Scientia Agricultura Sinica

Comprehensive Evaluation and Breeding Evolution of Early Maturing Upland Cotton Varieties in the Northwest Inland of China

WANG CaiXiang,YUAN WenMin,LIU JuanJuan,XIE XiaoYu,MA Qi,JU JiSheng,CHEN Da,WANG Ning,FENG KeYun,SU JunJi

Scientia Agricultura Sinica. 2023, 56(1): 1-16. doi:10.3864/j.issn.0578-1752.2023.01.001

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【Objective】This paper evaluated comprehensively early maturity upland cotton varieties in northwest inland cotton-growing region of China, and uncovered the evolution rules of the main traits from the breeding cultivars in the region. It provides a useful reference for recommendation and breeding of main cotton varieties in northwest inland region. 【Method】A total of 110 early-maturing upland cotton varieties which were approved in northwest inland cotton-growing region from 1988 to 2021 were applied, and phenotypic identification of 12 main traits related to early maturity, yield, fiber quality and plant architecture was carried out in Dunhuang, Gansu province and Shihezi, Xinjiang Production and Construction Corps in 2020 and 2021, respectively. Clustering analysis, correlation analysis and principal component analysis were respectively used to evaluate the phenotypic characteristics of the cultivars, and carry out the breeding evolution analysis of their main traits. 【Result】The phenotype value of 12 main traits had large distribution range from 110 early-maturing upland cotton varieties in northwest inland cotton-growing region of China. The variation coefficient in descending order is: fruit branch length (FBL), boll number per plant (BN), boll opening rate (BOR), boll weight (BW), fiber micronaire (FM), fiber strength (FS), lint percentage (LP), fiber length (FL), flowering time (FT), fruit branch angle (FBA), fiber elongation (FE), and fiber uniformity (FU). By cluster analysis, these cultivars were divided into four groups: Ⅰ, Ⅱ, Ⅲ and Ⅳ, which were the population of high fiber quality, early maturity, loose plant type and high yield, respectively. Correlation analysis showed that early maturity was negatively correlated with yield and fiber quality related to traits, while the yield related to traits were positively correlated with the fiber quality traits, and most of them reached significant or extremely significant level. The 12 main traits were simplified into four principal components which had cumulative contribution rate of 66.92%. Based on the comprehensive evaluation results of these varieties by principal components, an excellent cultivar Zhongmian 113 ranked the first in the 110 early-maturing upland cotton varieties with a score of 0.67, which was characterized by early maturity, high yield and excellent fiber quality, and relatively loose plant type. Additionally, it was found that the early maturity character of cotton varieties approved in Gansu province was significantly better than that of varieties approved in northwest inland region and Xinjiang province, and the traits related to yield and fiber quality of cotton varieties approved in Gansu province were significantly lower than those of varieties approved in northwest inland region and Xinjiang province. During the breeding evolution of early maturing upland cotton varieties in northwest of China for more than 30 years, most of the traits related to yield and fiber quality showed significant upward trends, and the plant type was becoming more compact. BN was negatively correlated with BW, LP, FL and FS in early cultivars, while BN was positively correlated with BW, LP, FL and FS in recent cultivars. 【Conclusion】In the process of breeding and improvement of early maturing upland cotton varieties in northwest of China, the main traits related to yield and fiber quality had wide distribution, and they had significant improvement trends year by year. In the recently developed varieties, BN, FL and FS had been improved cooperatively.

Genome-Wide Association Analysis of the Changes in Oil Content and Protein Content Under Drought Stress in Brassica napus L.

HU Sheng,LI YangYang,TANG ZhangLin,LI JiaNa,QU CunMin,LIU LieZhao

Scientia Agricultura Sinica. 2023, 56(1): 17-30. doi:10.3864/j.issn.0578-1752.2023.01.002

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【Objective】 Drought is one of the most adverse abiotic stresses that hinder growth and development of Brassica napus L., threatening its yield and quality. In this study, we identified the candidate genes that influence oil and protein content changes according to the results of the genome wide association mapping (GWAS) and differentially expressed genes (DEGs) under drought stress in B. napus. 【Method】 A natural population comprising 183 B. napus lines under drought stress across two years was genotyped with a Brassica 60K Illumina Infinium SNP array to perform GWAS. Combined with drought stressed seed transcriptome data from different treatment periods, the candidate genes related to the changes in oil and protein contents under drought stress were identified. 【Result】 The analysis showed that the best model for phenotypic data analysis in 2 years was mainly the Q or naïve model, and a total of 38 significant associated loci were detected (P<1/31597 or P<1/31278). By integrating GWAS and transcriptome results, 256 common genes were selected, and by comparing the database information of B. napus and Arabidopsis thaliana, 25 related candidate genes were identified. These genes, including transcription factors (e.g., bZIP transcription factor GBF6, TALE transcription factor ATH1, MYB-like Domain transcription factor MYBD, NAC transcription factor ANAC029, ERF transcription factor ERF111), related kinases (e.g., PIP5K1, PFK7), related proteins (e.g., seed stores protein CRU3, chloroplast protein DG238 and CP12, stress-responsive proteins HUP26 and M10), are involved in multiple biological processes such as photosynthetic reaction, substance transport, and stress response. For instance, oil-related protein kinase CIPK9, ABCA9 transporter, storage-related protein CRU3, may provide basis for explaining the changes in oil and protein contents of B. napus under drought stress. 【Conclusion】 The 25 identified candidate genes may affect the accumulation of protein and oil contents under drought stress during the seed development in B. napus.

