Scientia Agricultura Sinica

Gene Function and Breeding in Cotton

HAO MiaoMiao, XIAO GuangHui

Scientia Agricultura Sinica. 2023, 56(19): 3709-3711. doi:10.3864/j.issn.0578-1752.2023.19.001

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Cloning and Functional Characterization of the Promoter of GhSLD1 Gene That Predominantly Expressed in Cotton Fiber

LIU Fang, XU MengBei, WANG QiaoLing, MENG Qian, LI GuiMing, ZHANG HongJu, TIAN HuiDan, XU Fan, LUO Ming

Scientia Agricultura Sinica. 2023, 56(19): 3712-3722. doi:10.3864/j.issn.0578-1752.2023.19.002

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【Objective】Cotton fiber is the main economic product of cotton. It is the epidermal cells of the ovule outer integument through polar elongation and secondary wall thickening. As one of the longest plant cells, the cotton fiber cells are regarded as an ideal material in the study of plant cell growth and development. Identification of promoters specifically or preferentially expressed in fiber cells is of great significance for basic research on fiber development and molecular breeding for improving fiber traits. 【Method】In this study, we cloned the promoter of GhSLD1 gene, which is predominantly expressed in fiber cells. Through the PlantCARE website for promoter sequence analysis, we identified the important cis-regulatory elements contained in the cloned sequence. According to the distribution of some important cis-regulatory elements, the cloned promoter fragments were deleted at 5′- end. A total of 4 promoter fragments were obtained and the corresponding plant expression vector was constructed. The constructed plant expression vectors were used for genetic transformation of tobacco and cotton. The transgenic plants were identified through molecular identification of transgenic tobacco and cotton. GUS activity in different tissues, organs and fiber cells of transgenic plants at different development stages was also investigated. 【Result】The longest promoter cloned was 2 900 bp in length. In addition to a lot of transcription regulatory elements in the promoter, the sequence also contained multiple abscisic acid response elements, the elements essential for the anaerobic induction, methyl jasmonate response elements, brassinolide response elements, the elements involved in seed-specific regulation, the elements involved in defense and stress responsiveness, and MYB transcription factor binding sites. Four promoter fragments with a length of 2 900 bp (GhSLD-P1), 2 178 bp (GhSLD1-P2), 1 657 bp (GhSLD1-P3) and 1 232 bp (GhSLD-P4) were obtained by the 5′-terminal deletion, respectively. The transgenic tobacco plants were generated after confirmed by molecular identification. GhSLD-P1, GhSLD1-P2 and GhSLD1-P3 did not express in transgenic tobacco, while GhSLD-P4 is widely expressed, and the expression level of GhSLD-P4 was similar to that of CaMV 35S promoter. The different sequence between GhSLD1-P3 and GhSLD-P4 contained four abscisic acid response elements, two brassinolide response elements, and three MYB binding sites. These cis-regulatory elements may be associated with the non-expression of GhSLD1-P1, GhSLD1-P2, and GhSLD1-P3 promoters in transgenic tobacco. The transgenic cotton plants of GhSLD1-P2 were obtained after confirmed by molecular identification. GhSLD1-P2 predominantly expressed in transgenic cotton fibers, and its expression level was higher at the elongation stage (10-15 DPA) of fiber cells while lower in the early developmental stage (5 DPA) of fiber cells and the stage of secondary cell wall deposition (20-30 DPA). 【Conclusion】The GhSLD1-P4 promoter was a widely expressed promoter, and the GhSLD1-P2 promoter was a fiber predominant expression promoter, which was highly expressed during the elongation of fibers. It could be applied to the study on the gene function involved in cotton fiber development and molecular breeding for improving fiber traits.

Genome-Wide Identification and Expression Analysis of NLP (NIN- Like Protein) Transcription Factor Gene Family in Cotton

DING GuoHua, XIAO GuangHui, ZHU LiPing

Scientia Agricultura Sinica. 2023, 56(19): 3723-3746. doi:10.3864/j.issn.0578-1752.2023.19.003

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【Objective】To explore the structure and evolution characteristics of cotton NLP transcription factors in the whole genome, and further understand their expressions patterns, so as to lay a foundation for the further function research and utilization of NLP genes. 【Method】The NLP transcription factor family members in the whole genomes of four cotton species, Gossypium arboreum (G. arboreum, Ga), Gossypium raimondii (G. raimondii, Gr), Gossypium barbadense (G. barbadense, Gb) and Gossypium hirsutum (G. hirsutum, GH), were identified using two strategies, BLASTP and HMM search. Further bioinformatics analysis was carried out on the confirmed cotton NLP family members. The molecular weights, theoretical isoelectric points and other physical and chemical properties were predicted using online software Expasy; the MEGA 7 software was used to build the phylogenetic tree; protein conservative motifs were analyzed through MEME website; online software GSDS 2.0 was used to analyze gene structures; TBtools was used to view the chromosome localizations; McscanX was used to analyze the replication genes of cotton NLP family members; the PlantCARE website was used to predict the cis-acting elements in the promoters of cotton NLP family genes. The heat maps of cotton NLP genes expression levels of different tissues and under abiotic stresses were drawn through TBtools to analyze the tissue expression characteristics and abiotic stresses response characteristics. The expressions of GHNLPs in cotton under nitrogen starvation and nitrogen resupply treatments were analyzed by RT-qPCR. 【Result】A total of 11, 11, 21 and 22 NLP members were screened from the four cotton protein databases of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. These NLP family genes encoded 693-996 amino acids. The relative molecular masses ranged from 76.92-110.02 kDa and the theoretical isoelectric points were 5.13-7.77. The subcellular localization prediction results showed that almost all the NLP members located in the nucleus. Promoter analysis found a large number of cis-acting elements related to phytohormone and stress response. Phylogenetic analysis showed cotton NLPs were divided into three groups, I, II and III. Gene replication analysis showed that fragment replication was the main force for NLP members expansion in cotton. All the Ka/Ks values were less than 1, indicating that evolution of NLP family in cotton mainly underwent purification selection. The results of expression analysis also confirmed that GHNLPs responded to nitrogen starvation and nitrogen resupply. 【Conclusion】From the whole genome of G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, 11, 11, 21 and 22 NLP transcription factor members were identified respectively. They had high conservatism and some degree of differences. The expression levels of GHNLPs changed significantly during nitrogen starvation and nitrogen resupply processes, which may play a role in the response of cotton to nitrate.

