Table of Content

01 January 2017, Volume 50 Issue 1

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CONTENTS

Scientia Agricultura Sinica. 2017, 50(1):  0-1. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Construction of an SSR-Based Standard Fingerprint Database for Corn Variety Authorized in China

WANG FengGe, YANG Yang, YI HongMei, ZHAO JiuRan, REN Jie, WANG Lu, GE JianRong, JIANG Bin, ZHANG XianChen, TIAN HongLi, HOU ZhenHua

Scientia Agricultura Sinica. 2017, 50(1):  1-14.  doi:10.3864/j.issn.0578-1752.2017.01.001

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【Objective】 It is of great importance to construct a shareable high-quality crop variety standard DNA fingerprint database for effectively managing the huge number of known varieties.【Method】 Based on fluorescence capillary electrophoresis detection platforms and the plant variety DNA fingerprint database management system, a database containing 3 998 maize authorized accessions was built with 40 SSR primer pairs. Multi-laboratories and multi-detecting platforms were used to conduct the quality evaluation of the database.【Result】 Allele frequency distribution graphs of the 40 corn primers were plotted as characteristic spectrums of each primer, which played the role of the similarity of reference samples in the database construction. A decuplet fluorescent capillary electrophoresis combination was formed and a set of system default PANEL was established in the SSR analyzer. Statistics were conducted on the experimental conditions of the database construction. Of the total samples, 61% of them were subjected to two group independent trials and 33% of them were subjected to three group independent trials. Each sample had 2-5 sets of original experimental data and the corresponding fingerprint maps. The accumulated loss and variable sites of the final built standard fingerprint database accounted for only 0.2%, the data integrity reached 99.8%. The assessment results in different laboratories and different electrophoresis platforms showed that the SSR fingerprint data obtained high agreement on the same electrophoresis fluorescence detection platform, but showed a certain bias in different electrophoretic detection platforms. In order to realize the sharing of SSR fingerprint data in different laboratories, a unified fluorescent primer, analysis software and electrophoresis detection platform were needed. Overall pairwise comparisons were conducted on all the fingerprint data, the results showed that there existed a relative big overall difference among the certification varieties of corn in China. Percentage of different sites among the authorized varieties was mainly concentrated between 80% and 95% (accounted for 78.28%), the percentage of different sites with more than 50% reached 99.21%, and less than 20% of only 0.09%. The average hybrid rate of the authorized varieties reached 64% and mainly concentrated between 50% and 80% (accounted for 89%). By using the corn variety standard fingerprint matching service platform (URL: http: //www.maizedna.org/), a shared fingerprint database is realized. 【Conclusion】 A standard procedure in constructing crop variety SSR fingerprint database was formed in this study and the SSR fingerprint database was constructed with a scale of nearly 4 000 corn authorized varieties. Through joint multi-laboratory comparison tests, the accuracy of database building and the sharing property of database were ensured. A service platform website for corn variety standard fingerprint matching was established in this study, thus achieving sharing of corn authorized variety fingerprint database in national seed identification system, and providing an important reference for other crop species in building high-quality SSR fingerprint database.

Genome-Wide Association Study of Root Length and Hypocotyl Length at Germination Stage Under Saline Conditions in Brassica napus

ZHANG Rui, DENG WenYa, YANG Liu, WANG YaPing, XIAO FangZhi, HE Jian, LU Kun

Scientia Agricultura Sinica. 2017, 50(1):  15-27.  doi:10.3864/j.issn.0578-1752.2017.01.002

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【Objective】 Identification of the regulatory loci and candidate genes governing salt stress tolerance in Brassica napus at germination stage could lay the foundation for improvement of B. napus salt resistance. 【Method】 In this study, 317 representative B. napus inbred lines were genotyped under normal and salt-stressed conditions in a sand culture system. Significant SNPs associated with root length and hypocotyl length in B. napus under normal and saline stress conditions and their linkage disequilibrium (LD) were determined by genome-wide association studies (GWAS), based on the Brassica 60 K SNP array. Candidate genes were selected based on the combination analyses results of functional annotation of genes within the LD blocks and transcriptome analyses of seedling roots and leaves in B. napus under saline stress treatments. Accuracy of candidate gene selection was improved by real-time quantitative reverse transcriptase PCR (qRT-PCR). 【Result】 Hypocotyl length and root length of B. napus showed large variation among accessions at germination stage under normal and salt-stressed conditions, and frequency distribution revealed that all the target traits were quantitative traits and controlled by polygenic genes. Comparison of different models showed that MLM+P+K model was the optimal model. Based on this model, GWAS identified 45 loci significantly associated with target traits, including 40 and 5 SNPs associated with hypocotyl length and root length, and each of SNP explained 9.12%-14.46% and 7.67%-8.93% of phenotypic variation, respectively. Among the significantly associated SNPs, rs8970 on chromosome C04 was the most notable, since it was the only SNP, which could be repeatedly detected between root length and hypocotyl length, and associated with four traits simultaneously, explaining 7.67%-12.35% of observed phenotypic variation. Of the 11 important significantly associated SNPs, 6 SNPs were distributed in 10 to 442 kb of linkage disequilibrium (LD) blocks. By combining differentially expressed genes detected by transcriptome analysis with LD block identification, 447 genes were identified within the 11 important LD intervals, of which 15 were activated by salt stress. BnaSRO1, BnaPAGR2, BnaNPH3, BnaMYB124, BnaSAM-Mtase, BnaBIN2, BnaUMAMIT11, BnaEXPA7, BnaRPT3, BnaEF-hand and BnaF3H were most likely the candidate genes within their LD blocks. Results of qRT-PCR detection showed that 10 candidate genes were induced by salt stress treatment in root or hypocotyl at germination stage, except for BnaNPH3. In addition, tissue-specificity detection of candidate genes also showed that BnaUMAMIT11, BnaPAGR2 and BnaEXPA7mainly expressed in the root and hypocotyl at germination stage, and BnaRPT3, BnaBIN2 and BnaMYB124 possessed the highest expression in hypocotyl, confirmed that these genes might be involved in development of root and hypocotyl and salt resistance of B. napus at germination stage. 【Conclusion】 A total of 45 significantly associated SNPs controlling development and salt resistance in root and hypocotyl of B. napus at germination stage were identified by GWAS. By combined LD block identification, transcriptome analyses and functional annotation, 11 important candidate genes were screened within different LD blocks.