Effects of Sowing Date on Yield of Double Cropping Rice and Utilization Efficiency of Light and Heat Energy in Hunan Province

ZHANG Wei, YAN LingLing, FU ZhiQiang, XU Ying, GUO HuiJuan, ZHOU MengYao, LONG Pan

Scientia Agricultura Sinica. 2023, 56(1): 31-45. doi:10.3864/j.issn.0578-1752.2023.01.003

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【Objective】By comparing the effects of different sowing dates on yield of double cropping rice and utilization efficiency of light and heat energy, and analyzing the sensitivity of different varieties to sowing date and light and heat resource efficiency, the best sowing date was investigated, which was conducive to double-cropping rice yield and resource utilization efficiency in Hunan province. 【Method】Using the main double-cropping rice varieties approved in Hunan province, the main double-cropping rice producing area, as the test materials, three sowing dates for early and late rice were set up for field experiments from 2018 to 2019. The rice growth period, yield and yield components as well as meteorological data were analyzed to compare the yield and the utilization efficiency of light and heat resources of each variety under different sowing dates. 【Result】With the delay of the sowing date, the early and late rice shortened the growth period by shortening the days of the sowing-ear start stage, while the yield of early rice showed an increasing trend. Late-maturing rice responded faster to late sowing than middle-maturing late rice; In 2018, with the delay of sowing date, the heat energy utilization rate of early rice decreased by 2.73% and 4.45% respectively, the light energy utilization rate decreased by 1.28% and -1.77% respectively, the heat energy utilization rate of late rice decreased by -2.64% and 8.15% respectively, and the light energy utilization rate decreased by 2.14% and 13.25% respectively. In 2019, with the postponement of sowing date, the heat energy utilization rate of early rice increased by 0.36% and 10.36% respectively, and the light energy utilization rate increased by 1.53% and 12.52% respectively. The heat energy utilization rate of late rice decreased by -0.72% and 6.09% respectively, and the light energy utilization rate decreased by 0.58% and 6.21% respectively. 【Conclusion】The sowing date had a significant impact on the utilization efficiency of light and heat resources of double-cropping rice. It was appropriate to delay the sowing date of early rice and advance the sowing date of late rice to improve the efficiency of light and heat resources. The comprehensive efficiency of the combination of the second sowing date of early rice+the second sowing date of late rice for double cropping rice is higher, and the combination of late-maturing early rice and medium-maturing late rice combination had higher comprehensive efficiency.

Effects of Meteorological Factors on Quality of Late Japonica Rice During Late Season Grain Filling Stage Under ‘Early Indica and Late Japonica’ Cultivation Pattern in Southern China

FENG XiangQian, YIN Min, WANG MengJia, MA HengYu, CHU Guang, LIU YuanHui, XU ChunMei, ZHANG XiuFu, ZHANG YunBo, WANG DanYing, CHEN Song

Scientia Agricultura Sinica. 2023, 56(1): 46-63. doi:10.3864/j.issn.0578-1752.2023.01.004

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【Objective】The responses of the rice grain qualities to the climate factors during the grain filling period were studied to facilitate the variety screening and the agronomic practices optimization for japonica rice during the late rice season in southern rice region in China. 【Method】 To evaluate the effects of different climate factors during the grain filling period on the qualities of late japonica rice, the field experiment was conducted in Fuyang and Wenzhou, Zhejiang province, in 2018, using three late indica rice as control varieties and 20 japonica rice (including 10 inbred japonica rice, 3 japonica hybrids and 7 indica-japonica hybrids) as evaluating varieties. 【Result】(1) Based on the results of clustering with grain qualities, the late indica rice varieties were classified as an unique category due to its highest aspect ratio (3.18) and high amylose content (19.40%). Compared with the late indica rice, the most of hybrid varieties had greater brown rice rate (4.31%-5.28%), milled rice rate (6.51%-9.33%), head rice rate (25.83%- 28.34%), gel consistency (1.81%-4.27%), alkali spreading value (11.62%-50.85%), and tasted value (2.31%-2.85%), with lower amylose content (20.98%-28.14%) and protein content (1.16%-14.85%), showing obvious improvement of rice quality. Whereas, the rice quality performance response to late season were differentiated within inbred japonica varieties, and some inbred japonica rice varieties originating from southern Jiangsu and Jiaxing (4 varieties) were similar to the those in the hybrid-japonica category, while the rest inbred japonica varieties from Jiangsu and Shanghai (6 varieties) belonged to another category due to their relatively poor rice quality performances (high chalkiness, chalky grain rate and protein content) in the late season. (2) The rice grain qualities were closely related to climate factors during grain filling period of late rice season. The stage of 10-20 days after full heading was identified as the sensitive period of climate factors on rice qualities. The brown rice rate was negatively correlated with daily solar radiation (R: -0.40--0.19, P<0.05) and daily temperature difference (R: -0.45--0.28, P<0.05), and positively correlated with daily minimum temperature (R: 0.24-0.53, P<0.05) and precipitation (R: 0.38-0.45, P<0.05). The chalkiness rate and chalkiness grain rate were significantly increased with the rainfall and night temperature (R: -0.37--0.16, P<0.05; R: -0.43--0.12, P<0.05), resulting in reducing rice appearance quality. Meanwhile, the rainfall and night temperature were negatively correlated with rice tasted value (R: -0.37--0.16, P<0.05; R: -0.43--0.12, P<0.05). The daily maximum temperature at grain filling stage was positively correlated with rice tasted value (R=0.37, P<0.05), while the daily minimum temperature was positively correlated with rice protein content (R=0.19, P<0.05), chalkiness (R=0.16, P<0.05), and chalkiness grain percentage (R=0.12, P<0.05). 【Conclusion】 The stage of 10-20 days after full heading was the key period for affecting rice grain quality by climate factor, and the improvement of rice qualities for late japonica varieties in southern rice region should focus on the integration of high-quality japonica genes with an indica gene of local adaptation, and the choice of hybrid rice (including indica-japonica hybrids and japonica hybrids) was more reliable and convenient than inbred japonica rice. The breeding of inbred japonica varieties should consider their ecological adaptability in combined with local climatic conditions.

