according to the accumulation of Burr-III type distribution model, e.g. the result showed that rice X-42 was the most sensitive species to Cd stress with the minimum toxicity threshold among the rice cultivars, on the contrary, rice X-45 showed a strong tolerance to Cd stress in soils. The maximum concentration of half (50%) inhibiting concentration of Cd to rice cultivars with protection of the 95% rice species (HC550%) was 4.93mg•kg-1 as determined by Burr-III model.

HORTICULTURE

Analysis of Genetic Effects of the Cross Combinations of Tagetes patula

PAN Chen-1, HU Yan-2, BAO Man-Zhu-1, AI Ye-1, HE Yan-Hong-1

Scientia Agricultura Sinica. 2014, 47(12):  2395-2404.  doi:10.3864/j.issn.0578-1752.2014.12.012

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【Objective】Tagetes patula belongs to Tagetes genus of the Asteraceae family and it is an extremely popular annual plant for its great commercial and ornamental value. Heterosis breeding is an important approach to breed new cultivars. The heterosis and the genetic effects of horticultural traits of F1 hybrids of tetraploid T. patula were analyzed to establish a heterosis breeding system. 【Method】 Eighteen cross combinations of T. patula were made using six inbred lines K2, K3, K4, K5, K15, and K17 as female parents crossed with three male parents K6, K8, and K13 by following the North Carolina Design II incomplete diallel cross design method, then eleven horticultural traits of first flowering date, full-bloom stage, plant height, crown size, branch number, inflorescence number, pedicel length, diameter of inflorescence, diameter of disk florets, and number of ray florets, number of disk florets were investigated. Heterosis of horticultural traits was calculated using Excel software and combining abilities and heritabilities were analyzed using DPS software to chose primary parents and excellent combinations. 【Result】 The results of heterosis analysis showed that the crown size, number and diameter of inflorescence were improved over best parent and the full-bloom stage, plant height and ray floret number were improved over mid-parent, but the traits of first flowering date, branch number, pedicel length, diameter of disk florets and number of disk florets conferred negative best parent and mid-parent values. Analysis of general combination ability (GCA) showed that the line K6 was chosen as the preferred male parent due to the dwarf, compact and multiple flower traits conferred to the hybrid progeny; the line K13 was judged to be a good male parent for the production of hybrids with early flowering, large and double flower and long ornamental period; the line K3 was the preferred female parent for the production of hybrids of compact plant, early flowering, double flower and long ornamental period; the line K5 was the good female parent for the production of dwarf, compact, early flowering, multiple flower and long ornamental period hybrids; the line K17 was judged to be a good female parent for breeding dwarf, large and double flower hybrids. Analysis of special combination ability (SCA) suggested that SCA effects exhibited significant differences both in the combinations of the same parent and in the traits of the same combination. The crosses K2×K6 and K3×K8 were determined as the excellent combinations for the production of dwarf, multiple and double flower cultivars, exhibiting negative SCA effects in plant height and diameter of disk florets and large positive SCA effects in inflorescence number and number of ray florets. The cross K4×K13 was determined as the excellent combination for the production of compact, early flowering, large and double flower and long ornamental period cultivars, exhibiting negative SCA effects in first flowering date, plant height, crown size and branch number and positive SCA effects in full-bloom stage, diameter of inflorescence, number of ray florets. Analysis of heritability suggested that the traits in first flowering date, full-bloom stage, branch number, diameter of inflorescence, diameter and number of disk florets were primarily controlled by GCA variance and their broad sense heritability and narrow-sense heritability are above the average, whereas the GCA and SCA exhibited insignificant difference in the traits of plant height, inflorescence and ray floret number and the broad sense heritability performed higher, respectively. 【Conclusion】 The F1 hybrids of T. patula have quite distinct heterosis and it was of great value to breed early flowering, large, double and multiple flower, and long ornamental period cultivars. Among the parental lines, K6, K13, K3, K5, and K17 were chosen as primary parents. The crosses K2×K6, K3×K8 and K4×K13 were determined as the excellent combinations. The traits of first flowering date, full-bloom stage, branch number, diameter of inflorescence, diameter and number of disk florets were primarily controlled by additive effects, whereas plant height, inflorescence and ray floret number were mainly controlled by non-additive effects. In addition to first flowering date, horticultural traits were vulnerable to the environment.