Genetic Variation of Four Physiological Indexes as Impacted by Water Stress in Sugarcane

ZHAO PeiFang, ZHAO Jun, LIU JiaYong, ZAN FengGang, XIA HongMing, P.A. Jackson, J. Basnayake, N.G. Inman-Bamber, YANG Kun, ZHAO LiPing, QIN Wei, CHEN XueKuan, ZHAO XingDong, FAN YuanHong

Scientia Agricultura Sinica. 2017, 50(1):  28-37.  doi:10.3864/j.issn.0578-1752.2017.01.003

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【Objective】Understanding of the genetic variations in response to drought conditions of four physiological indexes, namely stomatal conductance (Gs), original light energy transformation efficiency of PSII (Fv/Fm), leaf elongation (LE) and leaf relative water content (RWC) could help their adaption to the current breeding program.【Method】The genetic variation (s_g²), broad sense heritability (h_b²) under rainfed and irrigated conditions for four physiological indexes, namely Gs, Fv/Fm, LE and RWC were measured for 13, 18, 15 and 10 times, respectively, from two sets of field trials in 22 and 18 genotypes which were consistently conducted at two locations in Kaiyuan and Yuanjiang counties of Yunnan province in the crop growing periods of 2013 and 2014. In the trials, rainfed and irrigated treatments were set as the main plot, and genotype was used as the sub-plot. The s_g² and error variation (s_e²) for each index at each measurement was determined using software GenStat, and h_b² was calculated. Paired t tests for the differences of s_g² and h_b² between drought and irrigated treatments were processed using software SAS9.1.【Result】The mean differences between drought and irrigated treatments were all significant (P＜0.01) in 13, 18, 15 and 10 measurements of Gs Fv/Fm, LE and RWC, respectively. Under drought and irrigated treatments, the differences in Gs among the genotypes was significant in 10 and 11 out of 13 measurements with the ranges of h_b² at 0.19-0.68 and 0.19-0.82, and grand means of 0.49 and 0.53, respectively, overall significantly higher s_g² and h_b² were found under the irrigated treatment. The differences in Fv/Fm, among the genotypes were significant in 17 and 16 out of 18 measurements with the ranges of h_b² at 0.26-0.83 and 0.16-0.85, and grand means of 0.64 and 0.58, respectively, overall higher s_g² and h_b² were found under the drought treatment. The differences in LE among the genotypes were significant in 14 and 10 out of 15 measurements with the ranges of h_b² at 0.09-0.89 and 0.09-0.81, and grand means of 0.58 and 0.50, respectively, overall higher s_g² and h_b² were found under the drought treatment. The differences in RWC among the genotypes were significant in 8 and 6 out of 10 measurements with the ranges of h_b² at 0.10-0.76 and 0.16-0.77, and grand means of 0.57 and 0.47, respectively, overall higher s_g² and h_b² were found under the drought treatment. 【Conclusion】The s_g² and h_b² of Gs, Fv/Fm, LE and RWC were impacted by water stress, in general, much higher s_g² and h_b² of Gs could be obtained under irrigated conditions and that of Fv/Fm, LE and RWC under drought conditions. However, higher h_b² could be obtained under irrigated conditions for all the four physiological indexes.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Advances and Prospects of High-Yielding and Simplified Cotton Cultivation Technology in Xinjiang Cotton-Growing Area

BAI Yan, MAO ShuChun, TIAN LiWen, LI Li, DONG HeZhong

Scientia Agricultura Sinica. 2017, 50(1):  38-50.  doi:10.3864/j.issn.0578-1752.2017.01.004

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 In the past 20 years, cotton production in Xinjiang has developed considerably through a series of technological strategies such as “exploration of heat potential via earliness-stimulating cultivation”, “exploration of solar radiation potential via dense and dwarf planting”, “improvement of water and fertilizer use efficiency via fertigation”, and improvement of net returns with convenient and simplified cultivation through the integration of agronomic techniques and mechanization. These strategies have considerably reduced labor input by reliance on mechanization rather than manual operations, precision seeding after plastic mulching, simplified plant pruning and synchronized harvesting achieved through rational and high plant density combined with chemical regulation, and improvements in yield and net returns through water-saving irrigation techniques and fertigation. The efficient use of key agronomic techniques and related materials and machinery ensures not only a high or super-high yield, but also a convenient and simplified management, thus resolving the contradiction between high yield and simplification in Xinjiang. The northwest inland of which Xinjiang is the main region has become the country's largest cotton-producing region with the highest average unit yield. To ensure continued high yield and efficiency of cotton production in the future, the cultivation strategy should also advance with the times. On the one hand, it should evolve from achieving high yield and benefits of cotton by exploration of heat and water potential to achieving high yield, fine quality and high net returns by exploitation of the light potential, fertigation, and integration of agronomic techniques and mechanization; on the other hand, in order to achieve simplified management of cotton, the traditional cultivation and management with “30% input to seeding and 70% to field management after seeding” should be changed to that with “70% input to seeding and 30% to field management after seeding”. To simultaneously improve cotton yield and quality, it is important to ensure a high photosynthetic efficiency population through comprehensive regulation of cotton plants. More attention should be paid to seed quality and seeding, and thus to further save costs and increase economic benefits by reducing management processes in cotton production. Studies on mechanisms for achieving simplified cultivation with high economic benefits of cotton in Xinjiang should be strengthened to lay a theoretical foundation for sustainable production.

Effects of Exogenous Selenium on the Physiological Activity, Grain Selenium Content, Yield and Quality of Foxtail Millet

MU TingTing, DU HuiLing, ZHANG FuYao, JING XiaoLan, GUO Qi, LI ZhiHua, LIU Zhang, TIAN Gang

Scientia Agricultura Sinica. 2017, 50(1):  51-63.  doi:10.3864/j.issn.0578-1752.2017.01.005

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【Objective】At different growth stages of the foxtail millet, selenium was sprayed at different concentrations to study the change rule of millet yield components, quality, protective enzyme activity and grain selenium content to determine the optimum amount of selenium and spraying period for foxtail millet, so as to provide a theoretical basis for production of selenium-rich foxtail millet. 【Method】The varieties Changnong35, Jigu20 and Jingu50 were used as test materials, and 6 selenium concentrations were designed, 0 Na₂SeO₃ (T0), 16.96 g·hm^-2 Na₂SeO₃ (T1), 33.92 g·hm^-2 Na₂SeO₃ (T2), 67.84 g·hm^-2 Na₂SeO₃ (T3), 135.68 g·hm^-2 Na₂SeO₃ (T4), 271.36 g·hm^-2 Na₂SeO₃ (T5). Study on influence of selenium levels on different stages of different millet varieties physiological and biochemical indexes, yield, quality and the content of selenium, spraying in seedling stage, heading stage and filling stage. 【Result】The different development stages of the different concentration of exogenous selenium foliar treatment millet, SPAD, POD, SOD, MDA, GSH and GSH-Px 6 physiological and biochemical indexes difference reached extremely significant level among varieties (P＜0.01). In addition to the MDA, other physiological indexes increase at first and then descend with the Se concentration increasing, reached the maximum when selenium concentrations T3. The activity of SOD, POD and GSH-Px, the content of GSH and SPAD values respectively after spraying treatment and compared with the control increased by 11.8%, 44%, 94%, 97.4% and 9% more than CK under heading spraying. Exogenous selenium can significantly improve the millet grain selenium content, and spraying at different time, different varieties increased with spraying selenium concentrations increased. Spraying time impact on grain selenium content in filling stage＞heading stage＞seedling stage, filling stage spraying influence on selenium content of different varieties Jingu50＞Changnong35＞Jigu20. Filling stage T3 treatment, Jingu50 grain selenium content reached 0.297 mg·hm^-2, which was increased by 8.6 times compared to the CK within the scope of the safety of the intake of selenium. Foliar spraying selenium can improve crude protein, lysine, and folic acid content of the foxtail millet, different spraying period different millet varieties T3 treatment of crude protein, lysine and folic acid content is the highest, compared with the control increased by 13.9%, 17.9% and 7.5%. Different concentration of selenium can improve millet production at T3 treatment achieve maximum, increase first and then decrease with selenium concentrations increased. Heading spraying T3 selenium concentration, production of Jingu50, Jigu20 and Changnong35 compared with the control respectively increased by 4.9%, 4.7% and 1.2%. 【Conclusion】The right amount of exogenous selenium on foxtail millet can play a significant role in promoting physiological characteristics, quality and production, Se-rich millet production prospects. At heading stage spraying sodium selenite at 67.84 g·hm^-2 is an optimal concentration and best period of millet foliar spraying selenium.

