Table of Content

16 November 2022, Volume 55 Issue 22

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Construction of High Density Genetic Map for RIL Population and QTL Analysis of Heat Tolerance at Seedling Stage in Rice (Oryza sativa L.)

LIU Jin,HU JiaXiao,MA XiaoDing,CHEN Wu,LE Si,JO Sumin,CUI Di,ZHOU HuiYing,ZHANG LiNa,SHIN Dongjin,LI MaoMao,HAN LongZhi,YU LiQin

Scientia Agricultura Sinica. 2022, 55(22):  4327-4341.  doi:10.3864/j.issn.0578-1752.2022.22.001

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【Objective】 With global warming, high temperature has an increasing impact on food crop safe. Excavation of heat tolerance gene resources is the most direct green ecological method to cultivate new varieties of heat resistance and eliminate the harm of high temperature, which also the basis for clarifying the physiological, biochemical and molecular genetic mechanism of heat tolerance.【Method】Establishing the identification and evaluation method of heat tolerance at seedling stage, a set of RIL populations was structured from the extreme heat-tolerance Ganzaoxian58(GZX58) and heat-sensitive Junambyeo (JNB), and then the high density genetic map was constructed using genotyping by resequencing technology. To converting SNP information into Bin genotype of the RIL population using sliding window method, which predicting the recombination breakpoints on the chromosomes, finally a high density BinMap genetic map was constructed. Based on the genotype and phenotype data of the 171 lines, QTL mapping of the high temperature seedling survival rate (HTSR) and heat tolerance class (HTC) was performed by ICIM method of the QTL IciMapping software.【Result】A high-density genetic map containing 3 321 Bin markers was constructed, the number of Bin markers for each chromosome between 159 and 400, the average physical distance two markers was about to 106 kb; heat tolerance of the parents and RIL populations was identified by stepwise heat stress at seedlings stage, there have a significant negative correlation between HTSR and HTC, in addition, there has a significant positive correlation between HTSR and indica gene frequency (Fi), which the higher of the Fi, the heat tolerance is better; the bi-modal continuous distribution of phenotype traits from the RIL population showed that the heat tolerance is regulated by few major QTL. A total of 12 QTL controlling with heat tolerance at seedling stage, there have 8 and 4 QTL regulating for HTSR and HTC, respectively. There has a significant genetic overlap from HTSR and HTC, qHTS2, qHTS7 and qHTS8, three major QTL cluster play an important role in regulating the heat tolerance at seedling stage. Among these QTL, qHTS7 was a novel major QTL cluster, which has a strong effect on enhancing the heat resistance at seedling stage. 【Conclusion】 We constructed a high density genetic linkage map containing 3 321 Bin markers, which be used to analyzed the heat tolerance gene from the GZX58 at seedling stage, there have three key QTL cluster identified associated with the heat tolerance, a novel QTL cluster qHTS7 was discovered, efficient acquisition of target segments and candidate genes based on high-density genetic mapping, eight key candidate genes were selected by bioinformatics for regulation of the heat tolerance.

Construction of A Comprehensive Evaluation System and Screening of Cold Tolerance Indicators for Cold Tolerance of Cotton at Seedling Emergence Stage

SHEN Qian,ZHANG SiPing,LIU RuiHua,LIU ShaoDong,CHEN Jing,GE ChangWei,MA HuiJuan,ZHAO XinHua,YANG GuoZheng,SONG MeiZhen,PANG ChaoYou

Scientia Agricultura Sinica. 2022, 55(22):  4342-4355.  doi:10.3864/j.issn.0578-1752.2022.22.002