Accumulation Characteristics of Volatile Compounds in Wine Grape Berries Grown in High Altitude Regions of Yunnan

YANG Xiao-Fan, GAO Yuan, HAN Mei-Mei, PENG Zhen-Xue, PAN Qiu-Hong

Scientia Agricultura Sinica. 2014, 47(12):  2405-2416.  doi:10.3864/j.issn.0578-1752.2014.12.013

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【Objective】 Aroma is an important factor determining the quality of wine grape berries and wines. Terrior greatly affects the composition and concentration of volatiles. Study on aroma accumulation of grape fruit of Yunnan plateau region will not only help uncover the formation mechanism of grape flavor under the high altitude climate, but also provide some guidelines for the production of high-quality grape berries. 【Method】 Grape berries (Vitisvinifera L. Cabernet sauvignon) were collected from Meilishi (located at about 2 300 m above sea level) and Shuiri (about 2 900 m) vineyards of Deqin County, Yunnan Province in 2010 and 2011 vintages. Free volatile compounds were extracted using headspace solid phase micro-extraction (HS-SPME) method. The extraction of glycosidically-bound volatiles was followed by the separation using Cleanert PEP-SPE column, the hydrolysis of glycosidase AR2000 and the adsorption of HS-SPME. The qualitative and quantitative analyses were performed via the technology of gas chromatography-mass spectrometry. These volatiles were classified into three groups according to their biosynthetic pathways, including fatty acid-derived, amino acid-derived and isoprene-derived volatiles, and the effects of vintage and grape-growing region on the accumulation of the three groups of volatiles were assessed. 【Result】 The accumulation of soluble solid and variation of pH in grape berries were affected scarcely by vintages, either for Meilishi or Shuiri vineyards, but volatiles showed a great difference in the concentration between vintages. Compared with the 2010-vintage berries, regardless of the vineyard altitude, the berry samples of the year 2011 contained significantly higher concentrations of free-form straight-chain aliphatic alcohols, acids and esters that were produced from fatty acid metabolism, as well as norisoprenoids (free and glycosidically-bound) and free terpenes that were all generated from isoprene metabolism. As for amino acid metabolism-produced volatile compounds, the concentration of glycosidically-bound aromatic compounds was significantly higher in the 2010 grape berries compared with that in the 2011 berries, but the concentration of free aromatic compounds was significantly lower. In addition, there existed a great difference in the types of straight-chain aliphatic alcohols and aldehydes, aromatic volatiles branch-chain aliphatic volatiles and terprenes between two vintages, while almost no difference in the composition of volatiles was found between Meilishi and Shuiri berries in the same growing season. Combined analysis of two years of meteorological parameters showed that less rainfall and longer hours of sunshine were in favor of reducing the level of straight-chain aliphatic aldehydes and increasing the level of terpenes. Regarding vintage altitude, a continuous increase in the concentration of fatty acid-derived volatiles during berry mature was observed in the grape berries from the relatively high-altitude Shuiri vineyards, and a trend with first increase and then decrease was present in the berries from the Meilishi vineyards. At harvest of 2010, the grape berries from the Shuiri vineyards showed higher concentrations of free and glycosidically-bound straight-chain aliphatic aldehydes than the berries from Meilishi vineyards, but the concentration of straight-chain aliphatic alcohols in the berries growing in Meilishi was higher than that in the Shuiri-produced berries. Besides, the concentrations of norisoprenoids and pyrazines were relatively higher in the grape berries growing in high-altitude Shuiri. 【Conclusion】Climate variation affects the accumulation of volatile compounds of grape berries in the high-altitude wine-producing zones more significantly than the impact of vineyard altitude. Delay of harvest for Cabernet Sauvignon berries are suggested alone with an increase in vineyard altitude in order to reduce the level of pyrazines and straight-chain aliphatic aldehydes and herbaceous character of these grapes.