Global Sensitivity Analysis of AquaCrop Crop Model Parameters Based on EFAST Method

XING HuiMin, XIANG ShiYao, XU XinGang, FENG HaiKuan, YANG GuiJun, CHEN ZhaoXia

Scientia Agricultura Sinica. 2017, 50(1):  64-76.  doi:10.3864/j.issn.0578-1752.2017.01.006

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【Objective】Sensitivity analysis is an important link in crop model localization, and it plays an important role in AquaCrop model calibration and application.【Method】In this study, in order to identify the sensitivity parameters, the 2012-2013, 2013-2014 and 2014-2015 winter wheat experiments were conducted in National Precision Agriculture Demonstration Research Base in Beijing, China, the Extended Fourier Amplitude Sensitivity Test (EFAST) method was used to carry out sensitivity analysis of 42 crop parameters of AquaCrop model.【Result】The sensitivity parameters were: (1) For dry biomass: water and temperature stress (minimum growing degrees required for full biomass production (stbio), upper threshold of soil water depletion factor for canopy senescence (psen)), biomass and yield production (water productivity normalized (wp)), transpiration (crop coefficient when canopy is complete but prior to senescence (kcb)), canopy and phaenological development (GGD-increase in canopy cover (cgc), GDD-from sowing to emergence (eme), maximum canopy cover in fraction soil cover (mcc), GGD-decrease in canopy cover (cdc), total length of crop cycle in growing degree-days (mat), building-up of harvest index during yield formation (hilen)). stbio, kcb, wp and cgc were the four most sensitive parameters; (2) For canopy cover: canopy and phaenological development (cgc, mcc, number of plants per hectare (den), soil surface covered by an individual seedling at 90% emergence (ccs), mat and cdc), root development (maximum effective rooting depth (rtx)), water and temperature stress (psen), transpiration (kcb); (3) For yield: canopy and phaenological development (GDD-from sowing to flowering (flo), mat, cdc, hilen and GDD-from sowing to start senescence (sen)), water and temperature stress (psen), biomass and yield production (reference harvest index (hi) and wp), transpiration (kcb).【Conclusion】The results of first order and total order sensitivity analysis for AquaCrop model of winter wheat maximum dry biomass and dry biomass time-varying showed that there was a little difference in the choice of sensitivity parameters, but many differences in the ranking. The sensitivity analysis of maximum dry biomass was not comprehensive, which could not analyze the effect of crop parameters on dry biomass during the whole growth period. The results of the first order and total order sensitivity analysis for AquaCrop model of winter wheat canopy cover time-varying showed that there was a good consistency in the selection and ranking of sensitive parameters. The values of total order sensitivity indices of crop parameters were higher than first order, and the influences on canopy cover were more obvious. This study provides guidelines for AquaCrop model calibration and application in Beijing, China, as well providing guidance to simplify the AquaCrop model and improve its precision, especially when many parameters are used.

PLANT PROTECTION

Gene cloning and functional analysis of OhPbs2 in Oidium heveae

FENG Xia, LIN ChunHua, KANG Xun, JIN Yang, LIU Xiao, HE QiGuang, LIU WenBo, MIAO WeiGuo, ZHENG FuCong

Scientia Agricultura Sinica. 2017, 50(1):  77-85.  doi:10.3864/j.issn.0578-1752.2017.01.007

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【Objective】Pbs2 is one of the important members of HOG-MAPK pathway in MAPK signaling pathway, and plays an important role in osmotic regulation of plant pathogens. Oidium heveae is an obligate parasite. In this paper, the function and effect of Pbs2 of O. heveae were studied by using Colletotrichum gloeosporioides.【Method】The homologous cloning method was used to amplify the OhPbs2 by using the genomic DNA and cDNA as template. The domain of this gene was analyzed by bioinformatics. Phylogenetic analysis of seven homologous protein sequences of other fungi and OhPbs2 was conducted, and the phylogenetic tree was constructed by the maximum parsimony method in MEGA6 to further analyze and identify this gene. Using homologous recombination and protoplast transformation, OhPbs2 was transformed into the ΔCgPbs2 of C. gloeosporioides. The transformant was screened on PDA+1.5 mol·L^-1 sorbitol. At the same time, the genome of the transformant was extracted as a template and identified with the primer pairs of OhPbs2, and the correct transformant ΔCgPbs2+OhPbs2 was selected for subsequent phenotypic determination. The growth state of ΔCgPbs2, ΔCgPbs2+OhPbs2 and wild type strains was compared under different culture conditions. And the pathogenicity of the three strains was detected by inoculating the leaves of the rubber tree.【Result】The full-length of the OhPbs2 is 1 927 bp, cDNA of the OhPbs2 is 1 860 bp, and contains an intron that encodes a 619 amino acids protein. Bioinformatics analysis showed that the protein had the same S_TKc domain as CgPbs2. The phylogenetic tree showed that the Pbs2 protein was closely related to Aspergillus fumigatus Pbs2 protein, and the similarity was 55%. And the similarity to Colletotrichum gloeosporioides was 49%, also it was close to Pbs2 protein of Neurospora crassa, Magnaporthe oryzae, Saccharomyces cerevisiae and Fusarium graminearum, the similarities were 54%, 53%, 53% and 50%, respectively. ΔCgPbs2+OhPbs2 strain could grow white colonies on PDA+1.5 mol·L^-1 sorbitol medium, but ΔCgPbs2 strain could not grow. The sequencing results showed that the OhPbs2 had been successfully transferred into the ΔCgPbs2. The color of ΔCgPbs2+OhPbs2 in the MM medium colony was white with short aerial hyphae, which is different from the wild type strain. The growth rate of ΔCgPbs2, ΔCgPbs2+OhPbs2 and wild type strains decreased gradually with increasing concentration in MM medium containing different concentrations of NaCl, sorbitol, SDS, H₂O₂ and fludioxonil. OhPbs2 not only restored the ability of the wild type strain to tolerate osmolality, especially to sorbitol, but also to the susceptibility to fludioxonil, and even to enhance potency. But OhPbs2 complementary changed the color of C. gloeosporioides and inhibited the growth of aerial hyphae. CgPbs2 might be involved in the pathogenicity of C. gloeosporioides, however OhPbs2 did not restore its pathogenicity, but weakened its pathogenic ability to a certain extent.【Conclusion】OhPbs2 may be involved in the regulation of the vegetative growth, oxidative stress, osmotic pressure and cell wall formation of the pathogen, and enhance the corresponding function of the C. gloeosporioides. OhPbs2 may be involved in the pathogenicity of the pathogen, but pathogenicity may be different from CgPbs2.