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【Objective】 In this study, the purpose was to comprehensively evaluate the cold tolerance of cotton varieties (lines) at seedling emergence stage, establish a reliable evaluation model, screen and identify indicators, and provide a simple and effective evaluation method for the selection and identification of cold-tolerant varieties in cotton.【Method】200 upland cotton varieties (lines) were used to test hypocotyl length, root length and 100-grain weight, etc. under three treatments of constant chilling (CC), diurnal variation of chilling (DVC) and normal conditions. A combination of integrated cold tolerance coefficient difference analysis, frequency analysis, drop analysis, principal component analysis, cluster analysis, and multiple regression analysis were used to classify their cold tolerance types, establish cold tolerance prediction models, and screen evaluation parameters. 【Result】The variation of each parameters at normal conditions were minor fluctuations ranging from 3.12% to 18.89%. The seedling emergence rate was above 85.00%, which had high viability and could be used for subsequent cold tolerance analysis. The variability of each parameter within the accessions increased under chilling stress, ranging from 7.14%-108.33%, and the most variable parameter were root length under CC condition and germination index under DVC condition. Principal component analysis converted the 14 parameters under chilling stress and 100-grain weight measured into six mutually independent composite indicators, representing 74.98% of the total data information. The comprehensive cold tolerance evaluation value (D) was calculated by the affiliation function method and then clustering analysis was performed. 200 cotton varieties (lines) were divided into five categories according to their cold tolerance, with 2 of the group Ⅰ being strongly cold tolerant, 42 of the group Ⅱ being cold tolerant, 69 of the group Ⅲ being medium cold tolerant, 83 of the group Ⅳ being more sensitive, and 4 of the group Ⅴ being sensitive, of which Xinluzhong 16 was the most cold-tolerant material. A multiple regression analysis was applied to establish a prediction model for cold tolerance of cotton at seedling emergence as Y=-4.10+0.58X4+0.40X14+0.32X1+0.22X5 (R²=0.92), and four parameters for cold resistance evaluation were confirmed, namely total length of seedling, emergence rate, and dry weight under CC stress, germination rate under DVC stress. The cold-tolerant varieties (lines) had higher seedling emergence rates of early sowing experiment in the field, which were basically consistent with the results of the indoor results. 【Conclusion】 It is feasible to use CC and DVC stress combined with multivariate statistical analysis to evaluate the cold tolerance of cotton at seeding stage, and total length of seedling, emergence rate, and dry weight under CC stress, germination rate under DVC stress can be used as evaluation parameters.

Functional Analysis of AhNRT2.7a in Response to Low-Nitrogen in Peanut

WANG Juan,CHEN HaoNing,SHI DaChuan,YU TianYi,YAN CaiXia,SUN QuanXi,YUAN CuiLing,ZHAO XiaoBo,MOU YiFei,WANG Qi,LI ChunJuan,SHAN ShiHua

Scientia Agricultura Sinica. 2022, 55(22):  4356-4372.  doi:10.3864/j.issn.0578-1752.2022.22.003

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【Objective】 Nitrogen (N) plays a key role in determining biomass and yield in crop production. NRT2s, the high affinity nitrate transporter genes, are mainly activated under low nitrogen stress condition and have been implicated in nitrate absorption and remobilization. This study will screen NRT2 gene family responding to low-nitrogen condition (1/20 of the normal level) and conduct a preliminary functional analysis of AhNRT2.7a in order to provide target genes for breeding new peanut varieties with higher nitrogen utilization efficiency (NUE)，which will help to achieve the goal of to improve crop production with less N fertilizer demand and environmental degradation. 【Method】The spatio-temporal expression patterns under normal and low-nitrogen conditions of five peanut NRT2 genes, AhNRT2.4, AhNRT2.5b, AhNRT2.5c, AhNRT2.7a and AhNRT2.7b, were investigated. Using the cDNA of Huayu6309 as template, full length of AhNRT2.7a CDS was cloned and bioinformatic analyzed. Subcellular localization of AhNRT2.7a was conducted by construction of transient expression vector and transformation of Arabidopsis protoplasts. In order to explore the gene function of AhNRT2.7a, heterologous overexpression of the AhNRT2.7a gene in Arabidopsis were performed. Transgenic plants were used to determine chlorophyll content, nitrogen accumulation and the enzymatic activities of glutamine synthetase (GS), glutamate synthetase (GOGAT), nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) under normal and low-nitrogen conditions. 【Result】Four NRT2 genes of peanut were highly expressed in response to low nitrogen stress, and AhNRT2.7a was highly expressed in the stems and leaves. The total length of 1 380 bp was obtained, encoding a 459-amino acid protein with a molecular weight of 49.35 kD. The total of 12 typical transmembrane protein domains with hydrophobic regions was predicted. Bioinformatics analysis showed that the amino acid sequence had 99.56% sequence similarity with the cultivated peanut (Arachis hypogaea L.), followed by the wild-parents AA (A. duranensis) and BB (A. ipaensis). Subcellular localization analysis revealed that AhNRT2.7a was located in the cell membrane. Transgenic Arabidopsis plants for over-expressing AhNRT2.7a were conducted. Relative content of chlorophyll in mature and young leaves was significantly higher than that in wild-type Arabidopsis under different nitrogen supply. Meanwhile, the activity of five enzymes involved in nitrogen metabolism were examined. Furthermore, uptake, assimilation and re-mobilization of N, concentration of phosphorus and potassium were determined. The results have revealed that the activity of the two nitrogen metabolizing enzymes (NR and GS) and nitrogen accumulation in transgenic plants were significantly higher than in wild-type Arabidopsis. 【Conclusion】 These results indicated that AhNRT2.7a could enhance the nitrogen use efficiency (NUE) in plants, and also improve carbon metabolism. AhNRT2.7a seems promising as a candidate gene in breeding new peanut varieties with higher NUE.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATIONTECHNOLOGY

Discussions on Frontiers and Directions of Scientific and Technological Innovation in China’s Field Crop Cultivation

ZHANG HongCheng, HU YaJie, DAI QiGen, XING ZhiPeng, WEI HaiYan, SUN ChengMing, GAO Hui, HU Qun

Scientia Agricultura Sinica. 2022, 55(22):  4373-4382.  doi:10.3864/j.issn.0578-1752.2022.22.004

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China’s total grain output has exceeded 650 million tons since seven years ago, with the great achievement of ever-increased grain output for 18 consecutive years. However, China’s cereal production is facing many severe challenges, such as the shortage of agricultural resources per capita, higher cost for crop production per hectare, sharp decrease of labor population and progressively increase of grain import. Therefore, it is necessary to discuss the frontiers and the directions of scientific and technological innovation in China’s field crop cultivation. According to the existing problems in field crop production, the major scientific and technological requirements of China’s field crop were elaborated, such as the cooperative cultivation of high-quality and high-yield and high-efficient, the green cultivation of carbon fixation and energy conservation and emission reduction, and the “unmanned” intelligent cultivation. The scientific and technological frontiers and development status and trends of field crop cultivation at home and abroad were analyzed emphatically. The key scientific and technological breakthrough directions and paths were pointed out in three aspects: The coordination cultivation of high-quality and high-yield and high-efficient, the green cultivation of carbon fixation and energy saving and emission reduction, and the “unmanned” intelligent cultivation. Finally, the measures and suggestions for scientific and technological innovation of China’s field crop cultivation were put forward from the aspects of policy top-level design and effective investment, increasing the research and development of basic theories, the key technologies and practical products, and increasing the cultivation of compound talents. In the future, it is necessary to significantly improve grain quality and planting efficiency under the premise of continuously increasing grain yield through the collaborative cultivation of high-quality, high-yield and high efficiency in field crop cultivation. Through the green cultivation of carbon fixation and energy conservation and emission reduction, less material input and high efficient utilization of resources can be achieved. Through integration and innovation of various aspects of agronomy and agricultural machinery intelligence, the “unmanned” intelligent cultivation can be constructed for greatly less labor input and higher efficiency of large-scale production. Finally, it is to realize Chinese style modernization of field crop production, to ensure grain security and effective supply of agricultural products.

Study on the Quality Parameters of Strong Gluten Wheat and Analysis of Its Relationship with Meteorological Factors in Shandong Province

YU WeiBao, LI Nan, KOU YiHong, CAO XinYou, SI JiSheng, HAN ShouWei, LI HaoSheng, ZHANG Bin, WANG FaHong, ZHANG HaiLin, ZHAO Xin, LI HuaWei

Scientia Agricultura Sinica. 2022, 55(22):  4383-4397.  doi:10.3864/j.issn.0578-1752.2022.22.005