Identification of Pathogenic Fusarium spp. Causing Maize Ear Rot and Susceptibility of Some Strains to Fungicides in Jilin Province

CHAI HaiYan,JIA Jiao,BAI Xue,MENG LingMin,ZHANG Wei,JIN Rong,WU HongBin,SU QianFu

Scientia Agricultura Sinica. 2023, 56(1): 64-78. doi:10.3864/j.issn.0578-1752.2023.01.005

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【Objective】The objective of this study is to clarify the population distribution of Fusarium spp. of maize ear rot in Jilin Province and the inhibitory effect of fungicides on the growth of Fusarium mycelium, and to provide a basis for the targeted control of maize ear rot. 【Method】149 samples of maize ear rot collected from 36 cities and counties in Jilin Province in 2020 were isolated and identified by tissue isolation and molecular biology methods. The specific toxin synthesis primers of related genes were synthesized using Fusarium graminearum species complex (FGSC) toxin. The toxigenic chemotypes were detected, and the pathogenicity of some FGSC was determined. The inhibitory effect of 7 fungicides on FGSC was determined by the mycelial growth rate method. 【Result】A total of 233 Fusarium strains were isolated, belonging to 4 Fusarium complex species, including 9 Fusarium species, which were F. verticillioides, F. boothii, F. graminearum, F. proliferatum, F. asiaticum, F. chlamydosporum, F. fujikuroi, F. equiseti and F. subglutinans. The isolation frequencies were 33.05%, 26.18%, 25.32%, 12.45%, 0.86%, 0.86%, 0.43%, 0.43% and 0.43%, respectively. The isolate frequency of FGSC was the highest, which was 52.36%, and it was the dominant pathogen of maize ear rot in Jilin Province. The proportions of F. boothii, F. graminearum and F. asiaticum in FGSC were 50.00%, 48.36% and 1.46%, respectively. The phylogenetic tree showed that the interspecific and intraspecific genetic diversity of FGSC was rich. The results of pathogenicity assay showed that 52.73% of FGSC were medium pathogenic strains. F. graminearum isolated from the main maize producing areas in the east had the strongest pathogenicity. Toxigenic chemotype detection showed that F. asiaticum produced nivalenol (NIV) chemotype, F. graminearum and F. boothii produced 15-acetyl-deoxynivalenol (15-AcDON) chemotype. The EC₅₀ of the 7 fungicides for inhibiting the growth of FGSC ranged from 0.02 to 19.45 μg·mL^-1. Fludioxonil (FS), imazalil (FS), flusilazole (EC), tebuconazole (TC) and myclobutanil (EW) had good inhibitory effects on FGSC and the difference was not significant. The EC₅₀ of FGSC was less than 1.20 μg·mL^-1 and EC₉₀ was less than 100 μg·mL^-1. The difference of EC₅₀ between F. graminearum and F. boothii was significant under 30% pyraclostrobin treatment. The EC₅₀ of F. graminearum was 10.24 times higher than that of F. boothii. 【Conclusion】The dominant pathogenic Fusarium of maize ear rot in different maize producing areas of Jilin Province is different. F. graminearum and F. boothii are dominant species in the east and west, and F. verticillioides is dominant species in the middle. The interspecific and intraspecific genetic diversity of FGSC is rich. Fludioxonil, imazalil, flusilazole, tebuconazole and myclobutanil have better antifungal effect on FGSC. There is no significant difference in the fungicides susceptibility among FGSC.

Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses

GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing

Scientia Agricultura Sinica. 2023, 56(1): 79-89. doi:10.3864/j.issn.0578-1752.2023.01.006

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【Objective】The wheat blossom midge Sitodiplosis mosellana (Diptera: Cecidomyiidae), one of the most important wheat pests, undergoes obligatory larval diapause to survive adverse temperature extremes during hot summers and cold winters. This study aims to explore the potential roles of small heat shock protein (sHsp) gene Hsp21.9 in diapause process of S. mosellana. 【Method】 RACE and RT-PCR technologies were used to clone the full-length cDNA of Hsp21.9 from S. mosellana pre-diapause larvae. Bioinformatics programs were used to characterize the nucleotide and amino acid sequence of cloned Hsp21.9. Real-time quantitative PCR (RT-qPCR) was used to determine the mRNA expression level of Hsp21.9 in pre-diapause, diapause, post-diapause quiescent and developing larvae of S. mosellana, as well as over-summering larvae exposed to short-term (≤120 min) heat stress (35-50℃) and over-wintering larvae exposed to short-term (≤120 min) cold stress (0 to -15℃). The recombinant Hsp21.9 protein was expressed by Escherichia coli prokaryotic expression technology, and then purified. The activity of bacterially expressed recombinant proteins to suppress thermal aggregation of pig heart mitochondrial malate dehydrogenase (MDH) was determined by colorimetry. 【Result】The full-length cDNA of S. mosellana Hsp21.9 (SmHsp21.9) obtained was 1 087 bp (GenBank accession number: KT749988), which contained a 582 bp open reading frame (ORF). The predicted ORF encoded a protein of 193 amino acids of which the content of glutamic acid (12.4%) was the most, and the content of cysteine (0.5%) was the least. The estimated molecular weight and isoelectric point were 21.9 kD and 5.67, respectively. The amino acid sequence of SmHsp21.9 contains typical α-crystallin domain of the sHsp family. The domain consists of six β-sheets, which forms a β-sandwich structure. Sequence alignment and phylogenetic analysis suggested that SmHsp21.9 displayed the highest amino acid identity and the closest relationship to Hsp27 from the Nematocera Chironomus riparius. RT-qPCR indicated that SmHsp21.9 expression differed significantly among different diapause stages. The expression level was decreased after the initiation of diapause, gradually increased in October, and peaked in early-to-mid phase of post-diapause (December and January). Compared with the untreated control, the expression level of SmHsp21.9 was significantly induced in over-summering larvae exposed to heat stress (35-45℃) or over-wintering larvae exposed to cold stressed (-5 to -10℃), but temperature extremes i.e. as high as 50℃ or as low as -15℃ failed to do so. The treatment duration also affected transcript levels of SmHsp21.9, with the maximum value at 30-60 min. Recombinant SmHsp21.9 proteins obtained significantly prevented heat-induced (43℃) aggregation of MDH, suggesting its significant molecular chaperone functionality. 【Conclusion】The expression of SmHsp21.9 is regulated not only by diapause development, but also by environmental temperature. SmHsp21.9 might be involved in initiation and termination of diapause, and heat/cold tolerance during diapause in S. mosellana.

Vertical Distribution of Vegetation Roots and Its Influence on Soil Erosion Resistance of Gully Heads on the Gullied Loess Plateau

LOU YiBao,KANG HongLiang,WANG WenLong,SHA XiaoYan,FENG LanQian,NIE HuiYing,SHI QianHua

Scientia Agricultura Sinica. 2023, 56(1): 90-103. doi:10.3864/j.issn.0578-1752.2023.01.007

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【Objective】The vertical distribution of gully vegetation root system and its effect on soil erosion resistance were explored, so as to provide a theoretical basis for optimizing the allocation of vegetation measures in gully erosion control. 【Method】 In this paper, the gully heads covered with different vegetation (weeds (farmland), Agropyron cristatum, Artemisia gmelinii, and Medicago sativa) were taken as the research object. The scouring experiment of undisturbed soils was carried out to determine the soil anti-scouribility. Moreover, the root characteristics and mechanical and physiochemical properties of root-soil complex were measured using root scanner and ZJ series strain controlled direct shear test apparatus, and so on, respectively. 【Result】(1) The root distribution characteristics at the gully head varied among different vegetation types. Agropyron cristatum had the greatest root characteristic indexes (root weight density, root length density, root surface area density, and root volume density), followed by alfalfa, Artemisia, and weeds in farmland. In addition, the root indexes in the gully head soil of farmland decreased with the deepening of soil layer, while those of Agropyron cristatum, Artemisia gmelinii and Medicago sativa firstly decreased and then increased with the deepening of soil layer. Furthermore, the roots with the diameter of <0.5 mm dominated in the root system of each vegetation. (2) The variability of soil bulk density among different vegetation type was low, ranging from 1.17 g·cm^-3 to 1.37 g·cm^-3. The contents of >0.25 mm water-stable aggregates of farmland and Agropyron cristatum land were higher than that of Artemisia gmelinii land and Medicago sativa land. (3) The average soil cohesions of gully heads under different vegetation types were as follows: 12.75 kPa for Medicago sativa land, 9.05 kPa for Agropyron cristatum land, 8.60 kPa for Artemisia gmelinii land, and 7.25 kPa for farmland, respectively. Additionally, the soil cohesion of the farmland, Agropyron cristatum land, and Medicago sativa land decreased first and then increased with the deepening of soil layer, while that of Artemisia gmelinii land showed a decreasing trend in the depth of soil. (4) The average anti-scouribility coefficients of 0-100 cm soil at the gully head under different vegetation types were as follows: 39.31 L·g^-1 for Medicago sativa land, 25.49 L·g^-1 for Agropyron cristatum land, > 22.39 L·g^-1 for farmland and 14.75 L·g^-1 for Artemisia gmelinii land. Moreover, the soil anti-scouribility coefficient of the 0-20 cm soil layer, varying between 34.91 and 53.30 L·g^-1, was larger than that of the lower soil layers. 【Conclusion】 The combination of plants with tap roots and the plants with fibrous roots was suggested for the control of gully head erosion, and the results provided a theoretical basis for the research of gully headcut erosion and gully erosion control.