Preparation of Monoclonal Antibody Against AlEcR-A Protein and Its Response Induced by Exogenous 20-Hydroxyecdysone in Apolygus lucorum

TAN YongAn, XIAO LiuBin, HAO DeJun, ZHAO Jing, SUN Yang, BAI LiXin

Scientia Agricultura Sinica. 2017, 50(1):  86-93.  doi:10.3864/j.issn.0578-1752.2017.01.008

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【Objective】The objectives of this study are to prepare the monoclonal antibody against ecdysone receptors protein and clarity the response of AlEcR-A under exogenous ecdysterone hormone (20E) in Apolygus lucorum.【Method】The vector containing AlEcR-A was double enzyme restricted by Nde Iand Xba I, then the cDNA identified by sequencing was constructed to pCzn1 vector and transformed into Arctic express of E. coli. The target recombinant protein has over expressed and has been purified by using Ni-NTA agarose. BALB/c mice were immunized with the purified recombinant fusion protein. The spleen cells of mouse were fused with SP2/0 cells. The specificity of hybridoma cell line was characterized by ELISA and Western blot analysis. Finally, the mRNA relative expression and protein content of AlEcR-A treated with exogenous 20E by RT-PCR and Western blot were analyzed, respectively.【Result】The recombinant plasmid pCzn1-AlEcR-A was high-efficient expression in Arctic express when induced by 37℃ and 5.0 mmol·L^-1 IPTG. The 55 kD target protein from the strain containing AlEcR-A was obtained by the Ni-NTA agarose. One hybridoma cell line, named 8H7, was obtained and characterized by ELISA and Western blot analysis, which could specifically combine with the total protein of A. lucorum and recombinant protein of AlEcR-A. By using RT-PCR and Western blot analysis, both the mRNA relative expression and protein content of AlEcR-A were remarkably increased after treating with 20E which compared with control.【Conclusion】the high specificity monoclonal antibody of AlEcR-A protein was obtained, and 20E could induce the mRNA and protein expressions of AlEcR-A.

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

PLFA Fingerprint Characteristics of An Anthropogenic Loess Soil Under Long-Term Different Fertilizations

GUO Yun, SUN BenHua, WANG Ying, WEI Jing, GAO MingXia, ZHANG ShuLan, YANG XueYun

Scientia Agricultura Sinica. 2017, 50(1):  94-103.  doi:10.3864/j.issn.0578-1752.2017.01.009

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【Objective】 The changes of soil microbial community structure and activity are the key indicators for evaluating soil fertility. It is important to study the effects of long-term different fertilizations and soil managements on soil microbial community structure for fertilization and soil management, and even on the sustainable utilization of farmland of the anthropogenic loess soil.【Method】This research was based on the long-term trial of “National Monitoring Base of Soil Fertility and Fertilizer Efficiency on Loess Soil” in Yangling, Shaanxi province. Soil samples were collected from treatments as farmland without fertilization (CK), farmland with N fertilizer (N), N and K fertilizer (NK), P and K fertilizer (PK), N and P fertilizer (NP), and NPK plus cattle manure (MNPK), fallow land (FL) and abandoned land (AB). The effects of long-term fertilization and soil management on soil microbial community structure and its relationship with basic soil physio-chemical properties were studied by PLFA and routine analysis. 【Result】Compared to CK, total PLFAs, bacteria, fungi, and actinomycetes PLFAs of MNPK, NP and AB were increased by 218.8%, 73.9% and 74.3%, 188.3%, 80.8% and 82.6%, 315.8%, 111.5% and 167.0%, 23.7%, 21.3% and 16.3%, respectively, and also the fungi/bacteria ratio (F/B) was significantly increased. Total PLFAs, bacteria, fungi of N, NK and PK were not significantly different, but PK reduced actinomycetes PLFAs significantly. Compared to farmland soil, FL and AB inhibited the growth of G⁺ and G^- significantly. Shannon-Winner richness index (H), Simpson dominance index (S), Pielou evenness index (J) and richness index (S) were all the highest in MNPK, and the lowest in FL. AB and NP could also increase Shannon-Winner richness index (H) and Pielou evenness index (J) significantly. The result of principal component analysis showed that MNPK, AB, NP and FL could significantly change soil microbial community structure. MNPK could increase the abundance value of G^- (18:1ω5c, cy19:0ω7c), (16:0, 10Me22:0 saturated fatty acid) and eukaryot (18:3ω6c, 16:3ω6c, 22:2ω6c). AB and NP could also increase the abundance values of bacteria (15:0, 18:0, 22:0, 17:0 saturated fatty acid). The result of redundancy analysis (RDA) showed that the importance of soil properties for microbial growth in the order were as organic matter＞total nitrogen＞soil moisture＞Olsen-P＞pH＞bulk density＞available-K, which are all important for microbial growth.【Conclusion】Farmland with NPK fertilizer plus manure, NP fertilizer and abandoned land could improve the diversity of soil microbial community structure, thus improving the ecological environment of soil, while long-term absolute bare fallow had a negative impact on soil health.

