Scientia Agricultura Sinica

Genome-Wide Association Analysis of Yield and Combining Ability Based on Maize Hybrid Population

LI ZhouShuai,DONG Yuan,LI Ting,FENG ZhiQian,DUAN YingXin,YANG MingXian,XU ShuTu,ZHANG XingHua,XUE JiQuan

Scientia Agricultura Sinica. 2022, 55(9): 1695-1709. doi:10.3864/j.issn.0578-1752.2022.09.001

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【Objective】By analyzing the yield of the hybrids from the inbred lines bred from the Shaan A and Shaan B group, the combining ability of the inbred lines were evaluated, genome-wide association analysis, and mining associated loci for yield and its combining ability conducted. It will provide references for improving maize inbred lines selected from Shaan A group and Shaan B group and applying them in varieties breeding. 【Method】Based on NCⅡ genetic design, 85 excellent inbred lines from Shaan A group and Shaan B group were used to construct a hybrid population containing 246 F₁. Then, the yield of the hybrid population was tested in three environments to evaluate their general combining ability (GCA) and special combining ability (SCA). Using the 6H90K maize array to detect the parental genotypes, 63 879 high-quality SNPs were obtained, which were used to analyze the genetic characteristics of parental lines. According to the parental genotypes, 55 951 high-quality SNPs were inferred in the hybrid population for genome-wide association analysis of hybrid yield, GCA, and SCA using additive model and non-additive model. Meanwhile, candidate genes around the significant SNPs were screened and annotated based on the maize B73 reference genome.【Result】The yield in the three environments accorded to the normal distribution with wide variation, the broad-sense heritability of yield was 59.04%, and the environmental effect was significant. There was significant positive correlation between hybrid yield and combining ability, and the correlation between hybrid yield and SCA (r=0.95) was higher than that between hybrid yield and GCA (r=0.62). The genetic characteristic of Shaan A group and Shaan B group was different, and inbred lines from Shaan A group have higher general combining ability. Totally, five, seven and nine significant SNPs were detected (-log₁₀(P)>3.86) for GCA, hybrid yield and SCA, respectively. Among them, four SNPs were co-located in hybrid yield and SCA. Ultimately, 17 associated SNPs were anchored. Dominant allele analysis of different trait-associated loci showed that four GCA-associated SNPs were controlled by additive effects, and the F₁ BLUE-associated loci could be divided into 4 types mainly by the dominant effect, and the heterozygous genotype is the favorite allele or sub-optimal allele for yield in F₁. Through functional annotation, the candidate genes were specifically expressed in maize growth and kernel establishment, for example, GRMZM2G165828 and GRMZM2G057557 were related to maize kernel development. 【Conclusion】Based on this study, we consider that GCA and SCA jointly affect the yield of hybrids, and the effect of SCA is greater. Moreover, GCA and SCA may have different genetic basis, and GCA can be increased with the accumulation of favorable alleles. Using the genome-wide association analysis in the F₁ hybrid population can carry out genetic analysis related to combining ability, mine the genetic loci related to yield and combining ability, and accelerated the application of the associated loci in molecular breeding.

Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars

DONG YongXin,WEI QiWei,HONG Hao,HUANG Ying,ZHAO YanXiao,FENG MingFeng,DOU DaoLong,XU Yi,TAO XiaoRong

Scientia Agricultura Sinica. 2022, 55(9): 1710-1722. doi:10.3864/j.issn.0578-1752.2022.09.002

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【Objective】 The establishment of apple latent spherical virus (ALSV)-induced gene silencing on domestic soybean cultivars will offer a simple, time-saving and operable system for gene function and genetic breeding of soybean cultivars in China.【Method】The infectious clones of ALSV which was used to infect plant through Agro-infiltration was constructed. A 327 bp cDNA fragment of phytoene desaturase (GmPDS) cDNA was amplified from soybean cultivar Williams 82 and inserted into the pALSV2 vector. The pALSV1 and pALSV2-GmPDS were used to co-infect Nicotiana benthamiana plant through Agro-infiltration, and the ALSV virus particle was purified from systemic infected leaves of N. benthamiana plants at 17 days post infiltration (dpi). The purified virions were mechanically inoculated onto the first-round true leaves of soybean and photobleaching phenotype on the upper non-inoculated leaves was monitored from 10 to 30 dpi. Using the empty ALSV vector as a control, the gene expression levels of ALSV coat protein (CP) gene and GmPDS in ALSV:GmPDS infected soybean were examined by RT-PCR and quantitative RT-PCR analysis, respectively. 【Result】At 20 dpi, soybean cultivar Nannong 1138-2 inoculated with ALSV:GmPDS showed photobleaching phenotype in the systemic infected leaves, however Williams 82 did not show gene silencing phenotype of PDS in the systemic infected leaves. The results of qRT-PCR confirmed that the RNA expression level of GmPDS in Nannong 1138-2 was significantly reduced but ALSV:GmPDS was unable to silence PDS in Williams 82. By using the same method, the gene silencing efficiency of other 9 domestic soybean cultivars mediated by ALSV:GmPDS was examined. Soybean cultivars including Nannong 47, Andou 203, Xiangdou 4, Zhonghuang 13, Shanning 29 and Qihuang 34 all displayed photobleaching phenotype in plant systemic infected leaves after inoculation with ALSV:GmPDS, while GmPDS was not silenced in Hedou 12, Zhonghuang 311 and Shanning 16.【Conclusion】In this study, an Agrobacterium-mediated ALSV viral VIGS vector was constructed. ALSV virions was propagated and purified from N. benthamiana plant. The purified virions were rub inoculated onto the first-round true leaves of soybean. Finally, an efficient gene silencing system was established on a number of domestic soybean cultivars in China.

Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat

ZHAO HaiXia,XIAO Xin,DONG QiXin,WU HuaLa,LI ChengLei,WU Qi

Scientia Agricultura Sinica. 2022, 55(9): 1723-1734. doi:10.3864/j.issn.0578-1752.2022.09.003

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【Objective】To develop a novel tool for functional verification and molecular breeding in tartary buckwheat, this study focused on establishing and optimizing an efficient callus genetic transformation system. 【Method】Callus induction factors including different explants, ratios of diverse growth regulators, and Agrobacterium tumefaciens types were systematically evaluated using “Xiqiao No. 2” as the derived plant. We further overexpressed FtCHS1, a key enzyme gene involved in the biosynthesis of tartary buckwheat flavonoids in obtained calli to validate the optimized genetic callus transformation system. The positive transgenic lines were confirmed by PCR and fluorescent observation. Subsequently, the content of anthocyanins and metabolites in flavonol branch pathway were determined by UV spectrophotometry and High Performance Liquid Chromatography (HPLC), respectively. Furthermore, quantitative real-time PCR was performed to analyze expression levels of genes involved in flavonoid synthesis, in order to compare the differences between the FtCHS1-overexpressed calli and the control. 【Result】The optimal explant was hypocotyls and the optimal induction medium was the Murashige and Skoog (MS) medium supplemented with the addition of 0.8 mg·L^-1 6-BA (6-Benzylaminopurine) and 3.5 mg·L^-1 2,4-D (2,4-Dichlorophenoxyacetic acid). The induction rate of calli grown on the above medium reached up to 72%. Moreover, the optimized subculture medium containing MS with the additives of 3 mg·L^-1 6-BA and 1 mg·L^-1 KT (Kinetin) increased the percentage and coefficient of callus proliferation to 98% and 1.09, respectively. Additionally, the best Agrobacterium tumefaciens in the transformation process was GV3101, and the transformation efficiency was up to 31.3%. The functional analysis of FtCHS1 overexpressing in transgenetic calli demonstrated that: (1) The accumulations of kaempferol and quercetin in transgenic calli overexpressing FtCHS1 were dramatically higher than those in control groups (P<0.01), and anthocyanin, rutin and myricetin contents were also remarkably higher (P<0.05); (2) Overexpression of the exogenous FtCHS1 did not affect the expression levels of 5 endogenous orthologous genes FtCHSs in the transgenic calli (P>0.05), whereas genes encoding key enzymes of the flavonoid synthesis pathway, such as FtCHI, FtF3H, FtFLS1, FtFLS2, FtFLS3, and FtDFR1, were up-regulated (P<0.05); (3) FtMYB5 and FtMYB6, the transcription factor genes that specifically positively regulated the flavonol synthesis, were up-regulated, while FtMYB8, a suppressor gene of anthocyanin synthesis, was down-regulated (P<0.05). 【Conclusion】In this study, the callus genetic transformation system of tartary buckwheat was successfully established from “Xiqiao No. 2”. FtCHS1 overexpression in the transgenic calli up-regulated genes related to flavonoid synthesis, resulting in flavonoids accumulation.

Effects of Different Nitrogen Application Levels on Photosynthetic Characteristics, Nitrogen Use Efficiency and Yield of Spring Maize in Sichuan Province

XIONG WeiYi, XU KaiWei, LIU MingPeng, XIAO Hua, PEI LiZhen, PENG DanDan, CHEN YuanXue

Scientia Agricultura Sinica. 2022, 55(9): 1735-1748. doi:10.3864/j.issn.0578-1752.2022.09.004

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【Objective】 In order to clarify the photosynthetic mechanism affecting the yield formation of spring maize under different nitrogen application levels, the effects of different nitrogen application levels on nitrogen use efficiency and soil nitrogen surplus were analyzed, so as to provide the theoretical reference for rational application of nitrogen fertilizer and promoting high yield and high efficiency of spring maize.【Method】Using the maize of variety Zhongyu 3 as experimental material, the field experiments were carried out in 2019 and 2020 at the long-term fertilizer effect experimental site of Ya’an Experimental Farm of Sichuan Agricultural University. Nitrogen supply included five levels, such as 0 (no nitrogen application), 90 (low nitrogen), 180 (appropriate amount of nitrogen), 270 (farmers’ habitual nitrogen application), and 360 kg·hm^-2 (high nitrogen), which were marked as N0, N1, N2, N3, and N4, respectively. The leaf area was measured at jointing period, silking period and grain-filling period, and the leaf area index and leaf area duration were calculated, respectively. The photosynthetic parameters, such as net photosynthetic rate of ear leaves were measured at grain-filling period, and chlorophyll content was measured at silking period and grain-filling period. The dry matter accumulation of aboveground population was measured at silking period, grain-filling period, and harvest period, the yield was measured at harvest, the nitrogen content of each part was analyzed, and the soil nitrogen surplus, nitrogen use efficiency of spring maize and economic benefit of nitrogen application were calculated.【Result】(1) The spring maize yield increased first and then remained flat with the increase of nitrogen application levels. In 2019 and 2020, the yield under N2 treatment was the highest, with an average of 9 746 kg·hm^-2, which was 179% and 28.7% higher than that of N0 and N1 treatments (P<0.05), respectively, but there was no significant difference among N2, N3, and N4 treatments. 2-year yield was fitted by linear + platform fitting, the platform nitrogen application level was 134.8 kg·hm^-2, the platform yield was 9 604 kg·hm^-2, and the output-input ratio of platform nitrogen fertilizer (134.8 kg·hm^-2) was the highest (12.6). (2) Compared with no nitrogen application, the appropriate amount of nitrogen application (N2) significantly increased chlorophyll content, net photosynthetic rate, stomatal conductance, transpiration rate of ear leaves, leaf area index and leaf area duration. However, with the increase of nitrogen fertilizer application, there was no significant difference or even decreased significantly in the above indexes. (3) Combined with the correlation analysis and partial least square analysis of photosynthetic characteristics and harvest yield, the yield was significant positively correlated with leaf area duration, net photosynthetic rate, stomatal conductance, transpiration rate, leaf area index, chlorophyll a+b of spring maize (P<0.01), and the main factor affecting spring maize yield was chlorophyll a+b. (4) During the harvest period, the grain nitrogen accumulation and total aboveground nitrogen accumulation increased significantly with the increase of nitrogen application level, and increased slightly or basically flat after N2 treatment (more than 180 kg·hm^-2) in the two years. The fitting results showed that the nitrogen application level was 139 kg·hm^-2 when the soil nitrogen surplus was 0 kg·hm^-2; The nitrogen apparent recovery efficiency of spring maize under N2 treatment was the highest in the two years, with an average of 73.7%, which was 10.8% higher than that under N1 treatment (P<0.05), the nitrogen apparent recovery efficiency decreased significantly with the continuous application of nitrogen fertilizer. Compared with N2 treatment, the nitrogen apparent recovery efficiency of N3 and N4 treatments decreased by 32.9% and 48.1%, respectively (P<0.05).【Conclusion】The proper amount of nitrogen application could obviously improve the photosynthetic performance of spring maize leaves, delay the degradation of total chlorophyll in ear leaves, prolong the duration of photosynthesis, and optimize the role among total chlorophyll, leaf area index and leaf area duration in the yield formation of spring maize. At the same time, the proper amount of nitrogen application could significantly increase the dry matter accumulation of aboveground population and grain yield, promote the absorption and accumulation of nitrogen to maize, reduce nitrogen residue in soil, and improve the nitrogen apparent recovery efficiency. Considering the factors such as yield, economic benefit of fertilization, apparent nitrogen use efficiency and nitrogen surplus, the nitrogen input of 139-180 kg·hm^-2 could maintain the goal of high yield and high efficiency of spring maize in the experimental area (Ya’an, Sichuan).