Effects of Straw Returning and Nitrogen Fertilizer Types on Summer Maize Yield and Soil Ammonia Volatilization Under Future Climate Change

ZHAO ZhengXin,WANG XiaoYun,TIAN YaJie,WANG Rui,PENG Qing,CAI HuanJie

Scientia Agricultura Sinica. 2023, 56(1): 104-117. doi:10.3864/j.issn.0578-1752.2023.01.008

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【Objective】 Returning straw to the field and applying nitrogen fertilizer can increase crop productivity. However, under the conditions of climate change, the different management measures have great uncertainty in the nitrogen utilization of summer maize farmland. It is very important to clarify the impact of straw returning and nitrogen fertilizer types on summer maize yield and soil ammonia volatilization under future climate conditions. 【Method】 This study used the DNDC model to predict the impact of returning straw to the field and different types of nitrogen fertilizers on summer maize yield and soil ammonia volatilization accumulation in Guanzhong area under different scenarios in the future. Through the verification of field soil temperature, moisture, yield and soil ammonia volatilization test data, the DNDC model could simulate crop yields and soil ammonia volatilization accumulations under different treatments under the future climate conditions well. 【Result】Both simulation and actual measurement results showed that returning straw to the field increased summer maize yields and promoted soil ammonia volatilization under the current climate conditions. Compared with ordinary urea, slow-release fertilizers had no significant effect on summer maize yield but would significantly reduce soil ammonia volatilization accumulation. Sensitivity analysis showed that both crop yield and soil ammonia volatilization accumulation were the most sensitive to nitrogen application. Under the RCP4.5 emission scenario, the single application of stable nitrogen fertilizer (NF1) treatment and single application of urea (NF2) treatment significantly reduced the summer maize yield in 2050s-2090s and 2070s-2090s, respectively. Both the treatment of straw combined with stable nitrogen fertilizer (SF1) and the treatment of straw combined with urea (SF2) significant increased summer maize yield in 2050s-2090s; under the RCP8.5 emission scenario, NF1 significantly reduced the summer maize yield from 2070s to 2090s, and NF2 showed no significant change. The summer maize yields under SF1 and SF2 were increased significantly from 2050s to 2090s. For NF1 under the RCP4.5 emission scenario in 2050s-2090s and under the RCP8.5 emission scenario 2030s-2090s, the soil ammonia volatilization accumulation significantly increased compared with current climate conditions; for the remaining treatments, the cumulative amount of soil ammonia volatilization in future periods under different emission scenarios would be significantly reduce compared with current climatic conditions. 【Conclusion】The DNDC forecast results showed that under the climate conditions of rising temperature and CO₂ concentration and changing precipitation in the Guanzhong area in the future, the returning straw to the field and applying stable nitrogen fertilizer would significantly increase the summer maize yield and reduce the accumulation of ammonia volatilization in the soil, and it was the best high-yield and emission-reducing farmland management plan. This research provided a theoretical basis for coping with climate change and the rational use of straw and nitrogen fertilizer.

Spatial Characteristic and Its Factors of Nitrogen Surplus of Crop and Livestock Production in the Core Area of the Baiyangdian Basin

ZHAO HaiXuan,ZHANG YiTao,LI WenChao,MA WenQi,ZHAI LiMei,JU XueHai,CHEN HanTing,KANG Rui,SUN ZhiMei,XI Bin,LIU HongBin

Scientia Agricultura Sinica. 2023, 56(1): 118-128. doi:10.3864/j.issn.0578-1752.2023.01.009