Cloning and Functional Characterization of GhCPR5 in Disease Resistance of Gossypium hirsutum

XU FuChun, ZHAO JingRuo, ZHANG ZhenNan, HU GaiYuan, LONG Lu

Scientia Agricultura Sinica. 2023, 56(19): 3747-3758. doi:10.3864/j.issn.0578-1752.2023.19.004

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【Objective】This study on the function and mechanism of GhCPR5 in response to Verticillium dahliae (V. dahliae) and Botrytis cinerea (B. cinerea) in cotton analysed nucleotide sequence, protein structure, expression pattern, and biological function to provide a theoretical basis and genetic resources for cotton disease resistance and breeding mechanism research. 【Method】GhCPR5 was identified from the unpublished transcriptome data of cotton responses to V. dahliae infection. The full-length coding sequence of GhCPR5 was amplified from upland cotton TM-1. The conserved domain, protein structure, and phylogenetic relationship of GhCPR5 and homologous genes were analysed using bioinformatics techniques. Real-time fluorescence quantitative PCR (qPCR) was used to analyse the GhCPR5 expression patterns in cotton roots, stems, leaves, ovules, fibres, and petals, and the induced expression of GhCPR5 by V. dahliae infection. The silencing fragments of GhCPR5 were amplified and inserted into the VIGS vector to generate the gene silencing construct TRV:CPR5. GhCPR5-silencing plants were created via Agrobacterium-mediated transformation methods. RT-PCR and qPCR were used to analyse the silencing efficiency of GhCPR5 in TRV:CPR5 plants. TRV:00 and TRV:CPR5 plants were inoculated with V. dahliae and B. cinerea, respectively, to analyse the difference in resistance in TRV:00 and TRV:CPR5 plants in response to pathogens. To analyse defence signal pathways involving GhCPR5, the expression levels of defence-related genes in TRV:00 and TRV:CPR5 plants were detected by qPCR. 【Result】The GhCPR5 cloned from G. hirsutum TM-1 is 1 683 bp long and encodes a 560 amino acid protein. The relative molecular weight and isoelectric point of GhCPR5 are 62.883 kDa and 9.01, respectively. Multiple-sequence alignment and phylogenetic analysis showed that GhCPR5 is highly homologous with the CPR5 of Durio zibethinus and Theobroma cacao. Furthermore, the C-terminals of GhCPR5 and CPR5 of other species are highly conserved and contain 4-5 transmembrane domains. The GhCPR5 expression level was induced by V. dahliae infection and is highest in leaves and lowest in stems. Under normal conditions, no significant developmental differences were observed between the GhCPR5-silencing plants, TRV:CPR5, and the TRV:00 control plants. After inoculation with V. dahliae, the rate of disease and the disease index of TRV:CPR5 plants were significantly higher than those of the control plants. Analysis of detached leaves inoculated with V. dahliae and B. cinerea and lactophenol-trypan blue staining showed that the lesions on the leaves of TRV:CPR5 plants were much bigger than those of TRV:00 plants, indicating that silencing GhCPR5 made cotton less resistant to V. dahliae and B. cinerea. In addition, the JAZ1 expression levels in TRV:CPR5 plants were significantly higher than in TRV:00 plants, whereas the PR3, PR4, and PR5 expression levels were markedly lower in TRV:CPR5 plants. 【Conclusion】GhCPR5 positively regulates cotton disease resistance; the downregulated expression of GhCPR5 significantly reduced cotton resistance to V. dahliae and B. cinerea.

Identification and Expression of CAD and CAD-Like Gene Families from Gossypium barbadense and Their Response to Verticillium dahliae

ZHANG YuJia, CUI KaiWen, DUAN LiSheng, CAO AiPing, XIE QuanLiang, SHEN HaiTao, WANG Fei, LI HongBin

Scientia Agricultura Sinica. 2023, 56(19): 3759-3771. doi:10.3864/j.issn.0578-1752.2023.19.005

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【Objective】Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis pathway, which plays an important role in enhancing plant mechanical strength and resisting pathogen invasion. The aim of this study is to identify CAD and CAD-Like (CADL) gene family members in Gossypium barbadense and to analyze their expression characteristics and their role in Verticillium wilt resistance, which provides reference for the mechanism elucidation and disease resistance breeding of cotton against Verticillium wilt. 【Method】The CAD and CADL gene family members in G. barbadense genome were identified by bioinformatics method, and their chromosomal location, phylogenetic relationship, gene structure and promoter cis-element prediction were systematically analyzed. The expression characteristics of GbCAD and GbCADL were analyzed by obtaining publicly released transcriptome data and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Functional analysis of GbCAD and GbCADL genes was performed by viral-induced gene silencing (VIGS) technique. 【Result】A total of 25 GbCAD and 34 GbCADL genes were identified from G. barbadense and distributed on 10 and 17 different chromosomes, respectively. GbCAD and GbCADL genes are divided into 3 and 4 subgroups, respectively. The genes in the same group contain similar exon-intron structures and conserved domains. GbCAD and GbCADL genes have different transcriptional expression characteristics, and the promoters of GbCAD and GbCADL genes contain various hormone response elements and stress response elements. Transcriptome data and qRT-PCR showed that the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL5A/D, GbCADL6A/D, and GbCADL7A/D were induced by Verticillium dahliae, especially the GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D indicated significant increased expressions under V. dahliae treatment. The genes of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D were respectively silenced in cotton by virus-induced gene silencing (VIGS) technology, to analyze the changes of VIGS plant lines against V. dahliae treatment. The results showed that, compared with the control plants, the VIGS plant lines indicated significant decreased resistance to V. dahliae. The results of diaminobenzidine (DAB) histochemical stain displayed that, both control and VIGS plants showed similar normal phenotype without V. dahliae addition; after 6 h treatment of V. dahliae, the VIGS plant lines silencing GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D expressions demonstrated a deeper brown coloring, indicating a higher reactive oxygen species (ROS) accumulation in the VIGS plant lines. The results of stem sectioning showed that, the stem vascular tissues of VIGS plant lines TRV:GbCAD10A/D, TRV:GbCADL4A/D, TRV:GbCADL6A/D, and TRV:GbCADL7A/D showed obvious dark brown enrichment after V. dahliae treatment, indicating the significant decreased resistance to V. dahliae. 【Conclusion】 Suppressing the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D could significantly reduce the cotton resistance to V. dahliae.