Effects of Reducing Nitrogen Application on Leaf Stay-Green, Photosynthetic Characteristics and System Yield in Maize-Soybean Relay Strip Intercropping

LI YiLing, PENG XiHong, CHEN Ping, DU Qing, REN JunBo, YANG XueLi, LEI Lu, YONG TaiWen, YANG WenYu

Scientia Agricultura Sinica. 2022, 55(9): 1749-1762. doi:10.3864/j.issn.0578-1752.2022.09.005

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【Objective】The aim of this study was to explore the characteristics of leaf green retention, photosynthesis and system yield of maize and soybean under different planting modes and nitrogen (N) application levels.【Method】The effects of planting methods (maize monoculture (MM), soybean monoculture (SS), maize intercropping (IM), soybean intercropping (IS)) and N application levels (0 N application (NN), reduced N application (RN: 180 kg N·hm^-2) and constant N application (CN: 240 kg N·hm^-2)) on leaf stay-green, photosynthetic characteristics, dry matter accumulation and system yield of maize and soybean leaves were studied by field positioning experiment.【Result】The maize yield increased with the increase of N application, and the soybean yield increased first and then decreased with the increase of N application; Under RN, the seed dry matter accumulation of IM was the largest, the total yield of maize-soybean intercropping system was the highest, and the system productivity index (SPI) was the largest too. Under intercropping, the leaf green period of each crop was longer, the photosynthetic characteristics were more stable than that of monoculture, and better than that of monoculture at seed formation stage; Under all N application levels, the percentage of green leaves under intercropping treatment was significantly higher than that under monoculture. The maximum green leaf attenuation rate of IM appeared 7 d, 5 d and 1d later than that of MM, respectively, while IS was 7 d, 0 d and 11 d later than SS, respectively. Compared with monoculture, the intercropping could significantly reduce the average attenuation rate of maize leaves, prolong the days of maximum attenuation rate and reduce the attenuation degree of green leaves. The photosynthetic rate of each crop was higher under intercropping than monoculture, and the reduced N application was higher than the constant N application. At R2 stage, the photochemical quenching coefficient (QP) under IM was 12.78% higher than that under MM, and the non-photochemical quenching coefficient (NPQ) was 21.30% lower; NPQ decreased with the increase of N application level, while the ratio of RN to NN decreased by 17.11%. The fluctuation range of SPAD value of intercropping was weaker than that of monoculture, and showed a stable upward trend. In maize R2 stage, IM was 34.52% higher than MM; In soybean R2 and R6 stage, IS was 10.39% and 29.48% higher than SS, respectively, and the SPAD value of RN was the highest. At R2 stage, IMRN was 17.46% higher than IMNN, and MMRN was 35.02% higher than MMNN; in soybean R6 stage, ISRN was 7.71% and 6.67% higher than that of ISNN and ISCN, and SSRN was 10.03% higher than that of SSCN.【Conclusion】Under reduced N application condition, the maize-soybean intercropping significantly prolonged the green holding period of leaves; After flowering, the photosynthetic rate of leaves, the function of PS Ⅱ photosynthetic mechanism and chlorophyll remained at a high level were more stable than that of monoculture, and the accumulation of seed dry matter was enhanced, which gave full play to the production potential of maize and increased the yield of soybean, so that the total yield of intercropping system was significantly increased.