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【Objective】 The nitrogen surplus caused by excessive nitrogen input of crop and livestock production is the major source of pollution load in surface waterbody. The spatial differentiation characteristics of agricultural unused nitrogen were quantified in this study, so as to provide a basis for the zoning management of nitrogen and the effective management of agricultural nitrogen source in the river basin. 【Method】This study was conducted in Baoding, the central area of the Baiyangdian Basin. The nitrogen input and output of the crop and livestock production system were analyzed in all counties of Baoding in 2016. The nitrogen surplus in the crop production system was defined as the differential value between each input item (chemical fertilizer, organic fertilizer, atmospheric dry and wet settlement, irrigation water, seeds, non-symbiotic nitrogen fixation and straw returning to the field) and the output item (crop grain and straw); the nitrogen surplus in livestock production system was defined as the differential value between the amount of manure and the amount of organic fertilizer; and the nitrogen surplus in the agriculture and animal husbandry was defined as the sum of crop production system and livestock production system. 【Result】(1) The level of nitrogen surplus in crop production was ranging from 90.27 to 581.73 kg·hm^-2, with the lowest value in Dingxing District and the largest value in Mancheng District. Vegetables contributed to the most nitrogen surplus of crop production (31.3%), following by fruit trees (29.0%), wheat (27.8%), and maize (11.9%). Fertilizer was the primary source of nitrogen surplus (61.8%), following by organic fertilizer (16.8%), straw return to the field (8.9%), atmospheric settlement (5.2%), irrigation (3.4%), non-symbiotic nitrogen fixation (3.0%), and seeds (0.9%). (2) The nitrogen surplus level of livestock production was ranging from 0.06×10⁴t to 2.48×10⁴ t with the lowest value in Lianchi District and the highest value in Xushui Distrct. Beef cattle accounted for 71.0% of the total nitrogen surplus of livestock production. (3) The unused nitrogen level in the agricultural and animal husbandry system was between 0.43×10⁴ t and 4.97×10⁴ t, among which the unused nitrogen amount was the highest in the Xushui area. In farming systems, farming was the main source of nitrogen (55.8% of unused nitrogen). 【Conclusion】The nitrogen unused space varied significantly in Baoding counties, the core area of Baiyangdian Basin, and the highest nitrogen unused in Xushui District was 10.4 times that in Jingxiu District. The largest contribution to the amount of unused nitrogen in the crop and livestock production is the crop production, of which vegetable production is the most important industry.

Effects of Irrigation Amount on Berry Development and Aroma Components Accumulation of Shine Muscat Grape in Root-Restricted Cultivation

ZHANG KeKun,CHEN KeQin,LI WanPing,QIAO HaoRong,ZHANG JunXia,LIU FengZhi,FANG YuLin,WANG HaiBo

Scientia Agricultura Sinica. 2023, 56(1): 129-143. doi:10.3864/j.issn.0578-1752.2023.01.010

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【Objective】The effects of different irrigation amounts on grape berry quality, aroma component accumulation, and the expression level of aroma compounds biosynthetic genes were studied to determine the relationship between irrigation patterns and sensory quality of table grapes, so as to provide a reference for choosing the optimal irrigation amount in root-restricted cultivation. 【Method】The table grape cultivar Shine Muscat was used as the test material, the control group (CK), the mild water deficit group (DI-1), and the severe water deficit group (DI-2) were set up to systematically compare the effect of different irrigation amounts on the morphological indicators, appearance color indicators, aroma components, and expression levels of terpene biosynthetic genes of grape berries. 【Result】Irrigation amount could affect the morphological and texture characteristics of grape berries. Comparing with other treatments, the longitudinal diameter of grape berries at harvest time was not significantly affected by irrigation amount, while the horizontal diameter and single berry weight of grape berries in deficit irrigation group were significantly reduced. The firmness of grape pulp also decreased under the influence of deficit irrigation, especially under DI-2 group, of which the pulp firmness was significantly lower than that under other treatment groups. Meanwhile, the glucose content in the grape berries under the deficit irrigation group DI-1 and DI-2 was significantly higher than that under the control treatment, and the fructose content under the severe deficit irrigation group DI-2 was significantly higher than that under other treatments. Mild deficit irrigation of DI-1 exerted little effect on the content of total soluble solids and titratable acid in grape berries. The contents of chlorophyll and carotenoids in grape skins were decreased under the deficit irrigation treatment, and the ratio of the chlorophyll content to carotenoids content in the skins of the DI-2 group was the lowest. Additionally, the amount of irrigation also affected the accumulation of aroma components in grape berries. The terpenes compounds reached the highest content in the berries of the DI-1 group, such as limonene, phellandrene, α-pinene, γ-terpinene, (E)-β-ocimene, terpinolene, (E)-furanoxylinalool, linalool, dihydrolinalool, α-terpineol, citronellol, nerol, and geraniol, followed by the content of terpenes under the DI-2 group, and the lowest under the control group. As for esters, the total content of those compounds under the DI-1 group was the highest, followed by the control group, and the content of the DI-2 group was the lowest. For the total amount of aldehydes, the content in the DI-1 group were significantly lower than those in the control group and DI-2 group. For the total amount of higher alcohols, the content of DI-1 group was the highest, followed by DI-2 group, and the control group was the lowest. There were differences in the expression patterns of terpene biosynthesis-related genes under different irrigation conditions. The expression of VvDXS1, VvDXS2, VvDXR, VvDHR, VvPNLinNer1, VvCSLinNer, VvGwbOci, VvCSbOci and VvGwGer were up-regulated in response to water deficit. 【Conclusion】According to the accumulation of aroma components and the comprehensive score of sensory quality, the mild water deficit (60%-70% of the maximum water holding capacity in the field) could better promote the formation of aroma quality of Shine Muscat grape berries and improve their commercial value.