Investigation on the Effects of Climate Change on the Growth and Yield of Different Maturity Winter Wheat Varieties in Northern China Based on the APSIM Model

SHI XinRui, HAN BaiShu, WANG ZiQian, ZHANG YuanLing, LI Ping, ZONG YuZheng, ZHANG DongSheng, GAO ZhiQiang, HAO XingYu

Scientia Agricultura Sinica. 2023, 56(19): 3772-3787. doi:10.3864/j.issn.0578-1752.2023.19.006

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【Objective】This study aims to clarify the impacts of climate change on the growth, development and yield of winter wheat of different maturity, so as to provide a theoretical basis for the sustainable production of wheat under future climate change. 【Method】The data about growth of two winter wheat varieties of Liangxing 99 (late-maturing) and Zhongke 2011 (early-maturing), soil, and meteorology, which were observed under different temperatures and [CO₂] treatments in the open top chamber in 2017-2020, were used to calibrate and validate the APSIM (agricultural production systems simulator) model. Then the verified model was used to simulate winter wheat yield, yield composition and phenology dates under different future climate conditions (RCP 4.5 and RCP 8.5) with a baseline period of 1986-2005. And the impacts of climate change and extreme high temperature on the production potential of different maturity winter wheat varieties were analyzed. 【Result】The APSIM model was able to well simulate the phenology, yield and biomass under different air temperature and [CO₂] treatments since the simulated and measured values of R² were higher than 0.614 and the values of nRMSE were all lower than 10.6%. However, the simulation result of leaf area index (LAI) was relatively poor. For the long-term simulation results, under different climate conditions, the days from sowing to jointing were shorter than the baseline for two wheat varieties. The shortened days of early-maturing variety were smaller than those of late-maturing variety. There was no obvious change in the days from jointing to maturity between the two varieties. The yield and potential yield of the two wheat varieties were higher under the future RCP conditions than under the baseline period. The yield and potential yield were the highest under the RCP 8.5 condition in 2100s. The yield and potential yield of early-maturing variety were more remarkably increased than those of late-maturing variety. Compared with the baseline, the LAI values of the two wheat varieties increased in the early growth stage. Then, the LAI of the late-maturing variety decreased obviously in the late growth stage, while the LAI of the early-maturing variety had no obvious difference. The aboveground biomass of the two wheat varieties both increased, and the early-maturing variety increased more remarkably than the late-maturing variety. Under different RCP conditions, extreme high temperature had negative impacts on the yield and 1 000-grain weight of the two varieties of winter wheat. Extreme high temperature at flowering stage had the greatest impact on 1 000-grain weight. Compared with the normal years, the 1 000-grain weight and yield of late-maturing variety decreased obviously in extreme-high-temperature years under the RCP 8.5 condition in 2100s, while the grain number also decreased slightly. Under different RCP conditions, compared with the normal years, extreme high temperature obviously reduced the 1 000-grain weight of early-maturing variety but slightly increased the grain numbers. Thus, yield reduction of early-maturing wheat variety in extreme high temperature years was not obvious. 【Conclusion】Early-maturing variety of winter wheat will be more adaptable to future climate change. Thus, breeding of wheat varieties to adapt to climate change is one of the effective measures to cope with future climate change.

Reforming the Cropping System to Achieve Maize Mechanical Grain Harvesting in Northern Huang-Huai-Hai Area of China

HOU LiangYu, ZHANG ZhenTao, HUANG ZhaoFu, LI LuLu, GUO YaNan, MING Bo, XIE RuiZhi, HOU Peng, XUE Jun, WANG KeRu, LI ShaoKun

Scientia Agricultura Sinica. 2023, 56(19): 3788-3798. doi:10.3864/j.issn.0578-1752.2023.19.007

