Scientia Agricultura Sinica

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Scientia Agricultura Sinica. 2018, 51(15): 0-0.

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Verification and Analyses of Soybean GmbZIP16 Gene Resistance to Drought

ZHAO WanYing, YU TaiFei, YANG JunFeng, LIU Pei, CHEN Jun, CHEN Ming, ZHOU YongBin, MA YouZhi, XU ZhaoShi, MIN DongHong

Scientia Agricultura Sinica. 2018, 51(15): 2835-2845. doi:10.3864/j.issn.0578-1752.2018.15.001

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【Objective】Soybean GmbZIP16 protein was screened by analyzing soybean drought transcriptome. Functions of soybean GmbZIP16 were verified by analyzing the phenotypic characterization of transgenic GmbZIP16 Arabidopsis and soybean hairy root complexes. On the basis of experimental result analyses above-mentioned, we could determine that GmbZIP16 was involved in the drought resistance process.【Method】soybean GmbZIP16 was found out by analyzing soybean drought transcriptome and cloned by PCR, which soybean cDNA as a template, and then ligated the fragment to pCAMBIA1302 and pCAMBIA3301 expression vectors by in-fusion ligase. The recombinant pCAMBIA1302-GmbZIP16 and pCAMBIA3301-GmbZIP16 vectors were transferred into Agrobacterium GV3101 and K599 competent cells by liquid nitrogen freeze-melt method, respectively. The transgenic Arabidopsis plants and transgenic soybean hairy root complexes were generated by Agrobacterium-mediated transformation method. The transgenic GmbZIP16 Arabidopsis plants were identified by semi quantitative PCR and quantitative real time PCR. Which demonstrated GmbZIP16 could over express in transgenic Arabidopsis and transgenic soybean hairy root complex plants. 2 weeks old transgenic Arabidopsis and WT plants grew under normal conditions were transferred into MS0 solid medium supplement with 6% and 8% PEG for 7d. Biomass differences between transgenic Arabidopsis and WT plants were investigated and analyzed. The different expression of stress- related genes between Arabidopsis and WT plants were analyzed by qRT-PCR. The transgenic GmbZIP16 soybean hairy root complexes and control group plants were treated with 25% PEG for 7 days, and then the leaf samples of transgenic GmbZIP16 soybean hairy root complex and control group plants were taken. The proline content, MDA content and chlorophyll content of leaf samples were measured by Multiskan Spectrum Microplate Spectrophotometer.【Result】The GmbZIP16 gene was isolated by PCR technology. The two transgenic GmbZIP16 Arabidopsis lines were obtained by Agrobacterium-mediated transformation method. Compared with the WT plants, the transgenic GmbZIP16 Arabidopsis lines had higher biomass (the fresh weight and the root length) and survival rate under drought stress by phenotypic characterization experiment. The expression levels of some relative genes such as RD29B, DREB2A and P5CS were improved in transgenic GmbZIP16 Arabidopsis, compared with the WT plants. The leaves of transgenic GmbZIP16 soybean hairy root complex plants had the higher proline and chlorophyll content and a lower MDA content than that of the control group plants under the deal with 25% PEG. 【Conclusion】The drought resistance of transgenic Arabidopsis thaliana was improved by expressing soybean GmbZIP16 in Arabidopsis thaliana. Over expression of GmbZIP16 could enhance the drought resistance of transgenic soybean hairy root complex. GmbZIP16 can improve the drought resistance of plants mainly by affecting the expression of genes related to stress tolerance.

Genetic Diversity and Genetic Relationship of Broomcorn Millet (Panicum miliaceum L.) Germplasm Based on SSR Markers

XUE YanTao, LU Ping, QIAO ZhiJun, LIU MinXuan, WANG RuiYun

Scientia Agricultura Sinica. 2018, 51(15): 2846-2859. doi:10.3864/j.issn.0578-1752.2018.15.002

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【Objective】The objective of this study is to analyze the genetic diversity and relationship of broomcorn millet landraces and wild materials by SSR markers, to provide available data for further evolutionary study of broomcorn millet.【Method】137 SSR primers are used to identify polymorphisms in six representatives which selected randomly from the total of accessions. A total of 103 primers produce clear and reproducible polymorphic fragments among the six accessions and then are used to amplify 146 broomcorn millet accessions. Genetic diversity and relationship between different individuals and populations is evaluated by analyzing genetic parameter, clustering, and genetic structure.【Result】 103 SSR markers detect a total of 308 alleles (Na) with an average of 2.99 for each SSR and the mean values of Shannon-Weaver index (I), Nei and PIC were 0.8478, 0.3642 and 0.5544, respectively. Their resolution range was 0.334-4.002 and more than 60% distribution at intervals of 1-4, indicated the moderate resolving power of these SSR. The observed number of alleles (2.9126), Shannon-Weaver index (0.8302), expected heterozygosity (0.5023), and PIC value (0.5278) of broomcorn millet accessions in China were all higher than those in abroad, indicated more abundant genetic diversity in Chinese samples. The genetic distance of the 12 populations ranged from 0.0783 to 0.5762 with a mean of 0.2938. The genetic identity ranged from 0.5620 to 0.9247 with a mean of 0.75. We found that the genetic similarity had a certain correlation with geographical distribution. The closer geographical distribution, the smaller genetic distance, the higher genetic identity. Cluster analysis divided 12 populations into 4 groups at a genetic distance of 0.15. Among them, resources in South America and Shanxi were each independently divided into one group, which had a far-distance relationship with other resources. In the inter-individual clustering, the division of resources at home and abroad was very significant. At a genetic distance of 0.63, 146 broomcorn millet accessions could be divided into three groups. Group Ⅰand group Ⅱ were foreign accessions, and group Ⅲ was domestic accessions. Further, group Ⅱ was divided into three subpopulations at a genetic distance of 0.39, and group Ⅲ was divided into five subpopulations at a genetic distance of 0.45. There had closer genetic relationship between Asia and European resources, as well as Hebei, Shanxi and Inner Mongolia in China resources than other populations. The result of genetic structure analysis showed that there is obvious genetic differentiation between the domestic populations and foreign populations. Five groups (Group 2, Group 5, Group 6, Group 7 and Group 9) were unique genotypes which owned by Chinese wild resources and distributed more widely, 2 groups (Group 1 and Group 4) were unique genotypes of foreign resources and have a relative narrow distribution. The population structures of Ningxia and South America tend to be independent, and the population structures of Hebei, Heilongjiang and Asia tend to be diversified. The UPGMA clustering results were consistent with the results of genetic structure analysis, and the genetic relationships were related to their geographical distribution.【Conclusion】The genetic diversity of wild accessions is higher than that of landraces, of which Hebei population has the most abundant genetic diversity, so we suppose Hebei province may be the sub-origin center of broomcorn millet.