The Possible Effects of Global Warming on Cropping Systems in China XIV. Distribution of High-Stable-Yield Zones and Agro-Meteorological Disasters of Soybean in Northeast China

GUO ShiBo, ZHANG FangLiang, ZHANG ZhenTao, ZHOU LiTao, ZHAO Jin, YANG XiaoGuang

Scientia Agricultura Sinica. 2022, 55(9): 1763-1780. doi:10.3864/j.issn.0578-1752.2022.09.006

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【Objective】 Soybean is an important oil-seeds crop, and Northeast China (NEC) is the main soybean productive region of China. It is important to investigate the distribution of zones both high yield and high yield stability of rain-fed potential yield and to identify the major agro-meteorological disasters, which could provide important scientific references for the reasonable layout of soybean.【Method】The year 1981 was taken as a time node wan divided the period 1961-2019 into two sub-periods (1961-1981 and 1981-2019). The well-calibrated DSSAT-CROPGRO-Soybean model was applied to simulate rain-fed soybean potential yield at each stations in possible planting area of NEC to clarify the distribution of zones both high yield and high yield stability. The distribution and temporal changes of cold damage and drought were identified in different high-yield and stable-yield zones. Coupled with statistical methods, the limitations of major agro-meteorological disasters on soybean high-stable-yield were further analyzed. 【Result】 (1) Compared with 1961 to 1980 (period Ⅰ), the soybean possible planting area was increased by 2.81×10⁶ hm² (3.57% of the whole land area in NEC). (2) Compared with periodⅠ, the percentage of high stability yield zones area of possible planting area increasing in periodⅡ. The percentage of high-stable zones area in possible planting area decreased from 17.67% to 17.11%, and the percentage of high-unstable yield zones area increased from 34.98% to 38.17%, while the percentage of low-stable area increased from 34.98% to 38.17% and the percentage of low-unstable zones area decreased from 18.58% to 18.49%. (3) During the study period, the frequency of cold damage during soybean growing season showed a trend of increasing and then decreasing. High-stable zones and high-unstable zones showed higher cold damage frequency, especially serious cold damage. The frequency of light drought and moderate drought increased, but the serious drought frequency decreased during the soybean growing season. (4) The yield changes of soybean in zones both high yield and high yield stability were negatively correlated with cold damage frequency, and yield variation changes of soybean were negatively correlated with both cold damage and drought frequencies. 【Conclusion】Under the background of climate warming, the soybean possible planting area expanded northward and westward in NEC, and the area increased. The area of high-unstable and low-stable zones increased, while the area of high-stable and low-unstable zones decreased. The main agro-meteorological disasters were different in zones both high yield and high yield stability. The frequency of cold damage in low yield zones was higher than that in high yield zones, while the frequency of drought in unstable yield zones was higher than stable yield zones. However, in areas with the change in high yield and high yield staiblity zones, the frequency of cold damage decreased and the frequency of drought increased. In summary, the cold damage was the main limiting factor in soybean yield increasing, while cold damage and drought were the main limiting factors in yield stability increasing.

The Role and Mechanism of Linoleyl Ethanolamide in Plant Resistance Against Botrytis cinerea in Tomato

SHAO ShuJun,HU ZhangJian,SHI Kai

Scientia Agricultura Sinica. 2022, 55(9): 1781-1789. doi:10.3864/j.issn.0578-1752.2022.09.007

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【Background】Gray mold caused by Botrytis cinerea is one of the important diseases of tomato and causes significant yield losses up to 30%-40%. Nowadays, chemical pesticide is usually used in tomato production, which is effective but increases the risk of food safety and results in environmental pollution. N-acylethanolamines (NAEs) are a kind of naturally lipid bioactive compounds in plants, which have been identified to have a variety of immune functions in mammals, however, its function and the underlying mechanism in plant immunity are still unclear.【Objective】The objective of this study is to investigate the effects of NAEs on tomato plant defense against B. cinerea infection, and to provide a basis for the development of green control technology of tomato gray mold.【Method】The B. cinerea was cultured in medium containing NAE 18:0, NAE 18:2, NAE 22:5, respectively, to evaluate their effects on B. cinerea growth. Tomato ‘Moneymaker’ plants were infected by B. cinerea with or without exogenous NAE 18:0, NAE 18:2, NAE 22:5, and disease index and fluorescence parameters of tomato leaves were measured. qRT-PCR was used to analyze the relative gene expression of B. cinerea Actin in tomato leaves that infected by B. cinerea with or without NAE 18:2 treatment. Transcript abundance of defense-related genes (e.g. PI I, PR-1, NPR1, Nr, ACO1, PYR1a), and contents of plant hormones (e.g. JA, SA, ETH, ABA, IAA) were measured. Fluorescence parameters of tomato leaves and the relative gene expression of B. cinerea Actin were analyzed in ethylene-insensitive mutant infected by B. cinerea with NAE 18:2.【Result】The growth of B. cinerea was not affected by exogenous NAEs treatment during in vitro culture. Exogenous application of NAEs could significantly improve the resistance of tomato plants to B. cinerea, and alleviate the decrease of photosystem II photochemical efficiency (Φ_PSII) caused by B. cinerea infection. NAE 18:2 had the best effect on tomato plant defense against B. cinerea among the NAEs, which obviously reduced the disease index and the Actin transcript level of B. cinerea by 60%. The expression levels of PI I, PR-1, NPR1, Nr and ACO1 could be induced by B. cinerea infection but not by NAE 18:2 treament. The expression levels of PI I, Nr and ACO1 were up-regulated when plants were pre-treated by NAE 18:2 before B. cinerea infection, and the expression level of ACO1 was the highest. Compared to the control, the contents of SA, JA, IAA and ETH in the leaves were increased significantly after B. cinerea infection, while only the contents of ETH were further increased when pre-treated by NAE 18:2. Moreover, exogenous NAE 18:2 pre-treatment could not improve the defense against B. cinerea in the ETH-insensitive mutant nr.【Conclusion】Exogenous NAE18:2 treatment can increase leaf photosynthesis, transcript abundance of defense-related genes, and the content of plant hormone ETH. It induce the resistance of tomato plants to gray mold, which may depend on the ETH signaling pathway.