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【Objective】Mechanical grain harvesting is the development direction of maize harvesting technique, however, under a crop rotation system of winter wheat and summer maize, which is hardly to be achieved in China, resulting from insufficient heat resource in limited growing season for maize. So far, the northern Huang-Huai-Hai area is the area where the mechanical grain harvesting for maize is most difficult to be applied and spread all over the world. Thus, a study on the feasibility of corn’s mechanical grain harvesting is considerably significant to the whole-process mechanization, quality, proceeds and industrial competitiveness of corn production in Huang-Huai-Hai area.【Method】 In northern Huang-Huai-Hai area, the dynamic observation for the accumulated temperature requirements for grain dehydration of 5 maize cultivars with different physiological maturity, including Zhengdan958, Xianyu335, Dika517, Jingnongke728 and Fengken139, were designed in Xiangxiang, Henan province from 2016 to 2017 and then in Beijing, in 2018 respectively. Then, based on the meteorological data from 2007 to 2018, after reserving 500 ℃·d for winter wheat growth, the northern Huang-Huai-Hai area was divided into 7 accumulated temperature zones according to a temperature gradient of 100 ℃, so as to spatially illustrate the heat resource distribution during maize growth season in this area. Hence, the different cultivars’ accumulated temperature requirements for grain dehydration were mapped under the heat resource distribution in this area. 【Result】 Under the conventional sowing condition of summer maize, each cultivar’s coverage that mechanical grain harvesting could be achieved after physiological maturity, with the advance of maturity, which were gradually extended northwardly, commonly 5-10 d later than the current sowing time for winter wheat in northern Huang-Huai-Hai area. However, the heat resource of accumulated temperature zonesⅠ-Ⅲ (1 900-2 800 ℃·d) were still hardly able to meet the requirements of Jingnongke728 and Fengken139 for a relative earlier physiological maturity than other 3 tested cultivars, not to metion reaching a grain moisture content of 25%. When the double cropping per year (winter wheat-summer maize) was reformed into the triple cropping per 2 years (summer maize-spring maize-winter wheat), the grain moisture content of all the tested cultivars, no matter planted in spring or summer, could reach 25% even less, achieving mechanical grain harvesting. Besides, for early-matured cultivars, by drying in filed and delaying harvesting, their moisture content might reach below 20% before winter wheat’s sowing, thus not only elevating harvesting quality, but also cutting drying costs.【Conclusion】In Northern Huang-Huai-Hai area, the problem, hindering maize production that insufficient heat resource in limited growing season issues in the difficult achievement of mechanical grain harvesting, could be effectively solved by reforming the current cropping system. Meanwhile, it also might provide a new horizon with theoretical foundation for the application of maize mechanical grain harvesting, further improving the quality and efficiency of maize production in Northern Huang-Huai-Hai area.

Screening and Evaluation of Non-Volatile Decision-Making Traits of Oviposition Resistance and Susceptibility of Bactrocera dorsalis

GONG QingTao, LI Miao, GAO XiaoLan, ZHANG KunPeng, LI GuiXiang, DONG XiaoMin, LI SuHong, ZHANG AnNing

Scientia Agricultura Sinica. 2023, 56(19): 3799-3813. doi:10.3864/j.issn.0578-1752.2023.19.008

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【Objective】The objective of this study is to analyze the relationship between the non-volatile traits of peach fruit and the oviposition of Bactrocera dorsalis, determine the best indicator of the risk of injury and the range of resistance and susceptibility, and to clarify the classification correlation characteristics of the main indicators.【Method】Using 13 representative peach varieties with different physical, villus, mineral elements and physiological characteristics as test materials, 60 non-volatile decision-making traits of 4 types were determined, and the selection test of the oviposition of B. dorsalis was carried out in the laboratory. The correlation and cluster analysis of the different traits and the average larval number in the fruit were carried out to determine the best indicator of the risk of injury and the range of resistance and susceptibility, and the performance of other main indicators was evaluated.【Result】The average absolute value of the correlation coefficient between the 4 types of non-volatile decision-making traits and the average number of larvae was in the order of physiological indicators (0.21, 18 species)>fruit physics (0.19, 18 species)>villus indicators (0.18, 7 species)=mineral elements (0.18, 17 species). Simple correlation analysis showed that there were 17 indicators above the low correlation level. The absolute values of the correlation coefficient from large to small were magnesium (-0.57)>hardness (-0.53)>calcium (-0.52)>selenium (-0.48)>long/short of villus (-0.45)>density of long villus (-0.44)>shady facets-L* (0.43)>free amino acid (-0.41)>transverse diameter (0.38)>amylum (0.37)>salt (-0.35)>single fruit weight (0.33) = pH (0.33)>tannin (-0.32) = anthocyanin (-0.32) = fruit water content (0.32) = Synthesis-L* (0.32). Shady facets-L and magnesium content showed the maximum positive and negative correlation, respectively, the correlation coefficient of nitrogen and protein was 0. The three indicators of magnesium, hardness and calcium reached a significant correlation level, indicating that the medium elements and hardness had the greatest impact on the oviposition resistance of B. dorsalis. The comprehensive analysis showed that magnesium was the best indicator of peach fruit damage risk. According to the absolute value of magnesium content and the degree of fruit damage, cluster analysis method was used to determine those ≥1.50 g·kg^-1as low-risk damaged varieties, that was the high-resistance variety, ≤0.92 g·kg^-1was judged as high-risk victimized varieties, that was the susceptible variety, and the middle were neutral varieties.【Conclusion】The non-volatile trait of peach fruit affects the oviposition selectivity of B. dorsalis. Medium elements (magnesium, calcium) and hardness significantly affect the oviposition selection of B. dorsalis, and all show negative correlation. Magnesium was selected as the best indicator of peach fruit damage risk. There are differences in the effects of 14 low degree related decision-making traits such as selenium on oviposition preference, with 7 positive and 7 negative. The positive correlated traits are all physical indicators except for amylum and pH, while the negative correlated traits are mineral element (1), villus (2), and physiological (4) indicators. Based on the situation of fruit damage, individuals≥1.50 g·kg^-1were preliminarily classified as high resistance varieties; ≤0.92 g·kg^-1were susceptible varieties, while the middle were neutral varieties.