Relationship Between Soil Water Variation, Wheat Yield and Grain Protein and Its Components Contents Under Sub-Soiling During the Fallow Period Plus Mulched-Sowing

ZHANG HuiYu, SUN Min, GAO ZhiQiang, LIANG YanFei, YANG QingShan, ZHANG Juan, LI NianNian

Scientia Agricultura Sinica. 2018, 51(15): 2860-2871. doi:10.3864/j.issn.0578-1752.2018.15.003

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【Objective】The objective of this paper was to clarify the effects of sub-soiling in fallow period (SS) and mulched-sowing on soil water storage, so as to contribute to explore the best tillage and mulched-sowing technique of winter wheat, which could further improve wheat grain yield and quality coordinately. 【Method】Field experiment was carried out from 2012 to 2014 in Wenxi, Shanxi province. Taking with or without SS as the main plots and three sowing methods (film-mulched soil hole sowing, FSH; film-mulched sowing, FM; drill sowing, DS) as the subplots, the effects of sub-soiling and mulched-sowing on soil water storage in the depth of 3 m, grain yield, grain protein content and yield were assessed.【Result】Compared with NT, the soil water storage in 0-300 cm soil layer from sowing stage to anthesis was improved significantly under SS, the yield was increased by 12%-30%, and the grain glutenin content and grain protein yield were improved significantly. Compared with DS, the soil water storage from wintering stage to anthesis stage was improved significantly, the grain yield was increased by 6%-24%, the grain gliadin content, grain protein content and grain protein yield increased significantly, the grain albumin increased. Compared with FSH, the soil water storage at anthesis was decreased significantly under the condition of SS combined with FM, yet showed no significant difference at each stage under the condition of NT combined with FM. The grain yield under FM was superior to that under FSH, showing significant difference under SS in 2012-2013, NT in 2013-2014. Under the condition of SS, the grain protein components content, grain protein content and yield were improved significantly under FM, compared with FSH. In addition, the soil water storage in the soil layer of 0-100, 100-200, 200-300 cm at anthesis showed positive effect by interaction, the soil water storage in the 100-200 cm soil layer showed closely relationship with grain protein in 2012-2013, and the soil water storage in the 200-300 cm soil layer showed closely relationship with grain protein in 2013-2014. Focusing on the contribution to grain protein yield, SS influenced the grain protein yield by affecting the soil water storage at anthesis; Under the condition of SS, the mulched-sowing influenced the grain protein yield by affecting the soil water storage at booting stage, and the contribution under FM was higher than under FSH. 【Conclusion】SS was beneficial to accumulate the precipitation. Mulched-sowing was conductive to reserve the soil water, such effect could last to anthesis. The soil water during the medium-term of the whole life related closely to the grain protein accumulation. In conclusion, SS plus film-mulched sowing could ultimately achieve the coordinated improvement of yield and grain protein content.

Regulation Effects of Different Mulching Patterns During the Whole Season on Soil Water and Temperature in the Maize Field of Loess Plateau

WU Yang, JIA ZhiKuan, BIAN ShaoFeng, WANG YongJun

Scientia Agricultura Sinica. 2018, 51(15): 2872-2885. doi:10.3864/j.issn.0578-1752.2018.15.004

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&【Objective】To indentify the synergy effects of different mulching patterns on soil water and temperature for improving maize (Zea mays L.) productivity sustainably, and to provide a theoretical evidence for establishing efficient and environmentally friendly mulching patterns in semi-arid areas of Loess Plateau.【Method】A 3-years located expeiment with four mulching patterns during the whole season were carried out in the experiment area: Full amount plastic film (PM), degradable film mulching (BM), ridge and furrow planting with plastic film mulched on the ridges (RH) and straw mulching (SM), and conventional flat planting without mulching (CT) was used as the control. Then the effects of different mulching patterns on soil water and temperature spatiotemporal dynamic variations and utilization characters were also studied thoroughly. 【Result】The results showed that mulching during the whole season significantly inhibited the invalid evaporation of soil during the fallow season and improved potential precipitation use efficiency (PPE). With PM, BM, RH and SM, the precipitation storage during the fallow season increased by 53, 51, 32 and 36 mm compared with CT, respectively, and the PPE increased by 14%, 12%, 11% and 10%, respectively. Film mulching significantly improved soil temperature, thereby improved the biomass accumulation rate and shortened the growth and developmental process of maize, and the jointing stage and maturity stage under PM, BM and RH advanced by 15, 8 and 7 d, and 17, 7 and 7 d, respectively, compared with CT. PM, BM and RH increased yield by 52%, 32% and 27%, improved accumulated temperature use efficiency (TUE) by 57%, 15% and 58%, increased water use efficiency (WUE) by 31%, 14% and 26%, increased precipitation use efficiency (PUE) by 53%, 27% and 29%, and reduced water consumption coefficient (WUC) by 33%, 21% and 22%, respectively, compared with CT. SM increased soil water storage during the maize growth season, but decreased soil temperature and presented retarded growth, the jointing stage and maturity stage with SM delayed by 4 d and 5 d, respectively, which decreased the yield, WUE and TUE by 21%, 18% and 9%, respectively, compared with CT. PM intensified the utilization of soil water, then the water supply capacity at the end of the season (WSC) under PM decreased by 19, 56 and 86 mm, compared with BM, RH and SM, respectively. The soil water balance under PM was greatly affected by the precipitation amount during the middle and late growth stage of maize. The precipitation amount from July to September during 2015 reduced 71 mm compared with the average value during many years, and the soil water storage in the 100-200 cm layer under PM reduced 28 mm compared with CT, and 5.4 mm compared with stable soil water storage, which caused water deficit in the deep soil layer.【Conclusion】PM would be a usefull method to improve the efficiency of soil hydrothermall resource utilization and to increase maize yield in semi-arid area of Loess Plateau, which accompanied with risks for maintaining deep soil water balance and stablizeing crop yield in the year with droughts during the middle and late growth stage of maize. BM and RH had weak effects on increasing soil temperature and maize yield compared with PM, whereas they were more benifical to maintain the balance of soil water. SM showed a significant effect on soil temeprature decreasing and yield reduction, which was not recommended in the semi-arid area with low temperatures.

Remote Sensing Estimation of Canopy SPAD Value for Maize Based on Digital Camera

HE Ying, DENG Lei, MAO ZhiHui, SUN Jie

Scientia Agricultura Sinica. 2018, 51(15): 2886-2897. doi:10.3864/j.issn.0578-1752.2018.15.005