Synthesis and Bioactivity of Sex Pheromone Analogues of Protoschinia scutosa

LI Hui,YIN ShiCai,GUO ZongXiang,MA HaoYun,REN ZiQi,SHE DongMei,MEI XiangDong,NING Jun

Scientia Agricultura Sinica. 2022, 55(9): 1790-1799. doi:10.3864/j.issn.0578-1752.2022.09.008

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【Background】In recent years, with the significant increase in the area planted with the newly introduced crop quinoa, Protoschinia scutosa, which prefers quinoa, has gradually increased and become a major pest of quinoa, posing a potential threat to the production of agriculture in China.【Objective】The objective of this study is to synthesize two different types of analogues modified with the polar group of the sex pheromone (Z)-hexadec-11-en-1-yl acetate (Z11-16:Ac), as the parent structure, and determine the biological activity, providing a theoretical basis for using sex pheromones and analogues for the green control of P. scutosa.【Method】The sex pheromone precursor (Z)-hexadec-11-en-1-ol (Z11-16:OH) was used as the raw material, and triethylamine was used as the acid-binding agent (4-dimethylaminopyridine was added as the catalyst for the reaction with the anhydride), and the esterification reactions with 2-bromopropionyl chloride, 2-chloroacetyl chloride, difluoroacetic anhydride, trifluoroacetic anhydride, methacryloyl chloride, and 2-butenoyl chloride were carried out respectively, the halogen atom modified analogues (K1-K4) and carbon chain end double bond modified analogues (K5-K6) were synthesized, separated and purified by column chromatography. Different concentrations (0.001, 0.01, 0.1, 1, 10, 100 μg·μL^-1) of sex pheromone analogues (K1-K6) were prepared by gradient dilution. The EAG response values of analogues K1-K6 were measured by direct stimulation on the male tentacles of P. scutosa using different concentrations of analogues. Sex pheromone analogue trap lures were prepared by adding different doses of analogues to the sex pheromone. The field trapping trials were conducted in the summer of 2021 in Yanqing, Beijing, and the analogue activity was analyzed by counting the male moth in the traps.【Result】As the stimulation concentration increased from 0.001 to 100 μg·μL^-1, the electrophysiological response values of male tentacles to analogues K1 to K5 showed a gradual increase trend. Male tentacles responded most strongly to the compound (Z)-hexadec-11-en-1-yl 2-bromopropanoate (K1), with a response value of 1.34 mV at 100 μg·μL^-1. The EAG activities of analogues K5 and K6 were relatively poor, with EAG response values of 0.67 and 0.57 mV at 100 μg·μL^-1, respectively. All the six analogues synthesized showed good biological activity in field trapping experiments. K5 had the highest average trapped number of 35.00 heads at an additive level of 100 μg, which was significantly higher than the sex pheromone control. The average number of trapped adults of analogue K1 was 32.33 heads at an additive level of 10 μg during the whole period (15 d) of field trapping tests, which was significantly higher than the sex pheromone control.【Conclusion】The analogues K1 and K5 showed potentiation activity and can be used as potentiators of sex pheromones as verified by EAG and field trials. The addition of different doses of analogues K2, K3, K4 and K6 exhibited comparable activity to sex pheromones. This study provides a scientific and theoretical basis for the green control of P. scutosa.

Optimal Management of Phosphorus Fertilization Based on the Yield and Grain Manganese Concentration of Wheat

WANG HaoLin,MA Yue,LI YongHua,LI Chao,ZHAO MingQin,YUAN AiJing,QIU WeiHong,HE Gang,SHI Mei,WANG ZhaoHui

Scientia Agricultura Sinica. 2022, 55(9): 1800-1810. doi:10.3864/j.issn.0578-1752.2022.09.009

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【Objective】To keep the manganese (Mn) nutritional balance of wheat grains and ensure the safety, yield and quality in wheat production region of eight provinces in Northern China, the changes of concentration and accumulation of Mn in wheat grains and grains yield at different levels of soil available phosphorus (P) and different treatments of P fertilization were investigated. 【Method】During 2018-2019, 34-site field experiments were conducted with three P treatments, including farmers’ fertilizer application (FF), recommended fertilizer application based on soil nitrate and P test (RF), and recommended fertilizer application without P (RF-P). The wheat yield, the concentration of Mn in wheat grain were tested, and the effects of P fertilization on wheat yield and the Mn concentration of grain at different levels of soil available P were studied. 【Result】In wheat production region of eight provinces in Northern China, the average wheat yield was 6 066 kg·hm^-2, and the average concentration of Mn in grains was 42 mg·kg^-1. Those test sites with concentration of Mn in grains less than 32 mg·kg^-1 or higher than 44 mg·kg^-1, accounted for 8.8% and 36.8%, respectively, which suggested that the problem of high concentration of Mn in grains should be paid attention to. With the increase of soil available P, both wheat yield and concentration of Mn in grains increased significantly. The wheat yield reached to the highest when the available P was in the range of 20-30 mg·kg^-1, while the concentration of Mn in grains reached to the highest when the available P>40 mg·kg^-1. P fertilizer was reduced with an average of 45.4% under the RF treatment. However, the wheat yields of RF and FF were 6 358 and 6 222 kg·hm^-2, respectively, and the concentration of Mn in grains were 42.8 and 43.6 mg·kg^-1, respectively, which showed no significant difference. At different levels of soil available P, RF could maintain a high wheat yield. When soil available P<10 mg·kg^-1, RF-P reduced not only the concentration of Mn in grains, but also reduced the wheat yield, while RF only reduced the concentration of Mn in grains. RF did not reduce the concentration of Mn in grains under other levels of soil available P. In addition, the concentration of diethylene triamine pentaacetic acid-manganese (DTPA-Mn) in soil increased following the increasing of soil available P. Furthermore, the concentration of Mn in grains were positively correlated with the concentration of soil DTPA-Mn. 【Conclusion】In wheat production region of eight provinces in Northern China, the soil available P should be maintained in the range of 20-30 mg·kg^-1 to achieve high wheat yield and suitable concentration of Mn in grains. The use of RF technology would not reduce the wheat yield. RF-P reduced the concentration of Mn in grains when the soil available P<10 mg·kg^-1, but there was a risk of reducing the wheat yield.