STORAGE·FRESH-KEEPING·PROCESSING

The Impact of Dilinolen on the Oxidative Stability of Soybean Oil

JU Xing-Rong, GAO Xiao-Hong, HE Rong, ZHENG Yi, YUAN Jian

Scientia Agricultura Sinica. 2014, 47(12):  2417-2424.  doi:10.3864/j.issn.0578-1752.2014.12.014

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【Objective】 Oxidative stability of oil is directly related to oil storage quality. Diacylglycerol, one of the trace components in soybean oil, affects the oxidative stability of oil. This article aims to study the influence of dilinolen on the oxidation stability of soybean oil and develop a new soybean oil antioxidant that will stabilize the oil quality and extend its shelf life. 【Method】 Experiments were carried out using original soybean oil (NSSO) and trace components-stripped soybean oil (TSSO) which was passed through chromatographic column. Dilinolen (LL) at different proportions of 0, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, and 2.0% was added to the soybean oils. A 30 ppm Chlorophyll B was also added into TSSO (Chlorophyll B/TSSO, W/W) and the control group was the soybean oil without dilinolen. All the samples were thoroughly mixed and stored at 25 oC and 1 700 lx under the open condition. Quality indicators of soybean oil were measured every seven days in the 6 weeks trial period according to the following methods. Iodimetry (GB/T5538-2005) was employed to evaluate peroxide value (POV) of samples. An oil oxidative stability instrument (Ominion, OSI-24）was employed to evaluate the oxidative stability index (OSI) of soybean oil using the GB/T21121-2007 method. Head-space gas chromatography and mass spectrum (GC/MS) were used to determine the composition and relative content of volatile aldehydes in the oils. Fatty acid composition was measured by gas chromatography spectrum. All assays were conducted in triplicate and variance was analyzed by IBM SPASS19.0. The means were compared using Duncan’s multiple range test.【Result】The results obtained during the 6 weeks trial period are as follows: The longer the storage time, the higher the POV value, indicating the increased oxidation of the sample, while the increased magnitude of POV values decreased with increasing amount of dilinolen. The OSI decreased with prolonged storage, reflecting the poor oxidative stability of the oils; OSI values were mitigated with the increase of the amount of LL; The reduced amplitude of OSI of TSSO was lower than that of NSSO at the same amount of dilinolen and storage time. The relative contents of volatile aldehydes in the oils were increased with prolonged storage time. The content of volatile aldehydes in oils with dilinolen added were lower than that of the control, which decreased with the increase of the amount of dilinolen added. The ratio values of unsaturated fatty acids to saturated fatty acids (U/S) decreased as storage time increased. During the same storage cycle, the U/S of the oil samples containing dilinolen was higher than that of the control group. The higher the amount of dilinolen added, the higher the linoleic content, and there was no significant change in the content of other fatty acids. 【Conclusion】 Under room temperature and light conditions, it is effective to inhibit the oxidation of soybean oil with the addition of dilinolen. The effect of dilinolen on TSSO is also significantly better than on NSSO.

Study of Lignification’s Delaying and Its Relationship with Energy Metabolism in Loquat Fruits after Nitric Oxide Fumigation

CHEN Fa-He, ZHANG Mei-Zi, WU Guang-Bin

Scientia Agricultura Sinica. 2014, 47(12):  2425-2434.  doi:10.3864/j.issn.0578-1752.2014.12.015