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【Objective】Chlorophyll is an important pigment in plant photosynthesis. The objective of this study is to investigate the inversion of chlorophyll content using crop spectrum information, so as to provide an important basis for real-time monitoring and diagnosis of crop growth.【Method】Based on the field environment under different nitrogen fertilizer application levels (0, 50% and 100%) of maize, the light and small UAV equipped with consumer level digital camera was used to obtain the RGB image of the test area, and then the soil adjusted vegetation index was used for image segmentation. 15 common visible vegetation indexes were extracted based on images before and after segmentation. Then the correlation between vegetation index and SPAD values were analyzed, besides single variable regression model, multiple regression model and random forest regression model based on visible vegetation indexes were established to estimate the SPAD values. And then, the indicators of accuracy evaluation, coefficient of determination, root mean square error, mean relative error and P<0.01 were used to select the best indicators and the optimal model.【Result】There was a significant correlation between VI_plot and VI_plantvegetation indexes and the SPAD value of maize canopy, for example, the correlation coefficient between normalized redness intensity (NRI), normalized pigment chlorophyll ratio index (NPCI), blue red ratio index (BRRI) and SPAD value of VI_plant was above 0.77. The univariate regression models were built, which took NRI,NPCI, BRRI and DVI as the independent variables and the measured SPAD as dependent variable, including linear, exponential, logarithmic, two degree polynomial and power function models, and among those models, the two polynomial model constructed by the NRI index was the best one with the decision coefficient R² of 0.7976, the RMSE of 4.31, and the MRE of 5.91%; the precision of the model using the random forest regression algorithm was the highest, in which the determining coefficient was 0.8682, the RMSE was 3.92, and the MRE was 4.98%; the multiple regression model had higher accuracy than any single variable regression model, in which the decision coefficient R² was 0.819, RMSE was 4, and MRE was 5.67%. The six inversion models of SPAD were used to make the distribution map of corn canopy SPAD value, and then the map using random forest regression model had the best result which was the closest to real SPAD distribution with R² of 0.8247，RMSE of 4.3，MRE of 5.36％, therefore which could be used as a main method of corn canopy chlorophyll monitoring information.【Conclusion】The results showed that the application of UAV digital imagery in retrieving SPAD of corn was feasible, which also added new means and experience to the application of UAV remote sensing system in agriculture.

Molecular Identification of Pratylenchus Species in 10 Samples Collected from Wheat Field in Huanghuai Region of China

LIU HaiLu, WANG Xuan, LI HongMei, LI YanXia, XUE BoWen, MA JuKui

Scientia Agricultura Sinica. 2018, 51(15): 2898-2912. doi:10.3864/j.issn.0578-1752.2018.15.006

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【Objective】The Pratylenchus species are migratory endoparasites of plant roots, causing root lesion of many crops and great damage of agricultural production all over the world. In order to clarify the species of genus Pratylenchus co-infection with Heterodera avenae on wheat from Huanghuai region of China, 10 samples were collected from wheat field in 4 provinces of the region and the Pratylenchus species were molecularly identified. The phylogenetic relationship of Pratylenchus species and genetic variation of intraspecific populations were analyzed. The results will provide valuable information for the integrated control of nematode diseases on wheat root. 【Method】The Pratylenchus nematodes were extracted from 10 samples collected from wheat fields infected with H. avenae in Jiangsu, Anhui, Henan and Shandong provinces. Fivenematodes were randomly picked up from each sample, and the DNA of individual nematode was extracted as the template for PCR amplification. The fragments of rDNA 18S region were amplified and the PCR products were sequenced. The BLAST alignment of 18S sequences revealed the different Pratylenchus species may present in these samples. The DNA templates of two representative specimens from each sample were selected for the further amplification of fragments from rDNA 28S D2-D3 region and mtDNA-COI gene. The fragments were sequenced and the BLAST alignments were performed. The phylogenetic trees were constructed on basis of rDNA 18S, 28S D2-D3 and mtDNA-COI sequences using neighbor-joining method by MEGA4.0 software, respectively. Thespecies identification was supported by the analyses of phylogenetic relationships and sequence similarities. The species-specific primers as the sequence-characterized amplified regions (SCAR) markers were used to validate the identification. 【Result】The fragments amplified from rDNA 18S region of 50 individual nematodes were sequenced and the sizes were 857-935 bp. The BLAST searching revealed that the mixed Pratylenchus populations might present in some samples. The sequence sizes of the rDNA 28S D2-D3 fragments amplified from 20 selected specimens were 771-784 bp, the sizes of mtDNA-COI were 415-417 bp. The phylogenetic analyses as well as the comparisons of sequence similarities both demonstrated that three Pratylenchus species including P. coffeae, P. neglectus and P. scribneri,were found in 10 samples collected from wheat fields in 4 provinces of Huanghuai region. Among 10 samples, the sample JS2 from Peixian of Jiangsu Province and SD1 from Weifang of Shandong Province were infected only with P. neglectus, HN2 from Yongcheng of Henan Province and AH3 from Huaibei of Anhui Province were infected only with P. coffeae. The samples AH2 and AH5 from Xiaoxian and AH4 from Huaibei of Anhui Province as well as HN1 and HN3 from Yongcheng of Henan Province were all co-infected with P. neglectus and P. coffeae, and JS1 from Xuzhou of Jiangsu Province was co-infected with P. neglectus and P. scribneri. The DNA templates from 20 representative specimens were amplified using SCAR primers. The results showed that a single band of 140 bp was amplified from JS1-2, JS2-1, JS2-2, AH2-2, AH4-1, AH5-1, HN1-2, HN3-2, SD1-1 and SD1-2 using specific primer PNEG-F1/D3B5 for P. neglectus, a single band of 630 bp was amplified from AH2-1, AH3-1, AH3-2, AH4-2, AH5-2, HN1-1, HN2-1, HN2-2 and HN3-1 using primer PC1/PC2 for P. coffeae,a single band of 130 bp was amplified from JS1-1 using primer PsF7/PsR7 for P. scribneri. The results of SCAR detection confirmed the species identification mentioned above.【Conclusion】The molecular identification demonstrated that three species, P. coffeae, P. neglectus and P. scribneri, were found in wheat fields infested with H. avenae from four provinces in Huanghuai region, and P. neglectus is the dominant species. The co-infection of different Pratylenchus species occurred quiet common in wheat field from the region. The phylogenetic tree based on mtDNA-COI gene can effectively distinguish the close related Pratylenchus species, therefore, it is more suitable to identify Pratylenchus species than rDNA 18S and 28S markers.

Heterodera sojae, a New Cyst Nematode Record in China and Its Parasitism to Legume Crops

ZHEN HaoYang, PENG Huan, KONG LingAn, HONG BaoYuan, ZHU GuiLan, WANG RuiHui, PENG DeLiang, WEN YanHua

Scientia Agricultura Sinica. 2018, 51(15): 2913-2924. doi:10.3864/j.issn.0578-1752.2018.15.007