Spatiotemporal Patterns in Nitrogen Response Efficiency of Aboveground Productivity Across China’s Grasslands

HOU JiangJiang,WANG JinZhou,SUN Ping,ZHU WenYan,XU Jing,LU ChangAi

Scientia Agricultura Sinica. 2022, 55(9): 1811-1821. doi:10.3864/j.issn.0578-1752.2022.09.010

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【Objective】This study was to investigate the temporal and spatial patterns of nitrogen (N) limitation (indicated by N response efficiency) on aboveground productivity of China’s grasslands, which was important for the adoptive management and accurately simulating of ecosystem N cycling under global environmental change. 【Method】A meta-analysis was performed to investigate N response ratio (lnRR) and N response efficiency (lnRR/N, the ratio of lnRR to N addition rate) of aboveground productivity across China’s grasslands. All data (423 groups) were collected from in-situ N addition experiments published over 1980-2020. Linear, double-linear and multi-step regressions were explored to estimate the spatial and temporal dynamics of lnRR/N and its driving factors. 【Result】In general, lnRR increased with N addition rates and saturated at (21.1±5.5) g N·m^-2·a^-1 (mean±95%CI) with the maximum of 0.60±0.08. lnRR/N was, on average, 0.043±0.004, i.e., aboveground productivity could increase by (4.36±0.38) % per unit N addition (1 g N·m^-2·a^-1). lnRR/N also differed significantly among grassland types, N addition rates, experimental durations, and years. Over the past four decades, lnRR/N significantly decreased, with a much (1.5-1.7 times) faster rate in the warmer (MAT>4.5℃) and wetter (MAP>450 mm) climatic regions than that in the cooler (MAT≤4.5℃) and drier (MAP≤450 mm) climatic regions. Regression analyses revealed that the spatial variation of lnRR/N was mainly driving by annual precipitation and soil fertility (i.e., soil N content). In general, lnRR/N increased along with MAP and decreased with soil N content. However, the driving factors varied by climatic regions, with both MAP and soil N content in the wetter regions, MAP in the drier regions and MAT in the warmer regions. 【Conclusion】 The aboveground productivity in China’s grasslands was still limited by N, but the extent of N limitation or N response efficiency decreased over time, especially in those wetter and warmer climatic regions. To accurately predict the response of grassland ecosystem to the ongoing global environmental change, the studies should pay more attention to the spatial and temporal shifting in driving factors for plant productivity.

Cloning of Alternative Splice Variants of LsPHYB in Lettuce and Its Expression Patterns Under Heat Stress

SUI XinYi,ZHAO XiaoGang,CHEN PengYu,LI YaLing,WEN XiangZhen

Scientia Agricultura Sinica. 2022, 55(9): 1822-1830. doi:10.3864/j.issn.0578-1752.2022.09.011

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【Objective】Phytochrome B (PHYB) is a receptor for both light and temperature. In this study, the biological functions of alternative splice variants of LsPHYB in lettuce in response to high environmental temperatures were investigated through cloning them and analyzing their expression patterns, so as to provide a theoretical basis for breeding heat-tolerant lettuce.【Method】The cDNA sequences of LsPHYB were searched from the genomic database of lettuce by bioinformatics method. The cloned LsPHYB1, LsPHYB2 and LsPHYB3 were subjected to multi-sequence alignment, alternative splice variants form analysis and phylogenetic analysis. Protein properties, such as molecular weight, theoretical isoelectric point, hydrophilicity and hydrophobicity, were predicted by online software. Secondary structure, tertiary structure and conserved domains were analyzed by bioinformatics software. Three alternative splice variants were characterized for expression after high temperature treatment by RT-PCR. 【Result】There were three alternative splice variants of LsPHYB obtained by cloning, namely LsPHYB1, LsPHYB2 and LsPHYB3, with their CDS lengths of 3 509, 3 877 and 2 690 bp, which encoded 1 094, 960 and 853 amino acids, respectively. Alternative splice forms of LsPHYB1 were alternative 3′ splice site and skipped exon. Alternative splice forms of LsPHYB2 were alternative polyA and retain intron. An alternative splice form of LsPHYB3 was skipped exon. Conserved structural domain analysis showed that the N-terminal of PHYB2 lacked the PAS and PHY domains. The N-terminal of PHYB3 lacked the PAS and PHY domains, and its C-terminal lacked the HisKA domain. Phylogenetic analysis showed that three alternative splice variants were clustered into a clade. qRT-PCR analysis showed that the expression of LsPHYB3 was the highest at the first day of high temperature treatment; LsPHYB2 had higher expression than LsPHYB1 and LsPHYB3 at days 5-9 of high temperature treatment; at day 11 of high temperature treatment, the expression of LsPHYB1 was higher than that of LsPHYB2 and LsPHYB3. The three alternative splice variants peaked at different times during the 11 days of high temperature treatment. 【Conclusion】There were three alternative transcript variants of LsPHYB, named LsPHYB1, LsPHYB2 and LsPHYB3. LsPHYB3 expression was the highest in the early stage of high temperature treatment, LsPHYB2 in the middle stage, and LsPHYB1 in the late stage, suggesting that the three alternative splice variants were functionally differentiated in response to high temperature stress.