Identification and Expression Analysis of Heat Shock Protein Superfamily Genes in Callosobruchus chinensis

ZHANG Xin, YANG XingYu, ZHANG ChaoRan, ZHANG Chong, ZHENG HaiXia, ZHANG XianHong

Scientia Agricultura Sinica. 2023, 56(19): 3814-3828. doi:10.3864/j.issn.0578-1752.2023.19.009

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【Objective】The purpose of this study is to identify the gene members of the Callosobruchus chinensis heat shock protein (HSP) superfamily, and to clarify the expression changes of HSP genes in C. chinensis after high and low temperature stress, so as to provide a theoretical basis for further exploration of HSP gene function.【Method】The CDS and protein sequences of HSP genes of different insects were downloaded from Insect Base 2.0 and used as a reference for local BLASTp and tBLASTn comparison search in the full-length transcriptome sequencing database of C. chinensis. At the same time, target sequences were screened again by combining HMMER and key words to complete the summary of search results. Bioinformatics analysis of HSP superfamily genes in C. chinensis was performed using CDD, MEGA, ProtParam, and other online analytical tools. Seven candidate HSP genes were screened out based on high and low temperature transcriptome sequencing data of C. chinensis adults and the expression characteristics of 7 CcHsps were compared and analyzed by qRT-PCR technique under different developmental stages and temperature stresses of C. chinensis.【Result】A total of 31 HSP genes were identified, including 3 HSP90s, 8 HSP70s, 8 HSP60s, and 12 sHSPs (small HSP). Physicochemical analysis showed that the proteins encoded by CcHsps contain 159-776 amino acid residues (aa), the molecular weights are about 18.4-88.9 kDa, and the theoretical isoelectric points are 4.95-9.17. Subcellular localization results showed that most CcHsps were located in the cytoplasm, while a few genes were located in the mitochondrial matrix, endoplasmic reticulum and nucleus. Phylogenetic analysis showed that different family members of HSPs in C. chinensis could integrate well with HSP in other insects, which indicating their evolutionary conservation. The results of qRT-PCR showed that the 7 candidate CcHsps were differentially expressed under different temperature stresses. After high temperature stress, the expression level of CcHsp20.102 in male and female adults was up-regulated by 1 000 and 500 times, respectively, and the expression level of CcHsp70-5 in male and female adults was up-regulated by 500 and 450 times. After the larvae undergoing high and low temperature stress, the expression level of CcHsp19.855 and CcHsp70-5 was significantly different.【Conclusion】A total of 31 complete HSP superfamily gene members were identified by the full-length transcriptome sequencing data of C. chinensis, which were divided into 4 subfamilies. Different HSP families had different gene structures, protein conserved domains and gene expression characteristics. The differential expression of 7 candidate CcHsps in different developmental stages and under different temperature stresses indicated that they played different functions and roles. It is speculated that CcHsp20.102 and CcHsp70-5 may perform important functions in the adult resistance to high temperature stress, and the high temperature tolerance of larvae may be related to the differential expression of CcHsp19.855 and CcHsp70-5.

Response of Paddy Soil Health to Continuous Amendments of Organic Fertilizer and Lime Separately Under Double-Cropping Rice Fields

YIN ZeRun, SHENG Hao, LIU Xin, XIAO HuaCui, ZHANG LiNa, LI YuanZhao, TIAN Yu, ZHOU Ping

Scientia Agricultura Sinica. 2023, 56(19): 3829-3842. doi:10.3864/j.issn.0578-1752.2023.19.010

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【Objective】 The aim of this study was to investigate the mechanism of the comprehensive soil health of double-cropping rice fields in response to continuous organic fertilizer and quick lime amendments, and to verify the current major soil health assessment methods in terms of the adaptability, sensitivity, and their sensitivity indicators in paddy soils. 【Method】 Here the double-cropping rice fields in the middle and lower reaches of the Yangtze River watershed were selected. Based on the principles of experimental design, the control and treatment fields were set up. It aimed at illuminating the changes in topsoil physical, chemical, and biological indicators (24 indicators) after 6-year biannual continuous organic and quick lime amendments, and assessing the holistic soil health index using the Cornell Soil Health Assessment (CASH), the Haney Soil Health Test (HSHT), and the principal component analysis combined with minimal data set construction (MDS) methods. 【Result】 Compared with the control treatment (CK), 6-year continuous organic amendment treatment reduced the soil bulk density, penetration resistance, and microaggregates (<0.25 mm) significantly by 14%, 25%, and 32%, respectively; however, which rose the soil aggregate (0.5-1 mm), ammonium N, activated C, water extractable organic C and N, respiration rate, and autoclaved-citrate extractable protein significantly by 100%, 37%, 54%, 21%, 44%, 59%, and 8%, respectively; in addition, the minimum data set (MDS), comprehensive assessment of soil health (CASH), 2015, 2018, and Ward Laboratory (SHS) versions of Haney soil health test (HSHT) indexes were significantly enhanced by 75%, 20%, 42%, 95%, and 55% under 6-year continuous organic amendment treatment than that under CK, respectively (P<0.05). After the 6-year continuous liming, the soil penetration resistance, microaggregates, water extractable organic N, and pH were significantly increased by 44%, 22%, 61%, and 0.57 units than that under CK, respectively; whereas 0.5-1 mm soil aggregates, available Zn, and respiration decreased significantly by 39%, 14%, and 52% under 6-year continuous organic amendment treatment than that under CK, respectively; meanwhile, the MDS, CASH and SHS HSHT indexes were significantly decreased by 59%, 15% and 47%, respectively. 【Conclusion】 The 6-year continuous biannual organic and liming amendments separately exerted positive and negative effect on the paddy soil health, and the soil respiration rate sensitively indicated the paddy soil health. However, the soil health index cannot reflect the change in soil Cd accumulation and its phyto-availability, and the assessment tools for paddy soil with excess Cd concentration remained to be further developed.