Isolation, Identification, Phylogeny and Growth Promoting Characteristics of Endophytic Diazotrophs from Tuber and Root Crops

LI YanXing, GUO PingYi, SUN JianGuang

Scientia Agricultura Sinica. 2017, 50(1):  104-122.  doi:10.3864/j.issn.0578-1752.2017.01.010

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【Objective】The objectives of this study are to isolate, identify and analyze phylogenetics of endophytic diazotrophs from tuber and root crops, test plant growth promoting (PGP) characteristics of the isolates, and to explore population property and host distributions of endophytic diazotrophs from tuber and root crops. 【Method】 Surface sterilization and low nitrogen medium were used to isolate endophytic diazotrophs. nifH detection based on PCR amplification to confirm isolates as nitrogen-fixing bacteria. 16S rRNA was amplified with PCR, blasted in EzTaxon after sequencing, and analyzed with Clustalx-MEGA to make phylogenetic tree. PGP characteristics were evaluated by testing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, indole acetic acid (IAA) production and antagonism to Fusarium spp. 【Result】 Total 219 endophytic bacterial isolates were obtained from 14 tuber and root samples including radish, carrot, potato, ginger, beet, lotus, yam, taro, cabbage, and sweet potato. All isolates were verified as nitrogen-fixing bacteria after nifH inspection and identified as 79 species of 24 genera based on 16S rRNA. The 79 species are Acinetobacter harbinensis, Arthrobacter arilaitensis, Ar. bergerei, Ar. nicotianae, Ar. nicotinovorans, Ar. nitroguajacolicus, Bacillus amyloliquefaciens, Ba. aryabhattai, Ba. circulans, Ba. fengqiuensis, Ba. firmus, Ba. flexus, Ba. halosaccharovorans, Ba. idriensis, Ba. licheniformis, Ba. litoralis, Ba. luciferensis, Ba. marisflavi, Ba. megaterium, Ba. methylotrophicus, Ba. oceanisediminis, Ba. safensis, Ba. simplex, Ba. sonorensis, Ba. stratosphericus, Ba. subterraneus, Ba. tequilensis, Ba. thaonhiensis, Ba. thioparans, Brevibacillus brevis, Br. formosus, Br. nitrificans, Br. frigoritolerans, Chryseobacterium indoltheticum, Ch. lactis, Citrobacter youngae, Delftia lacustris, Domibacillus indicus, Enterobacter asburiae, E. ludwigii, E. mori, E. xiangfangensis, Fictibacillus barbaricus, Fi. enclensis, Fi. nanhaiensis, Fi. phosphorivorans, Flavimonas oryzihabitans, Flavobacterium oncorhynchi, Microbacterium hydrocarbonoxydans, Microbacterium phyllosphaerae, Micrococcus endophyticus, Paenibacillus barengoltzii, Pae. cineris, Pae. glycanilyticus, Pae. lautus, Pae. tundrae, Pantoea agglomerans, Pan. anthophila, Pan. dispersa, Pan. rodasii, Pseudomonas azotoformans, Ps. beteli, Ps. brassicacearum, Ps. geniculata, Ps. hunanensis, Ps. koreensis, Ps. parafulva, Ps. seleniipraecipitans, Ps. simiae, Ps. syringae, Rahnella aquatilis, Rhizobium leguminosarum, Rh. massiliae, Rh. radiobacter, Sphingobacterium canadense, Sp. faecium, Staphylococcus sciuri, Stenotrophomonas rhizophila, Variovorax paradoxus. This result showed the biodiversity of endophytic diazotrophs from tuber and root crops. Of the 219 endophytic diazotrophs, 77 strains are identified as 23 species of Bacillus, and 29 strains are identified as 10 species of Pseudomonas. This makes up 106 strains of 33 species, in percentages of 48.40% and 41.77% of the 219 endophytic diazotrophs and 79 identified species. Indicating that Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops. Conducted with 79 representatives of the 219 strains, PGP test showed that 8.86% strains showed ACC deaminase activity ranging from 0.026 to 13.76 µmol α-ketobutyrate·mg^-1 protein·h^-1, 64.56% strains showed IAA production ranging from 0.34 to 28.99 µg·mL^-1, and 6.33%-13.92% strains showed antagonistic against phytopathogen Fusarium sporotrichioides ACCC37402, Fusarium xysporum MLS1 and Fusarium xysporum ACCC37438 with antifungal indexes of 41% to 63%. 【Conclusion】 Large number of endophytic diazotrophs habitat in the tuber and root of normally growing tuber and root crops. Endophytic diazotrophs from tuber and root crops phylogenetically belong 79 species of 24 genera showing wide distribution and huge biodiversity. Bacillus and Pseudomonas are dominant populations of endophytic diazotrophs from tuber and root crops. About 10%-60% endophytic diazotrophs have PGP property of producing ACC deaminase or IAA or antagonism. This might be the underlying reasons that endophytic diazotrophs are benefit to host plants.

HORTICULTURE

Crossability and Sclerotinia Resistance among Hybrids between Hexaploid (AⁿAⁿCⁿCⁿC^oC^o) and Brassica rapa

LI QinFei, CHEN ZhiFu, LIU Yao, MEI JiaQin, QIAN Wei

Scientia Agricultura Sinica. 2017, 50(1):  123-130.  doi:10.3864/j.issn.0578-1752.2017.01.011

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【Objective】 Utilization of parental species is helpful to broaden and improve genetic basis of Brassica napus. A strategy of using hexaploid derived from hybrid between B. napus and B. oleracea as bridge was evaluated to improve B. napus by crossing with B. rapa. 【Method】 Hexaploid derived from interspecific hybrid between B. napus (Zhongshuang 9) and B. oleracea (C01, high resistant to Sclerotinia sclerotiorum) was developed to cross with B. rapa by evaluating its fertility, meiotic chromosome segregation and resistance to S. sclerotiorum. Hybrids between hexaploid and 110 B. rapa were developed to evaluate crossability, seed setting percentage and resistance to S. sclerotiorum. 【Result】Hexaploid had higher resistance to S. sclerotiorum than Zhongshuang 9. Its pollen fertility was 90.6%-92.7%, seed setting was 3-7 seeds/pod. The 68.80% pollen mother cells at anaphase I was 28﹕28. The hybrids between 110 B. rapa and hexaploid could be obtained when hexaploid was used as either female or male parent, with an average seed setting rate of (4.25±3.91) seeds/pod (hexaploid used as female parent: 4.27 seeds/pod, male: 3.95 seeds/pod on average, P=0.69). Embryos 15 d after pollination grew well when crossing hexaploid with B. rapa. Although crossability among genotypes of B. rapa with hexaploid was different, no significant difference was detected in crossability among ecotypes of B. rapa (semi-winter: (4.35±3.77) seeds/pod, spring: (4.34±4.51) seeds/pod, winter: (4.01±3.43) seeds/pod，P=0.44). Hybrids owned kinds of morphology at the seedling stage, but were similar with B. napus. Seed setting rate of hybrids was (7.72± 4.45) seeds/pod on an average, without significant difference among hybrids derived from spring, winter and semi-winter ecotypes of B. rapa (seed setting rate of hybrids derived from winter: (8.07±3.43) seeds/pod, semi-winter: (7.88±4.64) seeds/pod, spring: (6.41±3.00) seeds/pod, P=0.95). Compared with Zhongshuang 9, 6 hybrids had higher resistance to S. sclerotiorum via two years identification (P＜0.05). 【Conclusion】It was concluded that it is an efficient way to improve B. napus by transferring elite traits from parental species via hexaploid strategy.