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【Objective】In a survey for cyst nematodes in Jiangxi Province, China, a cyst nematode population was collected and extracted from soybean roots and rhizosphere soil. The morphological characteristics of this population are different from that of the soybean cyst nematode (Heterodera glycines). The objective of this study is to research morphological and molecular characteristics and parasitism test of this population, identify their species and parasitism to legume crops, and to provide scientific basis for disease prevention and control.【Method】Soil samples were collected in Wuyuan County, Shangrao City, Jiangxi Province. The cysts were collected by sieving method or dissected from the roots. The 2nd stage juveniles (J2s) and males in soil were extracted by using the shallow disk method. The cysts with typical characteristics were selected to make a slice of the vulva cone, the J2s and males separated by shallow disk method were made into permanent slides. Morphological characteristics of cysts, J2s and males were observed and measured under microscope. For molecular studies, single cyst from soybean roots was picked and then ground to extract the genomic DNA. The ribosomal RNA-ITS region and the D2-D3 expansion segments of 28S large subunit rRNA gene were amplified with universal primers AB28, TW81 and D2A, D3B, respectively. The sequencing results were deposited in the GenBank database and compared with nematode sequences using the BLAST homology search program. The closest sequences were selected for phylogenetic analyses. The MEGA software was used to construct the ITS and LUS D2-D3 phylogenetic tree of cyst nematodes using neighbor-joining method. The parasitism of this cyst nematode to 11 legume crops and 40 soybean cultivars was tested by artificial inoculation in greenhouse. 【Result】 Morphological characteristics of cysts, J2s and males of population in this study agreed with Heterodera sojae reported in Korea. Sequence from the ITS-rDNA (MG859982) was 99% and 98% identical to those of H. sojae from Korea (KU160510 and KU160512) and only 81% identical to the soybean cyst nematode (H. glycines) (KY794762.1). The D2-D3 (MG859981) sequence was 99% identical to H. sojae from Korea (KU160511). In addition, analysis of phylogenetic trees constructed by the ITS and LUS D2-D3 both showed that the Jiangxi population and H. sojae were grouped in the same clade with a reliability of 100%. The cyst nematode population from Jiangxi was identified as H. sojae - a new record species in China. Inoculation of 11 legume crops showed that J2s of H. sojae could infect 10 crops (soybean, jequirity, cowpea, pea, lentil, mung bean, adzuki bean, sword bean, green beans and alfalfa), but could complete the life cycle only on soybean and jequirity. Soybean cultivars resistant identification test results showed that 19 cultivars of the 40 inoculated soybean cultivars were highly susceptible, 11 cultivars were moderately susceptible, 5 cultivars were moderate resistant and 5 cultivars were highly resistant.【Conclusion】The population of cyst nematode collected on soybean from Jiangxi, China was identified as H. sojae - a new cyst nematode record in China. Parasitism tests indicated that soybean and jequirity were suitable hosts for H. sojae, and 30 of the 40 soybean cultivars were susceptible to phenotypic disease of cyst nematodes.

Transcriptome Analysis of Disruption of Mating in the Peach Fruit Moth (Carposina sasakii) by Chlorantraniliprole

SUN LiNa, TIAN ZhiQiang, ZHANG HuaiJiang, LI YanYan, YAN WenTao, YUE Qiang, QIU GuiSheng

Scientia Agricultura Sinica. 2018, 51(15): 2925-2936. doi:10.3864/j.issn.0578-1752.2018.15.008

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【Objective】 The objective of this study is to identify the difference of transcriptome of peach fruit moth (Carposina sasakii) adults under the stress of chlorantraniliprole, and describe biological characteristics involved in functional classifications and metabolic pathways, which is necessary to understand functional genes for mating. 【Method】Biological experiments were carried out to observe the effects of chlorantraniliprole on mating and reproduction of C. sasakii. The high throughput sequencing platform Illumina HiSeqTM2500 was used to sequence the transcriptome of C. sasakii adults (including virgin female and male, female and male with courtship behavior in the mating season after emergence for 4-6 h, female and male exposed to chlorantraniliprole for 4-6 h). De novo assembling and assessment were carried out by Trinity software, and then the effective sequential data were assigned to the relevant databases to perform functional annotation analysis. The temporal and spatial expression level of the related genes of C. sasakii was analyzed by qRT-PCR after chlorantraniliprole treatment.【Result】After chlorantraniliprole treatment, the mating rate, adult longevity and fertility of C. sasakii decreased. 102 831 unigenes were assembled from the transcriptomes, and 34 526 unigenes were annotated. According to the screening criteria, in the process of chlorantraniliprole treatment, 122 differentially expressed genes (DEGs) and 147 DEGs were identified in female and male adults, respectively, of which 31 DEGs were co-existed. The results of gene ontology (GO) functional annotation and enrichment analysis of 234 DEGs showed that these genes were annotated into 5 dominated process, including catalytic activity and binding activity in the molecular functional process, metabolic process, single-organism process and cellular process in the biological process. By KEGG pathways identification, 234 DEGs were enriched in 25 pathways for metabolic, which included insect hormone biosynthesis, drug metabolism, etc. The c64662.graph_c0 was identified as ryanodine receptor from C. sasakii by BLAST, its length is 15 637 bp and it has 99.0% identity with the reported CsRyR. Furthermore, 3 carboxylesterase unigenes, 4 cytochrome P450 unigenes, 3 troponin unigenes, 1 odorant binding protein unigene and 1 timeless unigene were annotated. The P450 c40709.graph_c0 is involved in insect hormone biosynthesis. Gene expression levels of 12 DEGs based on the FPKM value showed different trends. Finally, qRT-PCR was used to identify the 12 DEGs and RyR. The expression of carboxylesterase c51998.graph_c0 gene from C. sasakii adult was up-regulated after treated with chlorantraniliprole. The expression of cytochrome P450 c57480.graph_c0 and c53794.graph_c0 from C. sasakii female and male adults was significantly up-regulated and down-regulated, respectively, while the expression of cytochrome P450 c40709. graph_c0 gene was significantly down-regulated only in males, the expression of cytochrome P450 c53281.graph_c0 gene was up-regulated only in females after 6 h treatment. The expression of three troponin genes was significantly down-regulated during the whole trial period. The circadian clock unigene c60883.graph_c0 and odor binding protein unigene c45675.graph_c0 showed the same variation trend, which were up-regulated immediately after dark period, but were inhibited by chlorantraniliprole. The expression of RyR was significantly up-regulated in females, but there was no significant difference between male and control before the first courtship peak, and the expression of RyR was down-regulated significantly at the second courtship peak. The expression of other unigenes in the males was higher than that of the females except cytochrome P450s c57480.graph_c0, c40709.graph_c0 and c53281.graph_c0. Moreover, the expression of antennal esterase gene c54944.graph_c0, clock gene c60883.graph_c0 and odor binding protein gene c45675.graph_c0 was significantly up- or down- regulated when the dark/light changed alternately.【Conclusion】By transcriptome sequencing, it was found that the mechanism of disruption of mating in C. sasakii by chlorantraniliprole was possibly coursed by the interaction of target gene, olfactory related gene, metabolic gene, circadian clock gene and so on.

Recommended Methods for Optimal Nitrogen Application Rate

ZHANG YiTao, WANG HongYuan, LEI QiuLiang, ZHANG JiZong, ZHAI LiMei, REN TianZhi, LIU HongBin

Scientia Agricultura Sinica. 2018, 51(15): 2937-2947. doi:10.3864/j.issn.0578-1752.2018.15.009

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In order to obtain high crop yield and low environmental nitrogen (N) pollution risk simultaneously, identifying the optimal N application rate is one of the most effective methods. Based on the theory of optimal N application and the present situation of N fertilizer application in China, we summarized the recommended methods for optimal N rate used in current research. The existing recommended methods for optimal N rate were soil nutrient regulation during crop growth, N application effect curve, balance of N input and output, and the critical N rate based on standard nitrate-N of leaching water from farmland. The first three methods, which focused on agronomy effect firstly and then assessed its environmental effect, were for the purpose of obtaining better agronomic benefits. All of these three methods were scientific and reasonable, which had proved their application in practice. The forth method focused on environmental effect firstly and then estimates its effect on yield intending to prevent nitrate pollution of groundwater, which could quantify the actual environmental effect of optimal N application rate. However, the critical N application rate of the forth method has some uncertainty because of many influencing factors, and its variation under different years, different regions and different soil types need be further studied.