Effects of Long-Term Synergistic Application of Organic Materials and Chemical Fertilizers on Bacterial Community and Enzyme Activity in Wheat-Maize Rotation Fluvo-Aquic Soil

ZHANG LingFei, MA Lei, LI YuDong, ZHENG FuLi, WEI JianLin, TAN DeShui, CUI XiuMin, LI Yan

Scientia Agricultura Sinica. 2023, 56(19): 3843-3855. doi:10.3864/j.issn.0578-1752.2023.19.011

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【Objective】This experiment studied the effects of long-term synergistic application of organic materials and chemical fertilizers on soil bacterial community and enzyme activity, and revealed the relationship between soil nutrients, extracellular enzyme activity and bacterial community, so as to provide a theoretical basis for formulating long-term and reasonable fertilization strategies under wheat-maize rotation system in fluvo-aquic soil. 【Method】 Based on a 10-year located experiment, five treatments were set up, including no fertilization (NF), chemical fertilizer (NPK), chemical fertilizer with straw return (NPKS), 50% chemical fertilizer with 6 000 kg·hm^-2 pig manure (NPKP), and 50% chemical fertilizer with 6 000 kg·hm^-2cow manure ( NPKC ). 【Result】 (1) The combined application of organic materials and chemical fertilizers ( NPKS, NPKP and NPKC ) could significantly improve soil fertility and extracellular enzyme activity, among which NPKC treatment had the most significant effect. Compared with NPK treatment, the contents of organic matter, total nitrogen, available nitrogen, available phosphorus and alkaline phosphatase activity were increased by 13.8%-15.4%, 9.7%-15.5%, 7.2%-15.9%, 13.6%-38.5%和2.5%-13.1%. (2) Long-term combined application of organic and inorganic fertilizer significantly changed the bacterial community structure and composition. In the wheat season, compared with NPK treatment, NPKS treatment significantly increased the abundance of Aggregatilinea and Parachlamydia, NPKP treatment significantly increased the abundance of Pseudomonas, Nonomuraea and Flexilinea, while NPKC treatment only significantly increased the abundance of Luteitalea. In the maize season, compared with NPK treatment, NPKS treatment significantly increased the abundance of Phycisphaera and Syntrophothermus, NPKP treatment significantly increased the abundance of Gemmatimonas, and NPKC treatment significantly increased the abundance of Aquipuribacter and Desulfosoma. (3) The results of functional prediction showed that combined application of organic and inorganic fertilizers could promote soil carbon and nitrogen cycling compared with long-term single application of chemical fertilizer. In particular, the NPKC treatment had a strong effect on nitrification, ureolysis, aromatic compound degradation, xylanolysis and cellulolysis. (4) Mental analysis showed that soil pH was the main factor regulating bacterial community structure and ecological function in fluvo-aquic soil. 【Conclusion】 Long-term application of organic and inorganic fertilizers (especially chemical fertilizers combined with cow manure) could improve soil fertility and extracellular enzyme activity, increase the abundance of beneficial bacteria, significantly change the structure and composition of bacterial communities, and promote the circulation of carbon, nitrogen and phosphorus, thus construct an environment suitable for crop and bacterial growth in fluvo-aquic soil.

Effects of Rhizodeposition on Straw Carbon and Nitrogen Sequestration in Soil Profile Under Different Fertilization Conditions

MEI XiuWen, ZHU TengXiao, LI YuPing, LI ShuangYi, SUN LiangJie, AN TingTing, WANG JingKuan

Scientia Agricultura Sinica. 2023, 56(19): 3856-3868. doi:10.3864/j.issn.0578-1752.2023.19.012

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【Objective】Straw returning is an important measure for protecting black soil in Northeast China. Straw and rhizodeposition coexisted in the practical agricultural production, while the sequestration characteristics of straw carbon (C) and nitrogen (N) in soils remained not very clear under this condition. The purpose of this study was to compare the differences in the content of soil organic C (SOC) derived from straw C (¹³C-SOC) and that of soil total N (TN) derived from straw N (¹⁵N-TN) among different soil layers, and to investigate the effects of rhizodeposition on straw C and N sequestration in soil profile under different fertilization conditions, so as to provide a basis for the protection and utilization of black soil in Northeast China. 【Method】Based on the long-term experimental station of Shenyang Agricultural University, the treatments of adding ¹³C¹⁵N double-labeled straw (S) and its combination with rhizodeposition (hereafter referred to as “rhizodeposition”) (SR) were designed under different fertilization plots (no fertilization, CK; single application of chemical fertilizer, NP), including four treatments: CK+S, CK+SR, NP+S, NP+SR. The contents of SOC and TN, and values of δ¹³C and δ¹⁵N at different soil layers were measured after the 50 and 150 days of in-situ experiment. 【Result】At the early stage of straw decomposition (the 50^th day), fertilization, rhizodeposition and their interactions significantly affected (P<0.05) the contents of ¹³C-SOC and ¹⁵N-TN in the topsoil (0-20 cm). On the 50^th day, compared with the CK+S treatment, the CK+SR treatment increased the contents of ¹³C-SOC and ¹⁵N-TN in the topsoil by 18.6% and 21.7% (P<0.05), respectively. The contribution percentage of ¹³C-SOC to SOC in the topsoil was, on average, 10.5% and 12.0% in the S (CK+S and NP+S) and SR (CK+SR and NP+SR) treatments under different fertilization, respectively. The contribution percentage of ¹⁵N-TN to TN in the topsoil was, on average, higher 27.6% (P<0.05) in the two treatments under CK (CK+S and CK+SR) than that in the corresponding treatments under NP (NP+S and NP+SR). On the 50^th day, the contribution percentage of ¹³C-SOC to SOC and that of ¹⁵N-TN to TN at deep soil (20-50 cm) were 1.0%-2.2% and 0.5%-0.9%, respectively. At the later stage of straw decomposition (the 150^th day), rhizodeposition and fertilization significantly affected (P<0.05) the contents of ¹³C-SOC and ¹⁵N-TN in the topsoil, respectively. On the 150^th day, compared with the treatment of rhizodeposition, the treatment of straw addition increased the ¹³C-SOC content in the topsoil, on average, by 12.6% (P<0.05). The ¹⁵N-TN content in the topsoil was, on average, higher 22.0% (P<0.05) in the two treatments under CK than that in the corresponding treatments under NP. The contribution percentage of ¹⁵N-TN to TN in the topsoil in CK and NP treatments was 5.5% and 4.0%, respectively. On the 150^th day, the contribution percentage of ¹³C-SOC to SOC and that of ¹⁵N-TN to TN at deep soil were 0.8%-3.2% and 0.7%-1.8%, respectively. 【Conclusion】Rhizodeposition had a negative feedback effect on the sequestration of straw C in topsoil during the later stage of straw decomposition. Straw derived C and N were constantly migrated and then accumulated from topsoil to deep soil, and their influences on the stabilities of soil organic C and N pools should be paid more attention.