The Albino Mechanism of a New High Theanine Tea Cultivar Fuhuang 1

LIN XinYing,WANG PengJie,YANG RuXing,ZHENG YuCheng,CHEN XiaoMin,ZHANG Lei,SHAO ShuXian,YE NaiXing

Scientia Agricultura Sinica. 2022, 55(9): 1831-1845. doi:10.3864/j.issn.0578-1752.2022.09.012

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【Objective】 The aim of this study was to analyze metabolic and transcriptional mechanism of etiolated variation in tea plant, and to explore the albino mutation of a new homotheanine tea cultivar Fuhuang 1 and its formation mechanism of homotheanine. 【Method】 The experimental materials were Fuan Dabaicha and Fuhuang 1. The combined analysis of ultramicroelectron microscopy, widely targeted metabolism, targeted metabolomics and transcriptomics was used to clarify the pigments, metabolism and transcriptome data related to the albino tea.【Result】The results of ultrastructure showed that the chloroplast thylakoids of Fuhuang 1 was filamentous, with irregular arrangement of basal granule lamellae and many abnormal vesicles. Chlorophyll a and chlorophyll b content, chlorophyll a/b ratio, SGR gene expression and light-harvesting chlorophyll a/b protein (LHC) expression were significantly reduced in yellow leaves. Although the total content of carotenoids did not change significantly, the content of each component changed significantly. Zeaxanthin was the only component with significant increase, and the expression of its regulatory gene VDE was significantly up-regulated, and the content of other components decreased. Compared with Fuan Dabaicha, 680 differentially expressed genes (DEGs) and 57 significantly changed metabolites (SCMs) were identified in Fuhuang 1. KEGG enrichment analysis showed that SCMs and DEGs were significantly enriched in pathways related to amino acid biosynthesis, glutathione metabolism and TCA cycle. In addition, the pathways related to carbon and nitrogen metabolism were also activated. A total of 19 free amino acids were identified by targeted determination. The free amino acid content of Fuhuang 1 was 97.13 mg∙g^-1, which was significantly higher than that of Fuan Dabaicha. Theanine content was 66.90 mg∙g^-1, accounting for 68.89% of amino acids, while the content of arginine was 8.46 mg∙g^-1, which was 56.4 times as more as that of Fuan Dabaicha. The expression levels of GOGAT and GLU, which regulated amino acid synthesis, were up-regulated in Fuhuang 1 by 1.17 and 3.17 times higher than that in Fuan Dabaicha. 【Conclusion】 The leaf color of Fuhuang 1 was mainly influenced by chlorophyll, carotenoids and flavonoids. The combined action of SGR and the four LHCs might also affect chloroplast biogenesis to regulate leaf color. The content of theanine in Fuhuang 1 was significantly higher than that in Fuan Dabaicha. The main reason was that the up-regulation of ubiquitination-related protein hydrolase activity and the enhanced protein degradation ability. The biosynthesis of chlorophyll and other nitrogen-containing molecules was reduced, the carbon skeleton in yellow leaves was lacking, amino and nitrogen resources were stored more effectively. The activation of nitrogen metabolism associated with amino acid synthesis led to the accumulation of glutamate, one of the precursors of theanine synthesis. Theanine accumulated significantly in yellow leaves.

Analysis of Lipolysis and Oxidation Ability of Fermentation Strains in Sterilized Pork Pulp

WANG Ji,ZHANG Xin,HU JingRong,YU ZhiHui,ZHU YingChun

Scientia Agricultura Sinica. 2022, 55(9): 1846-1858. doi:10.3864/j.issn.0578-1752.2022.09.013

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【Objective】This study aimed to screen out the fermentation strains with high lipolysis and antioxidant ability, which provided the theoretical basis for development of new starter cultures. 【Method】The sterilized pork pulp was inoculated with Staphylococcus xylosus YSZ11, Staphylococcus xylosus YCC3, Staphylococcus saprophyticus YCC2, Macrococcus caseolyticus YZC2, and Macrococcus caseolyticus YZC3, respectively. The sterilized pork pulp without inoculation of strains was used as the control group. The changes of pH, peroxide value (POV), thiobarbituric acid reactive substances (TBARS), lipase activity, lipid composition and free fatty acid content were measured after 4 d fermentation.【Result】5 fermentation strains could reduce the pH value of the pork pulp. The POV and TBARS values were 2.51-2.96 mmol∙kg^-1 and 0.21-0.24 mg/100g in pork pulp inoculated with fermentation strains, which were significantly lower (P<0.05) than that in CK group. The activity of neutral lipase, acid lipase and phospholipase were all detected in the groups inoculated with the fermentation strains, while higher activity of acid lipase and phospholipase were found in the groups inoculated with the fermentation strains than that of neutral lipase. After 4 d of fermentation, the phospholipid content decreased significantly (P<0.05), while the free fatty acid content increased by 21.1%-73.7% in the groups inoculated with the fermentation strains. At the same time, the amount of saturated fatty acids decreased significantly, while the amount of unsaturated fatty acids especially the content of palmitoleic (C_16:1), oleic (C_18:1) and linoleic acids (C_18:2) increased significantly, and the linolenic acid (C_18:3) was also detected. 【Conclusion】This study showed that five fermentation strains could inhibit lipid oxidation, and promoted lipid hydrolysis by secreting lipase, which led to the increase of the content of free fatty acids, especially unsaturated fatty acids. Staphylococcus saprophyticus YCC2 and Staphylococcus xylosus YCC3 had better lipolysis and antioxidant ability, and showed a more prominent promoting effect on improving the quality of fermented meat products.