Yield and Nutrient Efficiency Differences of Winter Oilseed Rape Between Farmer’s Practice and Recommended Fertilization in Yangtze River Basin: Based on Large-Number of Field Experiments

ZHU Yun, XU HuaLi, ZHANG YangYang, REN Tao, CONG RiHuan, LU JianWei

Scientia Agricultura Sinica. 2018, 51(15): 2948-2957. doi:10.3864/j.issn.0578-1752.2018.15.010

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【Objective】Reasonable fertilization is critical for improving winter oilseed rape (Brassica napus L.) yield. Rough management of fertilization would cause the nutrient efficiency decline under current intensive planting management mode, thus affecting rapeseed yield. To provide an optimizing management strategy for the rational fertilizer utilization to enhance nutrient use efficiency, yield and nutrient efficiency differences were compared between farmer’s practice (FP) and recommended fertilization (RF) for winter oilseed rapes in the Yangtze River Basin (YRB) in this study. 【Method】Total of 535 field experiments were selected in the YRB during 2005-2016, including Sichuan, Guizhou, Hubei, Hunan, Anhui, Jiangsu and Zhejiang provinces, China. Three fertilization treatments, i.e., no-fertilizer (CK), farmer’s practice (FP) and recommended fertilization (RF), were designed. The differences in yield and its distribution characteristics, as well as differences of yield increase rate, fertilization rates, and partial factor productivity, were compared among the treatments and regions. The differences of fertilizer amounts between RF and FP treatments were calculated, and the reduction space of nitrogen, phosphorus and potassium fertilizer in Yangtze River Basin was evaluated.【Result】The range of winter oilseed rape yield for the CK, FP and RF treatments were 500-1 500 kg·hm^-2, 1 500-3 000 kg·hm^-2 and 2 000-3 000 kg·hm^-2, respectively. For the RF treatment, the contribution rate of the soil indigenous fertility supply was only 45.1%-49.7%. Rapeseed yield showed highest in the lower reach, then the middle reach, and lowest in the upper reach. The average rapeseed yield of FP management in the upper, middle and lower reaches of the Yangtze were 2 033 kg·hm^-2, 2 182 kg·hm^-2and 2 542 kg·hm^-2, respectively, and the RF management produced more rapeseed (16.7%, 16.5% and 13.9% averagely) than the FP in 77.5%-94.9% of the 535 trails. With the increasing level of soil indigenous fertility supply, yield increase rate showed a decline trend, and RF treatment showed a higher increase rate than FP at different levels. Averaged fertilizer rates of FP were 162.5-239.5 kg N·hm^-2, 58.6-82.0 kg P₂O₅·hm^-2and 45.5-60.8 kg K₂O·hm^-2, and the values were 162.2-233.6 kg N·hm^-2, 67.2-94.1 kg P₂O₅·hm^-2and 73.6-108.5 kg K₂O·hm^-2 of RF treatment. There was no significant difference in the amount of N fertilizer used under the two treatments, and the amounts of P and K fertilizer were lower under the FP treatment than the RF treatment. Compared with RF, 45.6% and 25.6% of the 535 experiments could reduce N and P fertilizer rates, respectively. However, only 13.2% of the experiments could lower K fertilizer rates. Meanwhile, 37.8%, 60.0% and 75.9% of the trails needed more N, P and K fertilizers input in the YRB, respectively. Fertilization rates were more appropriate in the lower reaches as compared with the other two regions, in which optimum N, P, and K rates were accounted 25.0%, 22.8%, and 17.1% of the 535 trails, respectively. The average partial factor productivity of nitrogen (PFP_N), phosphorus (PFP_P) and potassium (PFP_K) for FP was 11.1-14.2 kg·kg^-1, 28.6-45.8 kg·kg^-1and 38.3-47.6 kg·kg^-1, respectively. Although PFP_N in the RF treatment were 12.9%-15.9% higher than that in the FP treatment across the regions, the values were still lower as compared with other developed countries. Meanwhile, the PFP_P and PFP_Kof RF were respectively 6.9% and 19.6% lower than the FP treatments, indicating that current recommended fertilization rate also had a potential for fertilizer reducing. 【Conclusion】Recommended fertilizations management could significantly improve rapeseed yield as compared with farmer’s practice, and the fertilizer rates of FP had a larger adjustment space.

Functional Analysis of SlSWEET7a Gene During Maturation of Tomato Fruits

CHENG Jie, ZHANG XinSheng, LI AnQi, JIANG Jing

Scientia Agricultura Sinica. 2018, 51(15): 2958-2968. doi:10.3864/j.issn.0578-1752.2018.15.011

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【Objective】 The SWEETs (Sugars Will Eventually Be Exported Transporters) is a kind of sugar transporters which are involved in plant biological processes and play key roles in plant growth and development in response to various stresses and host-pathogen interaction. SWEET7a was cloned and its function was analyzed to provide the theoretical foundation for exploring the function of SWEETs during fruit development in plant by constructing the silence and overexpression of SlSWEET7a.【Method】Using Micro-Tom (Solanum lycopersicum) as a test material, the 842 bp full-length SlSWEET7a cDNA was cloned in fruits. The phylogenetic tree of SWEET proteins from Arabidopsis was constructed by using MEGA6.0, and homology of SlSWEET7a protein sequences was analysed compared with Arabidopsis. RT-PCR was performed to analyse the spatiotemporal expression of SlSweet7a during the fruits development periods. The SlSWEET7a silence vector and overexpression vector were constructed and transformed into tomato by Agrobacterium-mediated genetic transformation. The efficiency of two vectors was examined by the transient expression of Agrobacterium tumefaciens injection method. The expressions of SWEET7a in T1 generation transgenic green fruits were studied by quantitative PCR. The changes of sugar composition and content in transgenic fruits and leaves were detected by high performance liquid chromatography.【Result】The bioinformatics analysis of protein sequence showed that the SlSWEET7a is consisted of seven transmembrane domains. SlSWEET7a belongs to the CladeⅡof the SWEETs gene family, which was highly homologous to AtSWEET6 and AtSWEET8 in Arabidopsis thaliana. The analysis of spatiotemporal expression indicated that SlSWEET7a expression level was the highest at stalks and vascular bundles of mature green stage in tomato fruits, while the relative expression level was lower in breaker fruits and red ripping stage. The transient expression of SWEET7a silencing (S7a) and overexpression (OE7a) vector in tomato fruits was observed. The expression level of SlSWEET7a in fruit of OE7a was 6 times higher than that of non-injected fruit, which was significantly up-regulated compared with control. However, the expression level of SlSWEET7a in S7a was significantly decreased 5 times. Ten overexpression lines were obtained by kanamycin resistance screening, and eight overexpression lines were obtained by PCR. Fourteen silencing lines were screened by phosphinothricin and 10 transgenic silence lines were obtained by PCR. Real-time quantitative PCR analysis of the SWEET7a expression in T1 generation lines revealed that gene silencing occurred in overexpressed plants. The expression level of SlSWEET7a-overexpressing transgenic plants was significantly lower than that in wild plants, so did the silencing plants. Those results explained that the overexpressing plants also had the phenomenon of gene silencing. The determination of contents of fructose, glucose, sucrose showed that the silencing and overexpressing plants were higher than that of the wild type in the leaves and fruits after reducing expression of SlSWEET7a in tomato. Especially, the sucrose content of leaves was significantly increased. This showed that SlSWEET7a plays an important role in the facilitated diffusion of sucrose in cells.【Conclusion】SlSWEET7a plays an important role in regulating the loading of sucrose into the phloem of fruit tissue, the transportation and unloading of fruit stalks and vascular bundles.