Analysis of VlCKX4 Expression Characteristics and Prediction of Transcriptional Regulation in Grape

LI XuFei,YANG ShengDi,LI SongQi,LIU HaiNan,PEI MaoSong,WEI TongLu,GUO DaLong,YU YiHe

Scientia Agricultura Sinica. 2023, 56(1): 144-155. doi:10.3864/j.issn.0578-1752.2023.01.011

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【Objective】The cytokinin dehydrogenase/oxidase VlCKX4 and its promoter in grape (Vitis vinifera) were cloned to analyze the expression characteristics, promoter activity and transcription factor prediction, so as to provide a basis for the molecular mechanism that VlCKX4 involved in cytokinin-mediated berry set in grape. 【Method】The gene sequence of cytokinin oxidase/dehydrogenase 4 (VlCKX4) in Kyoho grape (Vitis vinifera × Vitis labrusca) was studied by bioinformatics method. The gene and its promoter were cloned by PCR. The expression characteristics of VlCKX4 were analyzed by real-time quantitative PCR (qRT-PCR). GUS activity assay was used to analyze the promoter activity. The transcriptional regulatory relationship of VlCKX4 was predicted by PlantTFDB, CisBP databases, and the output results were visualized by Gephi software. 【Result】The total length of VlCKX4 was 1 582 bp, which contained 1 566 bp open reading frame (ORF), encoded 522 amino acids, and had the family characteristic FAD domain and cytokinin binding (CK-binding) domain. qRT-PCR results showed that VlCKX4 was highly expressed in inflorescence and leaf, but the expression of tendril was lowest. The expression of VlCKX4 decreased first and then increased after treatment with cytokinin (CPPU), while the expression of VlCKX4 increased first and then decreased after treatment with the cytokinin inhibitor lovastatin (Lov). Cis-element analysis showed that the VlCKX4 promoter contained response elements of methyl jasmonate (MeJA) and other hormones. GUS histochemical staining showed that VlCKX4 responded to the hormones. Predictive analysis of transcriptional regulation showed that MYB, DOF and WRKY transcription factors were involved in transcriptional regulation of VlCKX4. Combined with transcriptomic expression data and coexpression relation, WRKY20, DOF1.5 and MYB59 were identified as key candidate transcription factors. 【Conclusion】 VlCKX4 was induced by CPPU and participates in the process of promoting grape berry set, during which prediction was regulated by transcription factors, such as WRKY20, DOF1.5 and MYB59.

Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR

ZHAI XiaoHu,LI LingXu,CHEN XiaoZhu,JIANG HuaiDe,HE WeiHua,YAO DaWei

Scientia Agricultura Sinica. 2023, 56(1): 156-164. doi:10.3864/j.issn.0578-1752.2023.01.012

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【Objective】The aim of this study was to develop a rapid and accurate quantitative method for identifying porcine-derived materials. 【Method】Porcine microsatellites DNA were selected from GenBank nucleotide database. Primers specific for porcine were designed based on the sequences of microsatellite DNA. Genomic DNA from 10 kinds of common animals was amplified by PCR method. The specificity of selected microsatellite DNA to porcine-derived materials was judged by the amplification products. According to the microsatellite sequence, the specific primers and probes were designed to establish a Real-time PCR method for identifying porcine-derived materials. The double standard curve was used to quantify the porcine-derived materials and total animal-derived materials, respectively, and the percentage content of porcine-derived materials was calculated. 【Result】Porcine specific microsatellite DNA with the accession number EF172428 was selected. Only porcine DNA gave a fragment through PCR assay, while there was no amplification for other non-target animal species DNA. The limit of detection was 0.02 ng in a 25 μL reactive system using the Real-time PCR method. This method could accurately detect porcine-derived components in mixed DNA samples and mixed meat samples with 1.32% percent error and 1.06%-7.12% percent error, respectively. 【Conclusion】The quantitative detection method of porcine-derived materials by Real-time PCR in this research could be used to detect the percentage content of porcine-derived materials in mixed samples.

Expression Pattern of m⁶A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis

YANG XinRan,MA XinHao,DU JiaWei,ZAN LinSen

Scientia Agricultura Sinica. 2023, 56(1): 165-178. doi:10.3864/j.issn.0578-1752.2023.01.013