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【Objective】Loquat (Eriobotrya japonica Lindl.) fruit is a famous southern fruit in China with its special texture and high metabolism. High metabolism rates reflect fast consumption of energy and rapid depletion of reserves such as carbohydrates and organic acids, so that its senescence is quick after harvest, and they are very liable to spoilage under improper storage conditions (such as room temperature). The cold storage can effectively prolong the shelf life and reduce the decay of loquat fruits, but the phenomenon of the peeling difficulty, flesh lignification, browning and hardening will occur in cold-stored loquat fruits. The quality deterioration of cold-stored loquat fruit is a main reason causing postharvest losses, and has become a limiting factor in market distribution of loquat fruits. The objectives of present study were to investigate the effect of nitric oxide (NO) treatment on the flesh lignification of loquat fruits and the relationship between flesh lignification and energy metabolism, to provide a better foundation for further researches on molecular biological mechanism of regulation on quality deterioration in cold-stored loquat fruits and storage technology of postharvest loquat fruits.【Method】Loquat fruits（E. japonica Lindl.cv. Jiefangzhong）were fumigated with 0(control group), 15 and 25 μL•L-1 NO gas for 2 h in a sealed container and then ventilated for 20 min. The fruits were stored at 5℃ and 85% RH. The changes of membrane permeability, firmness, juice percentage, lignin content, adenosine triphosphate (ATP) content, adenosine diphosphate (ADP) content, adenosine monophosphate (AMP) content, energy charge and the activities of succinate dehydrogenase (SDH), cytochrome oxidase（CCO）, H+-ATPase and Ca2+-ATPase were determined during cold storage, and the correlation between lignin content and energy charge was analyzed after NO treatments. 【Result】 Membrane permeability and firmness of loquat fruits increased gradually and the juice percentage decreased during the storage. Lignin content increased rapidly 10 days later and the chilling injury symptoms of loquat fruits were obvious. As compared with the control group, NO treatments delayed the increase of membrane permeability and fruit firmness, postponed the decrease of juice percentage, and significantly inhibited the formation of lignin content, kept the integrity of the cell membrane, thereby reducing the chilling injury of loquat fruits. ATP content decreased during cold storage. ADP content dropped rapidly in the first 10 days and stayed lower level eventually. The activities of SDH, CCO, H+-ATPase, and Ca2+-ATPase dropped sharply at the middle and later storage (15-30 d). These results showed that mitochondrial function was impaired which lead to energy charge level of loquat fruits drop rapidly. As compared with the control group, NO treatments delayed the decline of ATP and ADP content, inhibited the decrease of the activities of SDH, CCO, H+-ATPase, and Ca2+-ATPase significantly at the middle and later storage, which maintained better mitochondrial function. After 30 days of storage later, energy charge of loquat fruits by 15 and 25 μL•L-1 NO treatments were 11.8% and 12.9%, respectively, higher than control group. Correlation analysis showed that energy charge was very significantly negatively related with lignin content of loquat fruits and the correlation indexes were -0.715** and -0.598**, respectively, in 15 and 25 μL•L-1 NO treatments.【Conclusion】Energy deficit had a closely relationship with lignification of loquat fruits during cold storage. All the results indicated that NO treatments maintained high energy level by regulating the activities of SDH, CCO, H+-ATPase, and Ca2+-ATPase of mitochondria metabolism-related enzymes, thereby enhanced the chilling tolerance and delayed the development of loquat fruits’ lignification, the treatment with 25 μL•L-1 NO was better.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Cloning and Bioinformatics Analysis of PPARG Gene from Lanzhou Fat-Tailed Sheep

JIN Fang-Yuan-1, XU Hong-Wei-2, DA Xiao-Qiang-1, BAI Jia-Lin-1, FENG Yu-Lan-1, ZANG Rong-Xin-1, YANG Ju-Tian-1

Scientia Agricultura Sinica. 2014, 47(12):  2435-2445.  doi:10.3864/j.issn.0578-1752.2014.12.016