Fine-mapping of QTL and Development of InDel Markers for Fusarium oxysporum Race 1 Resistance in Watermelon

LI Na, WANG JiMing, SHANG JianLi, LI NanNan, XU YongYang, MA ShuangWu

Scientia Agricultura Sinica. 2017, 50(1):  131-141.  doi:10.3864/j.issn.0578-1752.2017.01.012

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【Objective】In order to clone gene and provide technical supports for molecular assisted breeding for Fusarium oxysporum race 1 resistance in watermelon, combined QTL mapping and re-sequencing of parental lines, tightly linked InDel (insertion/deletion) markers were developed and finally the major QTL was fine mapped.【Method】Firstly, genome-wide QTL scanning for Fusarium oxysporum race 1 resistance in an F₂ segregating population derived from a cross between the cultivated resistance female parent ‘ZXG01478’ and the cultivated susceptible male parent ‘14CB11’ was performed using the composite interval mapping program of the WinQTL cartographer 2.5 software. Secondly, InDel markers were developed based on the InDel information on the QTL region by re-sequencing of parental lines. Finally, fine-mapping, genetic map and QTL analysis were re-performed using developed InDel markers. Moreover, a total of 130 watermelon germplasms with different Fusarium oxysporum race 1 resistances were used to perform validation analysis.【Result】In the F₂ population, the frequency distribution of susceptible plant rate deviated from normality and appeared to have a discontinuous bimodal pattern. Moreover, the ratio of resistance to susceptible to Fusarium oxysporum race 1 corresponded to the expected 3﹕1 segregation for a single-locus inheritance (c²=0.52，P=0.47). Preliminary QTL mapping only identified one QTL (fon1) for Fusarium oxysporum race 1 resistance on LG1, which showed peak LOD of 26.05 and could explain 80.18% of the phenotype variation. The confidence intervals of fon1 was 193 333- 2 775 577 bp on chromosome 1 (physical map). A total of 19 InDels with length more than 20 bp were detected on QTL region by re-sequence analysis. Of these, 12 showed polymorphism between two parents. Six primer pairs were selected to genotype in the F₂ population. Using four recombinant lines in F₂ population, preliminary fine-mapping narrowed the QTL region to upstream of InDel2_fon1. QTL re-analysis showed that one of the new developed markers (InDel1_fon1) located on the peak QTL region, which showed peak LOD of 31.65 and could explain 91.46% of the phenotype variation. The genotype of InDel1_fon1 and 7716_fon were consistent with each other for all of the 130 watermelon germplasms, and there was a relative high coincidence rate (70.8%) of genotype and phenotype. Target QTL region was narrowed to physical distance of 246 kb using new developed InDel markers and flanking SNP markers. 【Conclusion】 Major QTL (fon1) confirmed the existence of Fusarium oxysporum race 1 resistance gene Fon-1 and was finely-mapped to a relative small region. One of the new developed markers, InDel1_fon1, was tightly linked to gene Fon-1, which could better applied in molecular assisted breeding for resistance to Fusarium wilt in cultivated watermelon.

STORAGE·FRESH-KEEPING·PROCESSING

The Volatile Compositions from Rice Stored with Dynamic Temperature and Humidity Based on SPME-GC/MS and  Electronic Nose (e-nose) Technics

CAO Jun, LIU Xin, CHEN WenRuo, DAI BingYe, DONG Wen, CHEN YinJi

Scientia Agricultura Sinica. 2017, 50(1):  142-160.  doi:10.3864/j.issn.0578-1752.2017.01.013

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【Objective】 Through laboratory simulation of dynamic temperature and humidity conditions, the volatile compositions of rice were studied to find out some characteristic volatiles closely related to the rice quality. The objective of this paper is to provide reference for the construction of dynamic storage and transportation of rice. 【Method】 According to the actual transportation conditions of rice, rice was stored by simulating dynamic temperature and humidity of storage and transportation. Rice samples with 5 moisture contents (14%, 16%, 18%, 20% and 22%) were stored and transported for two months, respectively, at low temperature, middle temperature and high temperature (respectively fluctuates around 10℃, 20℃, and 30℃, humidity fluctuates around 80%). All rice samples were tested by SPME-GC/MS and electronic nose (e-nose) in this study. Principal component analysis (PCA) was used to analyze the data. 【Result】 Under different temperature conditions, the radar graphs of different moisture contents and time of rice all were different. The response values of the same moisture content rice had obvious differences under the different temperature conditions at 15 day. With the time, the differences of 14%-18% moisture contents rice samples reduced. Principal component analysis of e-nose could significantly distinguish all rice samples during storage. There were 275, 262 and 215 kinds of volatile compositions of rice samples including alkanes, olefin or alkyne, aromatic compounds, glycol ether, aldehydes, ketones, acid esters, and heterocyclic were detected, respectively, under low, medium and high temperature conditions, but only 46 kinds of which were detected in the original samples. Alkanes showed a bigger difference under low temperature conditions, and followed by medium temperature, high temperature. Olefin or alkyne, aromatic compounds, glycol ether and heterocyclic showed greater difference with temperature rising, and aldehydes, ketones, acid esters showed the largest difference under medium temperature conditions. With the time, characteristic alkane transformed from straight-chain into cycloalkane under low temperature or moisture content conditions. Haracteristic olefin or alkyne were mainly oxygen-containing or ring-shaped material at the late stage of storage. The 2,6-Di-tert-butyl-4-methylphenol, octyl phenol, and cinnamonitrile were aromatic compounds of fresh rice. With the deterioration of rice quality, methoxy or naphthalene ring materials were main characteristic aromatic compounds in rice. The 2-methyl-1-hexadecanol, benzyl alcohol, decanal, and piperitone were main characteristic glycol ether, aldehydes or ketones and had special fruity or irritating smell. Methyl-2-aminobenzoate, methyl salicylate, dihydroactinidiolide were main characteristic acid esters and had a sweet taste at early stage or under low temperature conditions, and myristic acid, decanoic acid were found at later stage and were tasteless or offensive. Furan, quinolone were characteristic heterocyclic substances with special flavor.【Conclusion】 E-nose could quickly and effectively distinguish rice samples under different moisture contents and temperatures conditions. The types and content of volatile compositions are affected by moisture content and temperature conditions of rice. Low moisture contents of rice (14%-16%) and short-term storage (30 days) are conducive to control the change of volatile compositions, and high moisture contents (20%-22%) could speed up the change of volatile compositions in rice.