SSR Markers Screening for Identification of Citrus Cultivar and Construction of DNA Fingerprinting Library

LI Yi, MA XianFeng, TANG Hao, LI Na, JIANG Dong, LONG GuiYou, LI DaZhi, NIU Ying, HAN RuiXi, DENG ZiNiu

Scientia Agricultura Sinica. 2018, 51(15): 2969-2979. doi:10.3864/j.issn.0578-1752.2018.15.012

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【Objective】The purpose of the present work aims at the construction of DNA fingerprint library of citrus cultivar by using a series of SSR core primers in order to provide references for citrus nursery plant identification, cultivars registration and plant variety protection. 【Method】SSR primers were primarily screened by analyzing 8 mainly cultivated species belonging to Citrus while the PCR products were separated by agarose gel electrophoresis, the primers that generated high polymorphic and stable PCR products were chosen for further PCR amplification with more citrus cultivars, and the PCR products were separated by polyacrylamide gel electrophoresis. the further screened primers were labeled with fluorescent and used for amplification of the all tested cultivars. The PCR products were detected by fluorescence capillary electrophoresis and the primers suitable for fluorescent capillary electrophoresis were screened according to their PIC, number of alleles identified, and employed to construct the citrus cultivar DNA fingerprinting library, and the selected primers were used to identify the tested cultivars.【Result】Firstly, 80 pairs of polymorphic and stable primers were chosen from 362 SSR primers by 4% agarose gel electrophoresis with representative cultivars of 8 species of Citrus genus(including Ota ponkan, Shatian pummelo, Yuanjiang soar orange, Dahong sweet orange, Duncan grapefruit, citron, Femminello lemon, Mexico lime); Secondly, 60 pairs of SSR primers with high polymorphism, stability and clear amplification products were further selected from the above 80 pairs of primers by 6% polyacrylamide gel electrophoresis with 64 citrus cultivars and labeled with fluorescent dyes; Thirdly, 21 pairs of SSR core primers were verified for the construction of fingerprint library from above 60 pairs of primers by capillary electrophoresis with 168 citrus cultivars on the basis of PIC, the allele number, peak reading difficulty, amplification stability and chromosome location. The corresponding reference cultivars were selected for 21 pairs of primers to eliminate the errors caused by different instruments and experimental batches. Through the multiple electrophoresis with the 5 combination groups of 21 primers according to the color and size of amplified fragment, we established 500 citrus cultivars fingerprinting library. Among the tested cultivars, 111 were identified by using only one pair of primers, 86 with the combination of two pairs of primers; and 24 with the combination of 3 pairs of primers, the remaining 279 cultivars were not able to be identified individually, they could, however, be divided into 87 groups, between which it was clearly distinguished but within which the cultivars were indistinguishable.【Conclusion】The DNA fingerprint library containing 500 citrus cultivars was constructed by using 21 pairs of screened SSR core primers, the specific markers for some cultivars were selected; 221 cultivars and 87 cultivar groups were identified; and a citrus cultivar identification system based on SSR fluorescence labeling was constructed.

Effect of Postharvest O₃Treatment on Ethylene Metabolism of Kiwifruit Preharvest Treated with CPPU

SU Miao, LUO AnWei, LI Lin, LI YuanYuan, BAI JunQing, LI Rui, FANG YiMeng, SONG JunQi, LIN ZhiYing, LIU ZhanDe

Scientia Agricultura Sinica. 2018, 51(15): 2980-2989. doi:10.3864/j.issn.0578-1752.2018.15.013

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【Objective】 The objective of this study was to investigate whether or not the ozone (ozone, O₃) could effectively reduce the negative effects of the swelling agent (N-2-chloro-4-pyridine benzene-N'- phenyl urea, CPPU) on kiwifruit, which would provide useful information for using of CPPU on the market for the storage.【Method】First, the Qinmei kiwifruit was treated with 20 mg·L^-1 CPPU in the growing season, then treated with 10, 40, 70 mg·m^-3 ozone respectively in postharvest. The material contents in kiwifruit of methionine (methionine, Met), S-adenosyl methionine (S-adenosyl methionine, SAM), 1-aminocyclopropane-1- carboxylic acid (1-aminocyclopropane-1-carboxylic acid, ACC) and the related metabolic enzyme activity of ACC synthase (ACC synthase, ACS) and ACC oxidase (ACC oxidase, ACO) were studied. 【Result】The decreasing rate of Met, SAM and activity of SAM synthase, ACS, ACO under CK (without the use of CPPU and O₃ treatment as CPPU control CK group) during storage was lower than that under CK1 (using CPPU without using O₃ treatment for ozone treatment control CK1 group). At 60 days after kiwifruit storage, the contents of Met under CK1 and each treatment group (10, 40 and 70 mg·m^-3ozone) were 1.36, 2.62, 4.41 and 2.60 mg·(100 g)^-1, respectively. The contents under O₃ treatment was significantly higher than that under CK1 (P＜0.05). The contents of SAM under CK1 and 40 mg·m^-3O₃ group were 15.48 mg·(100 g)^-1 and 20.73 mg·(100 g)^-1, respectively (P＜0.05), but the contents under CK1 10 mg·m^-3 O₃ treatment, and 70 mg·m^-3 O₃ treatment no significant difference (P＞0.05). The ACC contents under CK1 and each treatment group (10, 40 and 70 mg·m^-3ozone) were 0.068, 0.059, 0.038 and 0.055 nmol·g^-1, respectively. There were significant differences (P＜0.05) between 40, 70 mg·m^-3 O₃ treatments and CK1. The ACS activities in CK1 group and each treatment group (10, 40and 70 mg·m^-3) were 0.084, 0.069, 0.054 and 0.080 nmol·(g·h)^-1, respectively. The peak of ACO activity was 0.062, 0.046, 0.029 and 0.051 nmol·(g·h)^-1, respectively. There was significant difference between the treatment and the CK1 (P＜0.05). The peak values of ethylene in CK1 and each treatment (10, 40, and 70 mg·m^-3ozone) were 18.42, 15.99, 9.86 and 11.69 μL·kg^-1·h^-1, respectively, and the peak of respiration was 18.77, 16.15, 12.24 and 15.48 mg·kg^-1·h^-1, respectively.【Conclusion】CPPU increased the kiwifruit ethylene production and accelerated fruit softening, had a negative impact on kiwifruit storage, while O₃ could effectively inhibit the ethylene metabolism of kiwifruit. O₃ treatment could effectively slow down the kiwifruit ripening due to the use of CPPU.