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【Objective】The objective of this study is to reveal the biological function of peroxisome proliferator-activated receptor gamma (PPARG) gene in sheep and provide theoretical data for application by cloning PPARG and analyzing its sequence as well as genetic features.【Method】The specific primers on the CDS template of PPARG gene were designed and then the PPARG gene sequence of Lanzhou fat-tail sheep was cloned using RACE and RT-PCR technology. After that, its genetic characteristics were analyzed by bioinformatics methods. 【Results】 The 1 774 bp full-length cDNA of PPARG of Lanzhou fat-tailed sheep (Genbank No.: KF727439) was obtained, in which the length of CDS is 1 428 bp. It encodes 475 amino acids in total. The coding regions on both sides of the wing are respectively provided with 5 '-UTR 131 bp (1-131 nt) and 3' -UTR 214 bp (1560-1774 nt). It was predicted that the molecular weight of PPARG protein of Lanzhou fat-tail sheep is 54.40 kD and its theoretical isoelectric point is 4.94. In addition, its maximum hydrophobic value is 3.600 and minimum hydrophobic value is -2.744. It is a non-transmembrane hydrophobin with no signal peptide, which locates mostly in cytoplasm (65.2%), seldom in nucleus (21.7%), scaffold (4.3%) and peroxisome (4.3%) by the result of subcellular level prediction and does not belong to the secretory protein. Its amino acid sequence contains 26 phosphorylation sites, 0 glycosylation site, 1 ZnF_C4 domain, 1 HOLI domain and 1 LCR domain. Also, its secondary structure is mainly randomly curly. Nucleotide sequence analysis revealed that the gene nucleotide sequence of Lanzhou fat-tail sheep is similar with those of the capra (99%), bos (98%), bubalus (97%), ovis (99%), orcinus (94%), sus (92%) and homo (90%). The similarity of amino acid sequence of PPARG between Lanzhou fat-tailed sheep and capra, bos, bubalus, ovis, orcinus, sus and homo are 100%, 99.8%, 100%, 100%, 98.7%, 98.5% and 97.5%. The phylogenetic tree indicates that Lanzhou fat-tail sheep is close to capra, ovis and bubalus, but far from fish, human and mouse. Base transitions occur in the 486th and 828th sites, which are (C vs. T) and (C vs. A), respectively, but the corresponding amino acids do not change.【Conclusion】The fact that the structure of PPARG of Lanzhou fat-tail sheep highly similar with other species indicates that the gene is highly conservative. It was predicated that most of the PPARG protein synthesises at the ribosome and its function is related to peroxisome. The results are in good agreement with its status of peroxisome proliferator activated receptor. The amino acids sequence of PPARG has 26 protein kinase phosphorylation sites. After phosphorylation some sites will lead to a series of dysfunction and expression of obesity-related genes, such as decreased expression of insulin sensitizing hormone and adiponectin. In PPARG , HOLI (also named LBD ligand) structural domain plays an important role in the process of hormone signal transduction, while ZnF_C4 zinc finger structural domain has the function of combining with lipid, which means that the Ser112 in PPARG zinc finger domain, after phosphorylation by MAPK, can inhibit the PPARG binding with the ligand, thereby reducing the activity of the PPARG protein. Both domains are associated with the differentiation process of lipid. The paper has laid a biological foundation for further study on the function of PPARG in lipid differentiation.

Studies on Dietary Methionine Requirement of Jinghong Laying Hens from Hatch to the Age of 4 Weeks

SONG Dan-1, 2 , YUE Hong-Yuan-2, CHEN Xiu-Li-1, 2 , LI Lian-Bin-1, 2 , GAO Yu-Peng-1, WU Shu-Geng-2

Scientia Agricultura Sinica. 2014, 47(12):  2446-2454.  doi:10.3864/j.issn.0578-1752.2014.12.017