Electronic Nose and Electronic Tongue Combined with Fuzzy Mathematics Sensory Evaluation to Optimize Bacon Smoking Procedure

WANG Qiong, XU BaoCai, YU Hai, LI Cong

Scientia Agricultura Sinica. 2017, 50(1):  161-170.  doi:10.3864/j.issn.0578-1752.2017.01.014

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【Objective】Using Electronic Nose (E-nose), Electronic tongue (E-tongue) and Fuzzy Mathematics Sensory Evaluation to pick out a bacon smoked by a certain brand and concentration of smoking liquid which resembles the traditional wood chips smoking bacon in flavor, and study the feasibility and accuracy of optimizing the bacon smoking procedure with E-nose and E-tongue. 【Method】Bacons made with three brands and three concentrations of smoking liquid and traditional wood chips smoking were analyzed by E-nose and E-tongue and verified with fuzzy mathematics sensory evaluation.【Result】After analyzing the E-nose Principle Component Analysis (PCA) chart, the accumulating contribution of Principle Components (PC) 1 and 2 is 99.760%, much larger than 85%, which means PC1 and PC2 have contained enough information to reflect the whole sample information. The distribution areas of sample 4 (3‰ Minghua MH-SO152) and sample 1 (woodchips smoked) are obviously separated, which suggests that the odor of the two samples are clearly different. However, the distribution areas of sample 5 (1‰ Minghua SY-SO968), sample 7 (3‰ Minghua SY-SO968), sample 9 (2‰ Jinniushan Ⅱ), and sample 10 (3‰ Jinniushan Ⅱ) are close to that of sample 1, which means their smell is close to sample 1 as well. The distribution areas of sample 6 (2‰ Minghua SY-SO968) and sample 1 overlap partially, which means the smell of sample 6 (2‰ Minghua SY-SO968) and sample 1 (wood chips smoked) are basically the same, thus the bacon added with 2‰ Guangzhou Minghua SY-SO968 smoking liquid smells basically the same as the traditional one. By analyzing the E-tongue Principle Component Analysis (PCA) chart, the accumulating contribution of Principle Components 1 and 2 is 88.903%, larger than 85%, enough to reflect the general sample information. The distribution areas of sample 2 (1‰ Minghua MH-SO152), sample 3 (2‰ Minghua MH-SO152) and sample 4 (3‰ Minghua MH-SO152) are far away from sample 1, which shows that their taste can be effectively distinguished from woodchips smoked sample 1. And the distribution areas of sample 8 (1‰ JinniushanⅡ), sample 9 (2‰ Jinniushan Ⅱ) and sample 10 (3‰ Jinniushan Ⅱ) locate near sample 1, thus they taste relatively like sample 1. The distribution areas of samples 5 (1‰ Minghua SY-SO968), 6, and 7 (3‰ Minghua SY-SO968) are closest to sample 1, which means they taste closest to sample 1, thus the bacons added with Guangzhou Minghua SY-SO968 smoking liquid taste closest to the traditional one. In Fuzzy Mathematic Sensory Evaluation and flavor description, the comprehensive scores of samples 1, 6, 7 and 10 (3‰ Jinniushan Ⅱ) are close and higher than sample 8, and described as heavy smoking flavor and good taste. The Fuzzy Mathematic Sensory Evaluation score of sample 6 is closest to sample 1, and the difference is merely 0.05, which verifies the E-nose and E-tongue results.【Conclusion】After analyzing the E-nose and E-tongue Principle Component Analysis charts, the bacon added with 2‰ Guangzhou Minghua SY-SO968 smoking liquid smells basically the same as the traditional one, and tastes closest as well, which means it can be used to replace the traditional smoking technique. Fussy mathematics sensory evaluation scores verified the fact and proved E-nose and E-tongue can be used to optimize bacon smoking procedure efficiently, thus provides a reference method to optimize meat production technique from flavor perspective. 

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Association of the PPARγ and C/EBPα Gene Expression with Intramuscular Fat Content in Different Varieties of Pig

PAN DaoXing, WANG Zhen, YANG MaoLin, LIAO QiaoPing, YANG ChangPing, WU YuPing, WANG JinZhou, LIU RuoYu

Scientia Agricultura Sinica. 2017, 50(1):  171-182.  doi:10.3864/j.issn.0578-1752.2017.01.015

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【Objective】The objectives of this study were to investigate the developmental changes of the PPARγ and C/EBPα mRNA expression level in different tissues，months and breeds, and its effect on intramuscular fat accumulation in pig. 【Method】Jiangkou Luobo Pig, Congjiang Xiang Pig and Large White Pig were slaughtered at 3, 4, 5, 6, and 7 months to collect samples from heart, liver, spleen, lung, kidney, small intestine, longissimus dorsi, and subcutaneous adipose for the purpose of determing the IMF content in Longissimus dorsi and extracting total RNA in order to investigate the developmental changes of the PPARγ and C/EBPα expression by real - time PCR.【Result】By real-time PCR, PPARγ and C/EBPα had the mRNA expression in all tissues of Large White Pig at 3, 4, 5, 6, and 7 months, but highly in liver, small intestine, longissimus dorsi, subcutaneous adipose, lowly in heart, spleen, lung, and kidney. The expression levels of PPARγ in the small intestine, longissimus dorsi, and subcutaneous adipose at 3, 4 months were extremely different with that at 6, and 7 months (P＜0.01). The expression levels of C/EBPα in the liver, lung, small intestine, and subcutaneous adipose at 3 month was extremly different with that at 6, and 7 months (P＜0.01); The mRNA expression of PPARγ and C/EBPα were lower in heart, spleen, lung, kidney, and longissimus dorsi of Jiangkou Luobo Pig, higher in subcutaneous adipose with issue-specificity. The expression amount increased with the age, and the increase of subcutaneous fat was the most. The expression levels of PPARγ in the small intestine, longissimus dorsi, and subcutaneous adipose at 3, and 4 months also were extremely different with that at 6, and 7 months (P＜0.01). The expression levels of C/EBPα in the small intestine, subcutaneous adipose at 3 month were extremely different with that at 5, 6, and 7 months (P＜0.01). The mRNA expression of PPARγ and C/EBPα in the Subcutaneous adipose of Congjiang Xiang Pig was the highest. The expression of PPAR was not significantly increased with the age. The expression of C/EBPα was significantly increased with the age. The expression levels of PPARγ and C/EBPα in the small intestine, longissimus dorsi, and subcutaneous adipose at 3, 4 months were different with that at 6, and7 months (P＜0.05). With growing, the mRNA expression levels of PPARγ and C/EBPα were basically the same in the model, and showed an increasing trend, then maintained the higher expression levels in 6, and 7 months. The mRNA expression levels of PPARγ and C/EBPα were gradually increased with the increase of the age and maintained a relatively stable level at the late growth stage. The expression of PPARγ and C/EBPα was the highest in Subcutaneous adipose, the higher in Liver, Lung, Small intestine, and the lowest in heart, spleen, and kidney. The PPARγ and C/EBPα mRNA expression in different tissues of Jiangkou Luobo Pig, Congjiang Xiang Pig were higher than that of Large White Pig. The correlation of gene expression and IMF content showed that the PPARγ and C/EBPα expression level were positively related to IMF content. 【Conclusion】It was inferred that PPARγ and C/EBPα gene may be related to regulatory mechanism of lipid metabolism and fat deposition.