Evolutionary Relationship Between Transposable Elements and Tandem Repeats in Bovinae Species

ZHANG Rui,ZHANG TianLiu,FAN TingTing,ZHU Bo,ZHANG LuPei,XU LingYang,GAO HuiJiang,LI JunYa,CHEN Yan,GAO Xue

Scientia Agricultura Sinica. 2022, 55(9): 1859-1867. doi:10.3864/j.issn.0578-1752.2022.09.014

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【Objective】The repetitive sequence is an important part of eukaryotic genomes and plays an important role in species evolution, gene genetic variation, and transcriptional regulation. The purpose of this study was to reveal the characteristics of tandem repeats in bovinae by investigating the evolutionary relationship between transposons and tandem repeats, so as to provide the theoretical support for the study of tandem repeats in bovinae. 【Method】 In this paper, the six genomes were selected as research object, including Bos taurus, Bos indicus, Bos mutus, Bubalus bubalis, Bison bison and Bos frontalis. The transposable elements and tandem repeats in six genomes was identified through TRF and RepeatMasker software. Meanwhile, the sequence similarity between the two types of tandem repeats was analyzed by BLAST, and single-locus tandem repeats (single-locus TRs, mlTRs), multiple-locus tandem repeats (multiple-locus TRs, mlTRs) and the characteristics of tandem repeat for the transposable elements were investigated too. 【Result】 (1) In the six bovinae genomes, the percent of tandem repeats in Bos taurus was the highest (49.13%), followed by Bos frontalis (46.82%), Bubalus bubalis (46.23%), Bos indicus (42.70%), Bos mutus (42.53%), and Bison bison (42.36%), in which the content of transposable elements in the genome ranged from 40.57%-45.71%, and was higher than that of tandem repeats (1.50%-3.42%). (2) In the tandem repeats, the proportion of mlTRs (76%-99%) was significantly higher than that of slTRs(1%-24%), indicating that the mlTRs was the main component of tandem repeats in six bovinae species. (3) The proportion of TE-derived tandem repeats was 43% to 84%, among them mutiple-locus tandem repeats could reach up to 94%. (4) The analysis of TRs-related transposable elements and their activity showed that these transposable elements were mainly from non-Long Terminal Repeats (non-LTR, including SINE and LINE) and long interspersed nuclear element (LINE), among which SINE/core-RTE (mainly BOV-A2) had the highest number (14 423-24 193) and relative number (4.06%-6.77%), which was considered to be the youngest and the most dynamic transposable elements. (5) The study on transposable elements of tandem repeats’ characteristics indicated that BovB and L1_BT contained a large number of tandem repeats in 0-600 bp and 1 500 bp-2 700 bp, respectively, which were more than 93% and 87% consistent with the consensus sequence, respectively, and the sequences were located in the non-coding region. 【Conclusion】 The repetitive sequence had similar distribution characteristics, non-LTR was an important source of TRs-related TEs, and SINE/Core-RTE(mainly BOV-A2) was the youngest and most dynamic transposable elements. At the same time, the tandem repeats could be used as internal structure component of transposable elements, indicating that tandem repeats and transposable elements interacted with each other in the process of genome evolution.

miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep

LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing

Scientia Agricultura Sinica. 2022, 55(9): 1868-1876. doi:10.3864/j.issn.0578-1752.2022.09.015

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【Objective】BCL2L11 could promote apoptosis in mammals and is involved in the development of tissues and organs related to reproductive traits and in disease treatment. The aim of this study was to explore the effect of miR-221-3p on the regulation of granulosa cell apoptosis in Small-Tail Han sheep by targeting BCL2L11, so as to provide evidence for further study of the regulation of BCL2L11 in granulosa cell apoptosis and atresia of follicles.【Method】Based on the whole transcriptome sequencing analysis of the ovarian tissue of the previous study in our group, the differentially expressed gene BCL2L11 and its regulatory element miR-221-3p were obtained in this study. Analysis of the expression of BCL2L11 in different tissues of the Small-Tail Han sheep by using semi-quantitative and tissue fluorescence quantification (RT-qPCR). The expression of BCL2L11 and miRNA-221-3p was identified in the ovarian tissues of the Small-Tail Han sheep in the follicular and luteal phase by RT-qPCR. The miR-221-3p mimic, BCL2L11 wild-type and BCL2L11 mutant-type were co-transfected in HEK293T cells with negative control, the dual luciferase reporter gene detection system was used to determine the targeting relationship between miR-221-3p and BCL2L11. The miR-221-3p mimic and negative control were transfected into ovarian granulosa cells to achieve the overexpression of miR-221-3p. RT-qPCR was used to detect the effect of miR-221-3p on the expression levels of BCL2L11 and the marker genes of the apoptosis of ovarian granulosa cell gene XIAP and Fas at the mRNA level. At the same time, the changes in proliferation of granulocytes in the miR-221-3p overexpression and negative control groups were also analyzed using the EdU assay. 【Result】The results showed that the expression of BCL2L11 in ovarian tissue was the highest, followed by spleen and lung tissues. RT-qPCR results showed that the expression of miR-221-3p and BCL2L11 was significantly different in the ovarian tissues of Small-Tail Han sheep between follicular and luteal phases. The expression of miR-221-3p was higher in follicular than that in luteal phase ovaries, whereas BCL2L11 was less expressed in follicular than that in luteal phase ovaries, which showed the phenomenon of a negative regulation. Dual luciferase reporter analysis showed that overexpression of miR-221-3p significantly inhibited the activity of BCL2L11 3’UTR vector (P<0.05). The overexpression of miR-221-3p significantly reduced the mRNA level expression of target gene BCL2L11, while follicular granulosa cell apoptosis expression of marker genes XIAP and Fas were also significantly reduced (P<0.05). Analysis of the EdU assay showed that the proliferation rate of granulosa cells overexpressing miR-221-3p was 18.9%, which was significantly higher than that of the negative control group at 10.43% (P<0.01). 【Conclusion】BCL2L11 and miR-221-3p were important genes and regulatory elements that regulate ovarian development in Small-Tail Han sheep. BCL2L11 was one of the target genes of miR-221-3p, and overexpression of miR-221-3p could inhibit granulosa cell apoptosis by target BCL2L11.

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