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【Objective】Dietary methionine(Met) levels for China’s new cultivation of Jinghong laying hens from hatch to the age of 4 weeks were studied.【Method】The trial had adopted one-factor completely random design. Three hundred 1-day-old Jinghong laying hens from a commercial supplier in YuKou poultry were randomly allotted to 5 dietary treatment groups, and the birds in the treatment groups were fed a diet with different dietary methionine levels, respectively, i.e. 0.2% (0.2% Met), 0.3% (0.3% Met), 0.4% (0.4% Met), 0.5% (0.5% Met) and 0.6% (0.6% Met). Each treatment group consisted of 5 replicates with 5 pens per replicate and 12 birds per pen. The experiment lasted for 28 days. On 14th day, after being deprived of feed for 12 h, 2 birds selected randomly from each pen were slaughtered by neck cut and birds were eviscerated manually. The thymus, spleen and bursa of fabricius were weighed and were expressed as a percentage relative to live BW at processing. On 28th day, after being deprived of feed for 12 h, 2 birds selected randomly from each pen were slaughtered by neck cut and birds were eviscerated manually. The immune organs and digestive organs were weighed and were expressed as a percentage relative to live BW at processing.【Result】No significant differences were found in average daily feed intake (P＞0.05), but the average daily gain was significantly different (P＜0.05), and the birds in 0.4% Met group showed the maximum value with 8.31 g•d-1 and significantly higher than 0.2%, 0.5% and 0.6% Met groups (P＜0.05), with a quadratic curve of a rising trend. Besides, feed/gain ratios were also significantly affected (P＜0.05), and the 0.5% Met group was the best (2.13:1) and significantly lower than 0.2% and 0.3% Met groups (P＜0.05), with a quadratic curve of reducing trend. Body weight was significantly affected (P＜0.05), and the 0.4% Met group was the best (268.70 g) and significantly higher than 0.2%, 0.5% and 0.6% Met groups (P＜0.05), with a quadratic curve of rising trend. Community evenness also showed a tendency of a quadratic curve of rising, the 0.5%Met group (85.19%) was significantly higher than the other groups (P＜0.05). By the end of 2nd week, no-significant influence on the relative weights of the thymus and bursa of fabricius (P＞0.05) was found among different groups. There was a significant impact on the relative weights of the spleen (P＜0.05), the 0.4% Met group reached the maximum. By the end of the 4th week, the relative weights of the thymus, spleen and bursa of fabricius showed a significant positive correlation (P＜0.05) with dietary Met levels and all values had a rise-fall tendency. The relative weights of the spleen and bursa of fabricius in 0.4% Met group were the largest; the thymus index in 0.5% Met group was the largest. No significant differences were found in pancreas index and the relative lengths of the duodenum (P＞0.05), but the relative weights of the duodenum, jejunum and ileumor or the relative lengths of the jejunum and ileum were significantly different (P＜0.05). With the increase of methionine, except the relative weights of pancreas which showed a rise-fall tendency, the relative weights of the duodenum, jejunum and ileumor or the relative lengths of the jejunum and ileum had shown a fall-rise tendency. The relative weights of the duodenum in 0.3% Met group were significantly lower than 0.2%, 0.5% and 0.6% Met groups (P＜0.05). The relative weights of the jejunum in 0.4% Met group were significantly lower than other Met groups (P＜0.05). The relative lengths of the jejunum in 0.5% Met group were significantly lower than 0.2%, 0.3% and 0.6% Met groups (P＜0.05). The relative weights of the ileumor in 0.4% Met group were significantly lower than 0.6% Met group (P＜0.05). The relative lengths of the ileumor in 0.3% Met group were significantly lower than 0.2%, 0.5% and 0.6% Met groups (P＜0.05). There were significant differences among groups in the level of serum urea nitrogen, uric acid and alkaline phosphatase (P＜0.05). The level of serum urea nitrogen in 0.6% Met group was significantly higher than 0.2%, 0.4% and 0.5% Met groups (P＜0.05). The 0.5% Met group was the minimum of serum urea nitrogen, while alkaline phosphatase was significantly higher than the other groups (P＜0.05). Uric acid in 0.6% Met group was significantly higher than the other groups (P＜0.05), but no significant differences were found in albumin, total protein by different methionine levels (P＞0.05). According to quadratic curve estimation of the weight and feed/gain ratio, the optimal methionine levels were 0.466% and 0.507%, which could be averaged to 0.487%.【Conclusion】Combined with the average daily gain, feed/gain ratio, community evenness and other economic indicators, immune index, digestive system, index of blood, the optimal methionine requirement of Jinghong laying hens of 0-4 weeks is 0.49%.