Screening of Y Chromosome Specific Primers and Y-SNPs in Sheep

CAO XueTao, PEI ShengWei, ZHANG Jin, LI FaDi, LI Gang, LI WanHong, YUE XiangPeng

Scientia Agricultura Sinica. 2018, 51(15): 2990-2999. doi:10.3864/j.issn.0578-1752.2018.15.014

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【Objective】The male specific region of the mammalian Y chromosome (MSY)does not recombine with X chromosome during meiosis process, which is an important genetic resource for analyzing paternal genetic diversity due to its strict father to son inherited character. In addition, most of genes on MSY exclusively or predominantly express in the testis, indicating they may play essential roles in spermatogenesis and male reproduction. Since it is extremely difficult to sequence of entire Y chromosome, many species have very few Y chromosome sequences. Therefore, the current study was conducted to select ovine Y chromosome specific primers and Y-SNP based on the previous primer information used in bovidae species, and to compare the Y-fragment sequences similarity among sheep, bovine, goat, yak and bharal. Meanwhile, the Y-SNP was associated with sheep scrotal circumference to supply scientific basis for constructing ovine Y-haplotypes, identifying molecular markers for embryo sex and male reproductive traits in the future.【Method】Based on the investigation of available references about bovidae Y chromosome, 29 pairs of Y-primers reported in cattle, yak, goat were selected to amplify rams DNA using ewes DNA and ddH₂O as negative controls. Subsequently, the Y-SNPs within ovine Y-specific fragment identified of different sheep breeds were investigated by DNA sequencing of DNA pooling and PCR-RFLP methods, including Suffolk sheep (n=146),White Suffolk sheep (n=91), East Friesian sheep (n=6), Texel sheep (n=72), South African Mutton Merino (n=17), Dorper sheep (n=32), Hu sheep (n=55), Tibetan sheep (n=34), Tan sheep (n=43), and bharal (n=14). Chromas and DNASTAR were used to analyze the results of DNA-pool sequencing, and DNAman was used for homology analysis of yak, goat, cattle and bharal. Meanwhile, the correlation analysis between the SRY11 gene fragment polymorphisms and the testis size was performed by SPSS 19.0.【Result】The results showed that 6 out of 29 pairs of primers analyzed were ovine Y-specific, which could amplify ZFY3, SRY6, USP9Y, UTY, SRY11 and ZFY6 fragments, respectively. However, 17 pairs of primers failed to show amplification bands, and 6 pairs of primers showed amplified bands in the DNA of the ewes. The similarity of them among sheep, bharal, cattle, goat and yak ranged from 81.51% to 98.84%. In addition, a Y-SNP (G＞A) with in SRY11 fragment was first identified in the Suffolk and white Suffolk sheep. According to RCR-RFLP analysis, two genotypes (AA, GG) were detected in the Suffolk sheep and white Suffolk sheep, while only the GG genotype was found in the other seven sheep breeds. The genotypic frequencies of the GG and AA were 0.747 and 0.253 in White Suffolk sheep, respectively, while they were 0.986 and 0.014 in Suffolk sheep, indicating the dominant genotype was GG genotype in White Suffolk sheep and Suffolk sheep. Association analysis suggested that the testis size of the GG genotype was significantly higher than those of the AA genotype in the white Suffolk sheep population (P=0.029).【Conclusion】In this study, six pairs of ovine Y-specific primers were identified, and the Y-linked fragment identified in ovine showed a high similarity with cattle, goats and yaks, indicating certain conservation in the evolutionary process. In addition, a Y-SNP was found to be specific in White Suffolk sheep and Suffolk sheep, which was closely associated with the testis size of white Suffolk sheep.

Follicular Development Related Genes Screening and Differential Expressed Analysis by Transcriptome Sequencing in Bovine Ovary

LI PengFei, MENG JinZhu, JING JiongJie, BI XiLin, WANG Kai, ZHU ZhiWei, Lü LiHua

Scientia Agricultura Sinica. 2018, 51(15): 3000-3008. doi:10.3864/j.issn.0578-1752.2018.15.015

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【Objective】 The study focused on screening some genes involved in follicular development through high- throughput sequencing in bovine follicles at different physiological states. 【Method】Cows were selected for estrus synchronization, and the largest follicle and second largest follicle at predeviation during the first follicle wave were gathered by B-type ultrasonography. Thereafter, the RNA libraries were constructed and RNA sequencing was performed by Illumina platform. Compared with the database, parameters were set to screen high-expressed genes and differentially expressed genes, and then GO analysis was conducted. Further screened regulatory genes directly related to follicular development by Genecards, and qRT-PCR was performed to validate expression of screened genes associated with follicular development. 【Result】 Results showed that 263 differentially expressed genes were obtained from the two transcripts, which could be assigned into 90 groups under three categories by GO clustering analysis (biological processes, 64.4%; cell component, 17.8%; molecular function, 17.8%). Some important functional enrichment pathways were obtained, such as regulating signal transduction and cytokine response biological pathways; 10 high-expressed up-regulated and down-regulated genes were selected by gene expression analysis, CYP17A1 involved in estrogen synthesis and fetal gender development, LOC785462 participated in steroid hormone synthesis, DACH1 regulated cell apoptosis in process of cell development, and MAP3K3 regulated ERK and MEK1/2 signaling pathways. Six genes were found closely associate with follicular development by Genecards: up-regulated genes included PRSS35, PTGRF, ARID4B and GPR116, down-regulated genes included APOA1 and CPXM1. qRT-PCR results showed that expression of PRSS35 and ARID4B were significantly higher in DF than in SF (P＜0.05), and CPXM1 expression was significantly higher in SF than in DF (P＜0.01). There was no significant difference in the expression of PTGRF, GPR116 and APOA1 between DF and SF (P＞0.05), however, the expression variation trend was consistent with high-throughput sequencing results. 【Conclusion】The transcriptome sequencing results were accurate, PRSS35 and ARID4B played positive roles and CPXM1 played a negative role in regulating follicular development, genes related to bovine follicular development and important regulated pathways, which were of great significance to further study the regulation mechanism of follicular development.

Genome-Wide Identification and Expression Analysis of SRO Genes Family in Maize

ZHAO QiuFang, MA HaiYang, JIA LiQiang, CHEN Shu, CHEN HongLiang

Scientia Agricultura Sinica. 2018, 51(15): 3009-3019. doi:10.3864/j.issn.0578-1752.2018.15.016