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【Objective】 The role of RNA m⁶A methylation modification in muscle development has been continuously discovered in recent years. The aim of this study was to explore the mRNA expression of m⁶A methylases, including METTL3, METTL14, WTAP, FTO, and ALKBH5, in bovine muscle tissue and in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). Meanwhile, the changes of m⁶A level during SMSCs myogenic differentiation in vitro were analyzed. This study could provide a reference for clarifying the role and mechanism of m⁶A modification in skeletal muscle development. 【Method】 The expression of m⁶A methylases in skeletal muscle of newborn and adult cattle was detected by real-time quantitative PCR (RT-qPCR). Then, SMSCs were isolated from the Longissimus dorsi muscle of Qinchuan beef cattle. The proliferation and myogenic differentiation of SMSCs were verified by cell growth curve, immunofluorescence and RT-qPCR, and the temporal expression profiles of m⁶A methylases in proliferation (24 h, 36 h, 48 h, 60 h, and 72 h) and differentiation (Day 0, 2, 4, 6, and 8) of SMSCs were detected by RT-qPCR. Finally, the m⁶A levels during SMSCs differentiation were detected using LC-MS/MS and dot blot assays. 【Result】 The mRNA expression levels of m⁶A methyltransferases, including METTL3, METTL14, and WTAP, in the Longissimus dorsi muscle, forelegs muscle and hind legs muscle of adult cattle were significantly lower than those of newborn cattle (P<0.01). The mRNA expression of demethylases such as FTO and ALKBH5 in the hind leg muscle of adult cattle was higher (P<0.01), and ALKBH5 was higher in the Longissimus dorsi muscle of adult cattle (P<0.01). The isolated SMSCs had the functions of normal growth, proliferation and myogenic differentiation. The expression of METTL3 decreased gradually in SMSCs proliferation, but increased significantly at 72 h. The expression of METTL14 did not change significantly, while WTAP reached the highest level at 48 h, and then decreased gradually. The temporal expression profiles of FTO and ALKBH5 were similar in the proliferative phase. They did not change obviously before 60 h and increased significantly at 72 h. The expression patterns of METTL3, METTL14 and WTAP were consistent during SMSCs differentiation, with an increase in the early stage of differentiation, followed by a decrease, and an increase in the late stage of differentiation. The expression of FTO increased gradually with differentiation. The expression of ALKBH5 increased during the first 4 days of differentiation and then continuously decreased. Furthermore, the overall m6A level of mRNA declined during the myogenic differentiation in SMSCs (P<0.01). 【Conclusion】 The expression changes of m⁶A methyltransferases and demethylases in skeletal muscle of newborn and adult cattle were significantly different, suggesting that m⁶A modification might have an important role in the development of skeletal muscle in Qinchuan cattle. Meanwhile, these m⁶A methylases might regulate the proliferation and differentiation of bovine SMSCs. These results provide a theoretical basis for the study of the role and molecular mechanism of m⁶A methylation modifications in regulating skeletal myogenesis.

One-Step Multiple TaqMan Real-time RT-PCR for Simultaneous Detection of Swine Diarrhea Viruses

WANG YiDan,YANG FaLong,CHEN DiShi,XIANG Hua,REN YuPeng

Scientia Agricultura Sinica. 2023, 56(1): 179-192. doi:10.3864/j.issn.0578-1752.2023.01.014

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【Objective】 The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. 【Method】The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value.The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. 【Result】 Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1×10² copies/μL. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. 【Conclusion】In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.

Functional Analysis of MiZAT10A and MiZAT10B Genes in Mango

MO WenJing,ZHU JiaWei,HE XinHua,YU HaiXia,JIANG HaiLing,QIN LiuFei,ZHANG YiLi,LI YuZe,LUO Cong

Scientia Agricultura Sinica. 2023, 56(1): 193-202. doi:10.3864/j.issn.0578-1752.2023.01.015

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【Objective】Zinc finger protein (ZFP) plays an important role in plant abiotic stress response. Therefore, to provide a theoretical basis for stress resistance breeding, this study aimed to analyze the response of two zinc finger protein genes of MiZAT10A and MiZAT10B transgenic Arabidopsis to abiotic stresses, such as salt, drought, heavy metals and exogenous hormones. 【Method】 The promoter cis acting elements and motif of mango MiZAT10A and MiZAT10B genes were predicted and analyzed by online software PLACE and MEME, respectively. The chromosome location map was drawn by TBtools software and SiJiMi gene annotation file (GFF file and unpublished). Tissue expression patterns of MiZAT10A and MiZAT10B genes were analyzed by qRT-PCR. The overexpression vectors of MiZAT10A and MiZAT10B genes were constructed and transformed into Arabidopsis thaliana by Agrobacterium floral-dip method. The phenotype of MiZAT10A and MiZAT10B transgenic plant were observed and recorded under salt, drought, heavy metals, abscisic acid and gibberellin treatments. 【Result】 Promoter cis element analysis showed that there were many light response elements, hormone response elements and abiotic stress response elements in the promoter region of MiZAT10A and MiZAT10B genes. Expression analysis showed that MiZAT10A and MiZAT10B were highly expressed in buds and flowers. 9 of MiZAT10A and 14 of MiZAT10B transgenic Arabidopsis strains were obtained. Overexpression of MiZAT10A and MiZAT10B significantly resulted early flowering compared with the control lines. The root length of MiZAT10A and MiZAT10B overexpressing transgenic Arabidopsis was significantly longer than that of control lines under salt stress, drought stress, heavy metal stress, GA₃ and ABA hormone treatments. 【Conclusion】 Overexpression of MiZAT10A and MiZAT10B not only promoted transgenic Arabidopsis flowering early but also improved salt, drought, heavy metals and exogenous hormones GA₃ and ABA resistance.

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