RESEARCH NOTES

Improvement of the Saccharinity by Additive Action of Sucrose Transporters VvSUC11 and VvSUC12 in Transgenic Sugar Beet

YAN Tian-Tian, GUO Xin-Yong, XIANG Ben-Chun, ZHU Jian-Bo

Scientia Agricultura Sinica. 2014, 47(12):  2455-2464.  doi:10.3864/j.issn.0578-1752.2014.12.018

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【Objective】Sugar beet is an important sugar crop in the world. The sucrose content in roots is the key determinant of sugar beet quality. In order to study the improvement of saccharinity of sugar beet by two sucrose transporter genes from grape, VvSUC11 and VvSUC12, were introduced into sugar beet root using plant genetic engineering. The work will lay a foundation for using sucrose transporter genes to increase crop yield and quality. 【Method】 A bivalent root specific expression vector called pCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 containing grape sucrose transporters VvSUC11 and VvSUC12 genes was constructed and transformed into sugar beet variety KWS-9103 by Agrobacterium tumefaciens mediated transformation. Then PCR and RT-PCR analyses were used to confirm the integration and expression of the two genes in transgenic sugar beets. The leaf characters, physiological targets, tuberous root weight and saccharinity of transgenic and control plants were measured after transplanting to the field.【Result】Thirteen transgenic sugar beet lines were obtained by PCR and RT-PCR analyses. Results indicated that the average number of leaf width, petiole length and leaf number of transgenic plants were 19.31 cm, 29.17 cm and 38.33, which increased obviously by 31.81%, 16.61% and, 17.04%, respectively, the average number of leaf length were 25.16 cm, which increased insignificantly by 1.68%. However, there were no significant difference in the content of Chlorophyll a（1.31 mg•g-1）, Chlorophyll b（0.562 mg•g-1） and total Chlorophyll（1.87 mg•g-1） between transgenic and control plants. Compared to control, soluble sugar content (14.59 mg•g-1) of leaf was reduced markedly by 13.04%, but soluble sugar content (21.90 mg•g-1) of petiole was only increased by 3.36%. The average root weight and sugar content of individual plants were 3.067 kg and 183.2 g•kg-1, respectively. Compared with control plants, the root weight of transgenic plants increased by 5.91%, while saccharinity increased significantly by 9.41%. These results demonstrated that larger leaves and higher leaf number lead to the expansion of photosynthetic leaf area, and shorter petiole in transgenic sugar beets was not only helpful to accelerate the rate of transport of sugar molecules from the source leaves to the sink, but also promotes the formation of tuberous root and the accumulation of sugar.【Conclusion】The sugar transporters VvSUC11 and VvSUC12 genes transformation could effectively improve saccharinity in transgenic sugar beet.

Cloning and Expression Analysis of Nitrate Reductase Gene (MaNR) of Morus L.

WANG Qian-Ling, YU Ya-Sheng, YANG Yan, LI Jun, LIU Chang-Ying, Lv Rui-Hua , YU Mao-De

Scientia Agricultura Sinica. 2014, 47(12):  2465-2475.  doi:10.3864/j.issn.0578-1752.2014.12.019

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【Objective】 The objective of this study is to investigate the relationship between nitrate reductase (NR) and growth and regeneration of mulberry. A full length cDNA named MaNR was cloned from mulberry cultivar Guiyou No.62 and the sequence characteristics of MaNR were analyzed. Furthermore, the expression mode and influence factors during regeneration of mulberry hypocotyl regeneration in vitro was studied.【Method】 Specific primer sequences were designed according to the annotated gene scaffold 570 released in Morus notabilis genome database http://morus.swu.edu.cn/morusdb. Total RNA and DNA were extracted from mulberry leaf treated by KNO3, using RNAiso Plus (TaKaRa) and a modified CTAB method. cDNA and DNA were used as templates, RT-PCR was used to amplify MaNRcDNA sequence from RNA pools generated from KNO3-treated mulberry leaves. Bioinformatical tools were employed to analyze its deduced aimino acid sequence. Mulberry hypocotyls used as explants were inoculated in different nitrogen sources and growth regulating substances of the medium under aseptic conditions. Real-time PCR was used to investigate its relative expression level during hypocotyls regeneration in vitro and effects of different nitrogen sources and growth regulating substances. 【Result】 The obtained cDNA sequence of NR was 2 730 bp in length, encoding a polypeptide of 909 amino acid residues consisting of five classic conserved domain, protein molecular weight was 102.84 kD and the isoelectric point was 6.76. It showed that the sequence of NR has 95% identity compared with Morus notabilis and 78% indentity with other Rosaceae trees by NCBI BLAST. The obtained DNA sequence of NR was 5 142 bp including five exons and four introns. Cluster analysis showed that monocotyledons were clustered to one group, Mulberry and Rosaceae were clustered to one group, and the GenBank number was KF992020.1 and KF992021.1 respectively. Quantitative RT-PCR showed that this gene was highly expressed in roots, low expressed in stems. Glutamic acid significantly raised the expression of MaNR in leaves and GA3 significantly raised the expression of MaNR in stems. Hypocotyls could not be induced callus and multiple shoots in the single NH4+-N media but could be in the single NO3--N media. These nitrogen sources played a less part in subculture of shoot clumps. The shoots could grow in the single NH4+-N media and in the single NO3--N media. Real-time PCR exhibited that MaNR was highly abundant in cytoledons and hypocotyls than radicals. The relative expression level increased during hypocotyls regeneration in vitro and reduced to stable after shoot clumps formed. As time went on, the relative expression level of MaNR was reduced in shoot clumps with single NO3--N media or single NH4+-N. Glutamic acid showed a little inhibition of NR in the shoots subculture. 【Conclusion】 The full length of MaNR was cloned from mulberry trees. NO3--N was necessary for hypocotyls regeneration in vitro and the expression of NR affected by mulberry cell dedifferentiation and differentiation.