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【Objective】The SRO (similar to rcd one) is a group of plant-specific proteins which have important functions in plant growth and development, and response to abiotic stress. Based on maize genome information, the SRO family genes were identified, and the sequences, gene localization, protein structure and their evolutionary relationships were analyzed. Meanwhile, the SRO family genes tissue-specific expression profiles and expression pattern under salt and drought stresses of ZmSROs were analyzed. This research can lay a foundation for researching the function of SRO family genes in the growth and response mechanisms of stress in maize. 【Method】The Arabidopsis SRO protein sequences were used as queries to identify SRO in maize genome and download all the SRO gene sequences, which information regarding the maize SRO genes, including CDS sequences , amino acid sequences and chromosome location were obtained from MaizeGDB. The gene structure, protein molecular weight, isoelectric point, secondary structure, subcellular localization, conserved domain, and conserved element were also prospected and analyzed by GSDS2.0, Expasy-protparam, SOPMA, Plant-mPLoc, EMBL-EBI, MEME bioinformatics tools. Meanwhile the homologous sequence alignment and phylogeny were analyzed by Clustalx (1.83) and MEGA6.0 software. The expression levels of ZmSROs genes in different maize tissues and under high salt and drought stresses were analyzed by real-time fluorescence quantitative PCR. 【Result】Six SRO family genes were identified from Maize genome, and designated ZmSRO1a to ZmSRO1f. ZmSROs were mapped on 1, 4, 5 and 9 chromosome, and contained two to five introns. The CDS of 6 ZmSROs varied in length from 1,215 to 1,791 bp, and their predicted protein products ranged from 404 to 596 aa, with corresponding molecular masses of 45.23 kD and 66.78 kD, and with predicted isoelectric points in the range of 7.01 to 9.17. Subcellular localization analysis showed that ZmSRO1a/ZmSRO1b/ZmSRO1c/ZmSRO1d were located in the chloroplast, ZmSRO1e was located in the peroxisome, and ZmSRO1f was located in the nucleus. Phylogentic analysis revealed that ZmSROs can be divided into three subgroups. Subgroup Ⅰ a contains ZmSRO1a/b/c, subgroups Ⅰ b contains ZmSRO1f, and subgroup Ⅰ c contains ZmSRO1d and ZmSRO1e. Conservative structure domain analysis showed that ZmSRO1a/ ZmSRO1b/ ZmSRO1c/ ZmSRO1d/ ZmSRO1e contain the PARP and RST domain, lack the WWE domain. ZmSRO1f contains the PARP and WWE domain, lacks the RST domains. A total 5 motif were identified from ZmSROs proteins，named motif 1—motif 5. ZmSRO1a/ZmSRO1b/ZmSRO1c contain all of motif, ZmSRO1d/ ZmSRO1e lack the motif 3，ZmSRO1f lacks the motif 5. Different tissues expression assay showed that all ZmSROs had specific expressions in root. The expression of ZmSRO1a/ZmSRO1b/ZmSRO1c/ZmSRO1d/ZmSRO1e in root were significantly up-regulated under high salinity treatment for 1 h. ZmSRO1a/ZmSRO1b/ZmSRO1c/ZmSRO1d/ZmSRO1e were down-regulated in shoots, However, the expression of ZmSRO1f in shoot was significantly up-regulated under high salinity treatment for 6 h. ZmSRO1e in root was up-regulated under drought stress for 1 h and that of ZmSRO1f was up-regulated for 24 h. Meanwhile, the expressions of ZmSRO1a/ ZmSRO1b/ZmSRO1d/ZmSRO1e in shoot were down-regulated under drought stress and that of ZmSRO1c and ZmSRO1f had no significant changes in shoot. 【Conclusion】Maize SRO family contains six genes, and ZmSROs genes could be classified into threes subgroups. Six of ZmSROs genes had specific expressions in root and could respond to high salt and drought stresses in varying degrees.

Molecular Detection of R. turanicus and Its Eggs Carrying R. raoultii in Southern Xinjiang

LIU YongHong, LI BeiBei, LI KaiRui, HE Bo, ZHANG JingBo, PU XiaoFeng, CHEN MeiJuan, PAN JiaoJiao1, LI Fei, ZHANG LuYao, ZHAO Li

Scientia Agricultura Sinica. 2018, 51(15): 3020-3028. doi:10.3864/j.issn.0578-1752.2018.15.017

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【Objective】 There are 899 species of ticks in the world, belonging to 18 genera and 3 families, and there are 117 species in China, belonging to 2 families, 10 genera. There are at least 45 species in Xinjiang, accounting for more than 1/3 of the national ticks, and its distribution is extremely wide. Ticks can directly harm and spread many kinds of pathogens, and some pathogens can propagate vertically through eggs, causing huge economic losses to animal husbandry, and seriously threatening public health security. Rhipicephalus is one of the common species and dominant species in the desert and semi-desert region of southern Xinjiang. It is of great significance to confirm whether Rhipicephalus turanicus and its eggs are carrying the Rickettsia in the southern Xinjiang of China, so as to prevent and control of Rhipicephalus turanicus and its transmission of Rickettsia. 【Method】 The satiated blood female Rhipicephalus were collected from a sheep farm in alar, southern Xinjiang, and oviposited eggs at a certain humidity and temperature. A random sample of 5 independent eggs and the corresponding 5 female Rhipicephalus were as the research object. The genomic DNA were extracted from the female Rhipicephalus and eggs, respectively. In this study, ticks 12S rRNA gene and Rickettsia 16S rRNA gene were amplified by conventional PCR, and the amplification products were sequenced and underwent sequence analysis by using BLAST online platform and multiple molecular biology software. 【Result】 The 12S rRNA gene PCR amplification of 5 female ticks were all positive, and 4 segment of 12S rRNA gene sequence obtained by sequencing were identical. The similarity of Rhipicephalus 12s rRNA gene sequence and GenBank Rhipicephalus 12s rRNA gene sequences in the database were more than 99% using Blast analysis, and the top five gene sequences of the high similarity were Rhipicephalus, including Rhipicephalus from Xinjiang sheep. In this study, the Rhipicephalus 12S rRNA gene sequence was submitted to the GenBank database and obtained the login number of MG744514. The phylogenetic tree from GenBank database Rhipicephalus turanicus, Rhipicephalus sanguineus, Boophilus microplus, Dermacentor marginatus, Dermacentor nuttalli, Haemaphysalis longicornis, Hyalomma anatolicum, Hyalomma asiaticum, Hyalomma detritum, Ixodes persulcatus and peripheral-dust mites 12S rRNA gene sequence display, the Rhipicephalus 12S rRNA gene sequence obtained in this study was closest with Rhipicephalus sanguineus in the evolutionary relationship and clustered in the same small branch. Rhipicephalus was identified as Rhipicephalus turanicus. PCR amplification of all eggs Rickettsia 16S rRNA gene and 5 oviposit female Rhipicephalus, 1 Rhipicephalus and its eggs samples were positive, and the tick carrying rate was 20%. The sequencing results of 16S rRNA in female Rhipicephalus turanicus and its eggs were identical. The similarity was more than 99% of the Rickettsia raoultii 16S rRNA gene sequences in the GenBank database using Blast analysis. The top five genes of high similarity were 4 Rickettsia raoultii and 1 Rickettsia sp, including the rickettsia of the Hyalomma asiaticum and Dermacentor nuttalli from Xinjiang in 2011. In this study, the Rickettsia 16S rRNA gene sequence was submitted to the GenBank database and obtained the login number of MG744513. Evolutionary tree of 37 Rickettsia 16S rRNA gene sequences belonging to 24 species from the GenBank database showed, the Rickettsia 16S rRNA gene sequence obtained in this study was closest with Rickettsia raoultii in the evolutionary relationship and clustered in the same small branch, belonging to the speckle thermal gro up rickettsia with the other 15 kinds of rickettsia. Rhipicephalus turanicus and its eggs carried Rickettsia raoultii of spotted fever group Rickettsia. 【Conclusion】 R. raoultii were first found in Rhipicephalus turanicus and its eggs.

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