Nutritional Effects of Liquid Digestate on Tomatoes Grown in Facility Substrates

TENG YunFei, SHANG Bin, TAO XiuPing

Scientia Agricultura Sinica. 2023, 56(19): 3869-3878. doi:10.3864/j.issn.0578-1752.2023.19.013

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【Objective】 The nutritional effects of liquid digestate on tomatoes in facility soilless cultivation were investigated, so as to provide scientific basis for the substrate cultivation application of biogas slurry. 【Method】 Here, tomato was used as subject, and the dual inputs of fertilizers (liquid digestate (D) and mineral fertilizer (M)) and soilless substrates (peat substrate (P) and cinder substrate (C)) consisted of four treatments. The parameters of tomato growth, photosynthesis and nutrients uptake were recorded during the experimental period, as well as the properties of substrates. 【Result】The tomato dry biomass under DP treatment was higher than that under MP treatment by 12.6%, and DC treatment was higher than MC treatment by 70.9%. However, the dry biomass under DP and MP treatments were significantly higher than that under DC and MC treatments (P<0.05). The Cond, Ci and Tr of tomato leaf under DP treatment was significantly higher than that under other treatments (P<0.05), and the greatest leaf photosynthetic rate was also observed under DP treatment of (18.17±0.47) µmol·m^-2·s^-1. Liquid digestate significantly increased the dry biomass and photosynthesis of tomato plants. The total N and total P content of plant under DP treatment were higher than that under MP treatment by 46.9% and 19.7%, respectively, and DC treatment were 1.38 and 2.45 times higher than MC treatment, respectively. Moreover, liquid digestate treatments significantly increased the pH value, organic matter and available phosphate content of substrates compared with chemical fertilizer treatments (P<0.05). The highest fruit yield was obtained under DP treatment of (6.0±0.4) kg·m^-2, and the yields between DP and MP treatments had no significant difference. The liquid digestate treatments could significantly increase the soluble sugar content and vitamin C content in tomato fruits (P<0.05), and improve fruit quality. Meanwhile, the results of principal component analysis showed that DP treatment had the best overall performance, followed by MP and DC treatments, while MC treatment was the worst. 【Conclusion】The application of liquid digestate into peat substrate increased the photosynthetic efficiency of leaves and the absorption of nitrogen and phosphorus of tomato plants, as well as the properties of substrate and the yield and quality of fruits. Therefore, the combination was recommended for use in facility tomato cultivation.

Differences and Genesis of Grape Phenolic Compounds Among Different Altitudes in Yunnan Shangri-la

ZHANG KeNan, YIN HaiNing, WANG JiaKui, CAO JianHong, XI ZhuMei

Scientia Agricultura Sinica. 2023, 56(19): 3879-3893. doi:10.3864/j.issn.0578-1752.2023.19.014

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【Objective】Phenolic compounds are important secondary metabolites of wine grape, which have an important influence on the quality of grape and wine. In this study, the differences and genesis of phenolic substances in grape skins among different altitudes were studied combined with soil and climate factors, so as to provide a theoretical basis for the planting management of wine grapes at high altitudes region. 【Method】In the present research, Merlot wine grape was used as the test material. For three consecutive years (2020, 2021 and 2022), the differences of total phenols, flavonoids, tannins, total anthocyanins, the content of individual and non-individual anthocyanin components in grape skins at two altitudes (2 181, 2 300 m) at maturity stage were analyzed. Meanwhile, climate factors such as light, temperature and humidity at different altitudes were monitored during grape development, and the influences of climatic factors on phenolic substances of grape skins were analyzed. 【Result】There were no significant differences in the main mineral nutrients of the soils of the two altitude vineyards, and some differences in climatic factors, such as light, UV intensity, temperature and humidity. Altitude had a significant effect on the content of phenolic substances in grape skins. In the years of 2020-2022, the higher altitude was conducive to the accumulation of phenolic substances in grape skins. the content of total phenols, total tannin, total anthocyanins, most of the individual anthocyanins and the quercetin in berry skins were higher at the altitude of 2 300 m; compared with that at 2 181 m, the content of total tannin in grape skins at 2 300 m increased by 56.27%-174.49%. The flavonoid content at 2 181 m altitude were significantly higher than that at 2 300 m, with an increase of 32.25% to 79.48%. OPLS-DA analysis showed that, the main different compounds of phenolic compounds between the two altitudes were total tannin (TTC), total flavonoids (TFo), malvidin-3-glucoside (Mv), malvidin-3-acetly-glucoside (Mv-Ace), cyanidin-3- glucoside (Cy), and peonidin-3-glucoside (Pn). Grey correlation analysis showed that day-night temperature difference in grape growing season had a great effect on the content of total phenols and total flavonoids in grape skins. The content of total anthocyanins, individual anthocyanins and quercetin in skins were significantly affected by light and ultraviolet intensity. The content of three anthocyanins (Pt, Pn-Ace and Pn-Cou) and quercetin were mainly affected by the light intensity during grape veraison (July).【Conclusion】The climatic conditions of different altitudes, especially day-night temperature difference, light and ultraviolet intensity were the main factors causing the differences of phenolic content. The larger day-night temperature difference, stronger light and ultraviolet intensity at higher altitude were conducive to the accumulation of phenolic substances in grape skins.