Effects of Fertilizer in Shallow Soils on Growth and Distribution of Rice Roots at Seedling Stage

SUN Hao-Yan, LI Xiao-Kun, REN Tao, CONG Ri-Huan, LU Jian-Wei

Scientia Agricultura Sinica. 2014, 47(12):  2476-2484.  doi:10.3864/j.issn.0578-1752.2014.12.020

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【Objective】 The aim of the research was to study the effects of different fertilization depths on growth and distribution of rice roots at seedling stage, and to ascertain the importance of fertilizer in shallow soils on the rice growth and thus providing information for appropriate fertilization. 【Method】 The trial was performed at the experimental base of Huazhong Agricultural University. Five treatments were designed, which were no fertilizer and fertilization depths of 1 cm, 5 cm, 10 cm, and 15 cm. According to the growth period of rice, samples were taken on 10, 20, 30 and 40 d after sowing. Several parameters were tested, including root biomasses, morphological index parameters, absorbing surface area and shoot biomasses of rice to assess the effect of different fertilization depths on rice seedling growth.【Result】 The results showed that no significant differences were observed among treatments on 10 d after sowing The root biomass, morphological index parameters and absorbing surface area of 1 cm treatment were significantly higher than those of others on 20 d after sowing. The increment effects were 1 cm＞5 cm, 10 cm, 15 cm＞CK, and the shoot biomass had same tendency. The biomasses of rice root and shoot of 1 cm treatment were increased by 163.8% and 121.5% compared to those of 5 cm treatment on 30 d after sowing. The shoot biomasses differed among the treatments, the order of which was 1 cm＞5 cm＞10 cm, 15 cm> CK, and that of 1 cm treatment were 37.6% and 34.6% higher than those of 10 cm and 15 cm treatment, respectively on 40 d after sowing. Additionally, the results of root distribution indicated that 60% of rice roots concentrated in the 0-10 cm soil layer on 40 d after sowing. The root biomass of each soil layer in the fertilization treatments was higher than that of no fertilization treatment. The lower root biomasses of 1 cm treatment were significantly increased, which was 26.1% and 84.0% higher than other treatments in the 10-15 cm and 15-20 cm soil layers, respectively. 【Conclusion】 Different fertilization depths had impacts on the root growth and distribution of rice at seedling stage. During the whole growth period, the root biomass, morphological index parameters and absorbing surface area in 1 cm treatment showed significant advantages compared to other treatments. The 1 cm treatment could improve the root activity, increase the proportion of the lower root and benefit of the root architecture building. In the early rice growth period, the method of appropriate fertilization in shallow soils could significantly promote root growth. The shoot biomass of shallow soil fertilization was more than other treatments, which also responded to the well root growth and distribution of rice.