The Whole Genome Data Analysis of Sanjiang Cattle

SONG NaNa, ZHONG JinCheng, CHAI ZhiXin, WANG Qi, HE ShiMing, WU JinBo, JIAN ShangLin, RAN Qiang, MENG Xin, HU HongChun

Scientia Agricultura Sinica. 2017, 50(1):  183-194.  doi:10.3864/j.issn.0578-1752.2017.01.016

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【Objective】 The objective of this paper is to study the genetic diversity of Sanjiang cattle group and discuss its genetic variation at the genome level.【Method】Fifty individual genomic DNA were extracted and mixed with isocratic and equal volumes, then the DNA pool of the mixed samples were constructed. Genomic DNA was interrupted randomly by using CovarisS2 and the DNA fragments of 500 bp were recovered by electrophoresis, and  DNA library was constructed at last. Finally, the sequencing data were obtained through the Illumina HiSeq 2000. The short reads were mapped to bovine reference genome (UMD 3.1) to detect the genomic mutations of Sanjiang cattle using BWA software. The analysis of the re-sequencing data was implemented using SAMtools, Picard-tools, GATK, Reseqtools, the SNPs and indels were annotated based on the Ensembl, DAVID and dbSNP database. 【Result】A total of 77.8 Gb of sequence data were generated by whole-genome sequencing analysis, 99.31% of the reference genome sequence was covered with an mapping depth of 25.32-fold, 778 403 444 reads and 77 840 344 400 bases were obtained, of which 673 670 505 reads and 67 341 451 555 bases covered 86.55% and 86.51% of bovine reference genomes (UMD 3.1) respectively, paired-end reads mapping were 635 242 898 (81.61%), paired-end bases mapping were 63 512 636 924 (81.59%). A total of 20 477 130 SNPs and 1 355 308 small indels were identified, of which 2 147 988 SNPs (2.4%) and 90 180 (6.7%) indels were found to be new. Of the total number of SNPs, 989 686 (4.83%) homozygous SNPs and 19 487 444 (95.17%) heterozygous SNPs were discovered, homozygous/heterozygous SNPs was 1﹕19.7. Transitions were 14 800 438, transversions were 6 680 058, transition/transversion (TS/TV) was 2.215. SNPs of splice site mutations were 727, the number of SNPs which the start codon converts into no stop codon were 117, SNPs of premature stop codon were 530, the number of SNPs which stop codon converts into no stop codon were 88. A total of 57 621 non-synonymous SNPs and 83 797 synonymous SNPs were detected, the ratio was 0.69. Non-synonymous SNPs were detected in 9,017 genes, 567 genes were assigned as trait-associated genes, which included meat quality, disease resistance, milk production, growth rate, fecundity with the number of 471, 77, 21, 10, and 8 respectively, the function of some genes were overlap. In detection of indels, 693 180 (51.15%) were deletions and 662 148 (48.85%) were insertions, 161 198 (11.89%) were homozygous and 1 194 110 (88.11%) were heterozygous. Most variations were located in intergenic regions and introns. Heterozygosity (H), nucleotide diversity (Pi) and theta W of Sanjiang cattle genome-wide were 7.6 × 10^-3, 0.0039, 0.0040, respectively, which indicated that Sanjiang cattle have an abundant genetic diversity. The Tajima'D of Sanjiang cattle population was-0.06 832, which speculated that the population exists an unbalanced selection.【Conclusion】Results of this research will provide valuable genomic data for further investigations of the genetic mechanisms underlying traits of interest and protection of Sanjiang cattle breeds genetic diversity.

Insight into Genetic Basis of Bombyx mori Resistant Strains with Resistance to BmNPV by Molecular Linkage Analysis

GAO Rui, LI ChunLin, TONG XiaoLing, CAO MingYa, SHI MeiNing, XU AnYing, LU Cheng, DAI FangYin

Scientia Agricultura Sinica. 2017, 50(1):  195-204.  doi:10.3864/j.issn.0578-1752.2017.01.017

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【Objective】Bombyx mori nucleopolyhedrovirus (BmNPV) can lead the B. mori nucleopolyhedrosis, which caused a huge loss in the sericulture industry. The objective of this study is to map the controlling genes and understand the genetic basis underlying the BmNPV resistance to serve the theory support for resistant variety breeding. 【Method】Backcross population BC₁F (using for linkage analysis) and BC₁M (using for mapping analysis) were derived from crosses between the highly resistant B. mori strain 99R and the sensitive strain Dazao-N. Concentration gradient of virus was used to treat the parents, and the number of infected individuals was recorded. Then by using SPSS17.0, the LD₅₀ for each parent was calculated. Based on appraisal of resistance performance of two parents, the virus adding concentration for BC₁ population was determined and then the virus was fed to larva quantitatively at the start of 4th instar one by one. The infected individuals in the BC₁F were chosen as linkage analysis materials. By using the screened polymorphism markers covering all B. mori autosomes, linkage analysis was conducted and then the genotype data were analyzed by T test to get the linkage significance level of each polymorphism marker to show whether it linked with resistance. Thereafter, polymorphism markers will be enriched on the chromosomes which include the resistance linked polymorphism markers to map resistance locus on them. 【Result】Median lethal dose (LD₅₀) of 99R and Dazao-N is 2.92×10⁶ and 9.78×10⁵ polyhedral bodies, respectively, and the dose 2.0×10⁶ was chosen as the infection dosage of BC₁ backcross population, which is slightly higher than the average of two parents’ LD₅₀. Two independent linkage analyses were conducted successively in the autumn of 2014 and spring of 2015, which got a discrepant result that the linkage analysis result could not be repeated in the following confirmation; i.e., one marker, S2205, on Chr 22 was identified to link with resistance in the 1st experiment, while no linked marker was identified in the following one. Comparison with the previous reports on the linkage and mapping analysis of BmNPV resistance, it was found that the nonrepeatability was ubiquitous in the related researches in B. mori. Previously, a molecular marker (AY380833) was found to highly link with the resistant trait in NB and 871C, which are both the highly resistance strains to BmNPV. However, the linkage relationship was not detected in the linkage analysis population of 99R and 871C here.【Conclusion】the genetic complexity of resistance by molecular linkage analysis was further proved, including the variation among different resistant strains, as well the plausible multiple resistant loci in one single strain. At the same time, it was proposed that the BmNPV resistance may be a complex trait, as a prerequisite to be one type of qualitative-quantitative traits, the quantitative trait characteristic of BmNPV resistance is very obvious.