Identification of Molecular Markers Associated with Goose Egg Quality Through Genome-Wide Association Analysis

GAO GuangLiang, ZHANG KeShan, ZHAO XianZhi, XU GuoYang, XIE YouHui, ZHOU Li, ZHANG ChangLian, WANG QiGui

Scientia Agricultura Sinica. 2023, 56(19): 3894-3904. doi:10.3864/j.issn.0578-1752.2023.19.015

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【Objective】 The objective of this study was to screen molecular markers and candidate genes related to goose egg quality traits, to provide a theoretical support for the analysis of the genetic mechanism of egg quality traits and marker-assisted selection. 【Method】 In this study, a batch of healthy Sichuan White Geese (209 individuals) was selected as the research subjects. Five eggs from each goose during the peak egg production period were collected, and then six egg quality traits were measured, including egg weight, egg shape index, eggshell strength, eggshell thickness, eggshell weight, and egg yolk weight. Based on the whole-genome resequencing data (2.896 Tb, 12.44×/individual) of 209 Sichuan White Geese (female geese), a genome-wide association analysis was conducted to identify SNP loci and important candidate genes associated with egg quality traits. The genotype frequencies of the SNP loci were determined using the nucleic acid flight time mass spectrometry method. 【Result】After filtering, a total of 9 279 339 SNPs and 209 individuals were included for further analysis. The GWAS analysis identified 48 SNP loci significantly or suggestively associated with six egg quality traits (thresholds: 5.43×10^-9 and 1.09×10^-7). These loci were annotated to 27 candidate genes related to egg quality traits, including Pregnancy-associated plasma protein A (PAPPA), Serine/threonine-protein phosphatase 4 regulatory subunit 2 (PP4R2), Ethanolamine phosphotransferase 1 (EPT1), and Glutamate receptor ionotropic, kainate 2 (GRIK2). Among them, the candidate gene PAPPA was involved in protein metabolism and promotes the generation of insulin-like growth factor. Five SNPs within the 11 bp range of PP4R2 were significantly associated with eggshell thickness. Additionally, six SNPs on the GRIK2 gene were significantly associated with yolk weight. GRIK2 and PP4R2 were respectively associated with blood calcium homeostasis and cholesterol metabolism in organisms. Functional enrichment analysis revealed that the candidate genes were mainly annotated to “response to growth factor” (GO:0070848), “intracellular chemical homeostasis” (GO:0055082), “response to hormone” (GO:0009725), and “regulation of monoatomic ion transport” (GO:43269). 【Conclusion】 The GWAS analysis showed that the PAPPA, GRIK2, ASCC3, and EPT1 are potential functional genes associated with various egg quality traits, such as egg weight, egg yolk weight, and shell strength, providing theoretical references for molecular genetic marker-assisted selection of goose egg quality traits.

Effect of Porcine ECR1-Like Immune Adhesion on PAMs Capturing GFP-Escherichia coli

ZHANG Zheng, LING XiaoYa, FAN KuoHai, SUN Na, SUN PanPan, SUN YaoGui, LI HongQuan, YIN Wei

Scientia Agricultura Sinica. 2023, 56(19): 3905-3916. doi:10.3864/j.issn.0578-1752.2023.19.016

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【Objective】The aim of this study was to investigate whether the immune adherence function of porcine erythrocyte complement receptor type 1-like (ECR1-like) could promote porcine alveolar macrophages (PAMs) to capture sensitized genetic engineering bacteria GFP-E. coli, in order to explain the molecular mechanism of porcine erythrocyte immunity and its role in innate immunity. 【Method】The level of GFP-E. coli captured by PAMs was detected by flow cytometry, colony plate counting and RT-PCR, and the effect of porcine ECR1-like immune adherence on the capture of GFP-E. coli by PAMs was analyzed. Flow cytometry and cellular immunofluorescence technique were used to detect the changes of immune adherence function of porcine erythrocytes, and the number of porcine ECR1-like after the sensitized GFP-E. coli with ECR1-like immune adherence was removed by PAMs. 【Result】Flow cytometry showed that the average fluorescence intensity of PAMs in porcine erythrocyte adhesion group was significantly higher than that in blank control group (P<0.001), while the positive rate of PAMs cells in porcine erythrocyte adhesion group was significantly higher than that in blank control group (P<0.05). Colony smear count showed that the capture of GFP-E. coli by PAMs in erythrocyte adhesion group was significantly higher than that in blank control group (P<0.05). RT-PCR showed that the relative quantity of GFP-E. coli in PAMs of erythrocyte adhesion group was significantly higher than that of blank control group (P<0.01). Further blocking CR1-like on the surface of porcine erythrocyte, flow cytometry showed that the average fluorescence intensity of PAMs decreased to 256 301.56±9 208.85 (P<0.001), and the positive cell rate of PAMs decreased to (88.32±0.92)% (P>0.05). Colony count showed that the capture of GFP-E. coli in PAMs decreased to (136 666±8 818) CFU/mL (P<0.05), and RT-PCR showed that the relative quantity of GFP-E. coli in PAMs decreased significantly (P<0.01). Using cell flow and circulation interaction technique, it was found that the average fluorescence intensity of GFP-E. coli sensitized by porcine erythrocyte immune adherence decreased from 2 892.18±47.76 before circulation to 2 407.43±141.78 (P<0.05), and the positive cell rate decreased from (20.58±0.36)% before circulation to (17.39±0.23)% (P<0.05). The adhesion level was significantly lower than that before circulation. Meanwhile, the results of indirect immunofluorescence test showed that the average fluorescence intensity of porcine ECR1-like decreased from 344.33±37.92 before to 291.56±11.99 (P<0.05), and the positive cell rate decreased from (30.20±1.24)% before to (28.27±0.64)% (P<0.05). 【Conclusion】Porcine ECR1-like promoted the capture of sensitized GFP-E.coli by PAMs through its immune adhesion function. After PAMs removed sensitized GFP-E. coli adhered to the surface of porcine erythrocyte, the activity of CR1-like of porcine erythrocyte decreased, and the immune adhesion function decreased too.

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