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【Objective】 In this paper, the dominant distribution areas of strong gluten wheat were clarified in Shandong province, and the influence of key meteorological factors on its quality parameters was analyzed. 【Method】The high-quality strong-gluten wheat Jimai 44 was selected as the research material, and 296 samples were collected from 44 counties and districts in Shandong province in the growing seasons of 2018 to 2020. The relationship of meteorological factors, such as light, temperature and water, in different growth periods with wheat quality parameters was analyzed by using the method of stepwise regression. The geographic information system (GIS) was used for spatial visualization analysis, and the possible distribution of high-quality strong-gluten wheat advantageous areas was explored in Shandong province. 【Result】There were differences in the performance of each quality parameter in the different regions in different years. The proportion of samples reaching the standard of strong gluten was shown as maximum pull resistance>water absorption rate>bulk density>stabilize time>protein content>tensile area>wet gluten content in two years, and the coefficients of variation of quality parameters from large to small were stabilize time, tensile area, maximum pull resistance, wet gluten content, protein content, water absorption rate, and bulk density. The bulk density in western and northwestern Shandong was generally higher than that in other regions, and decreased with the increase of longitude, which was mainly related to the influence of rainfall during the rejuvenation-jointing period. The protein content was positively affected by the accumulated temperature ≥5℃ during the anthesis-milk maturity period, while increased from southwest to northeast in 2018-2019 and from northwest to southeast in 2019-2020. The wet gluten content was higher in the eastern Shandong region, which was significantly related to the high rainfall during the anthesis-milk maturity period in this region. The stabilize time was significantly negatively correlated with the maximum temperature during the jointing-anthesis period, and positively correlated with the rainfall during the sowing-overwintering period, and this affected its high value distribution and regional variation; The tensile area was significantly negatively correlated with the accumulated temperature ≥5℃ during the rejuvenation-jointing period, and gradually decreased from the west to the east of Shandong province. The maximum pull resistance was significantly negatively correlated with the accumulated temperature ≥5℃ during the anthesis-milk maturity period; It was low in the east-west direction and high in the middle area of Shandong province. Taking into account comprehensively, the high-quality probability of high-gluten wheat planting in eastern and southern Shandong province was stronger than that in central and northern, and the lowest in western. 【Conclusion】 Eastern and southern regions were the optimal planting areas for strong-gluten wheat in Shandong province, with the greatest possibility of high quality. The high maximum temperature during rejuvenation-jointing period, jointing-anthesis period and grain-filling period was unfavorable to the rheological parameters of wheat dough, while the increase of effective accumulated temperature during anthesis-milk maturity period was beneficial to the increase of protein content. The rainfall during sowing-overwintering period was beneficial to the increase of dough stabilize time; When the rainfall was less than 14.5 mm during anthesis-milk maturity period, it was not beneficial for the wet gluten content to reach the strong gluten standard; The rainfall during the rejuvenation-jointing period was not conducive to the increase of bulk density. Therefore, in the production of strong gluten wheat, it was suggested that irrigation should be carried out in the overwintering period and early grouting according to the weather conditions, and the irrigation time during rejuvenation-jointing period should be postponed as far as possible.

PLANT PROTECTION

Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus

ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen

Scientia Agricultura Sinica. 2022, 55(22):  4398-4407.  doi:10.3864/j.issn.0578-1752.2022.22.006

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【Objective】 The objective of this study is to construct an expression vector based on citrus leaf blotch virus (CLBV) and systematically express antimicrobial peptides to improve plant disease resistance, which will provide a new technical means for the prevention and control of citrus canker, citrus Huanglongbing (HLB) and other diseases.【Method】The subgenomic promoter sequence and multiple cloning sites were inserted after the terminator of the coat protein gene of CLBV to construct the viral expression vector pCLBV202 based on the previously constructed infectious clone pCY-CLBV201. Then green fluorescent protein gene (gfp) was inserted into the multiple cloning sites, and the expression of GFP was verified by Agrobacterium-mediated inoculation and fluorescence observation. The cecropin B (CB) gene of Antheraea pernyi was cloned and the recombinant vector pCLBV202-CB was constructed. Nicotiana benthamiana and citrus seedlings were inoculated by pCLBV202-CB using Agrobacterium-mediated injection and vacuum infiltration, respectively. Positive plants were screened and subjected to inoculation of Ralstonia solanacearum by injection and root irrigation or Xanthomonas citri subsp. citri (Xcc) by pricking the detached leaves. At the same time, the plants inoculated with the empty vector were set as the control. The resistance to tobacco bacterial wilt of the inoculated plants was evaluated by symptom observation, incidence rate and disease index. The citrus resistance to canker was evaluated by the detriment, incidence rate and colony concentration on leaves. 【Result】After inoculated by pCLBV202-GFP, green fluorescence could be observed on the systemic leaves of the N. benthamiana and Citrus limon, which was brighter in N. benthamiana, indicating that the expression vector based on CLBV was successfully constructed. After inoculation of R. solanacearum, the onset time of treatment group (pCLBV202-CB) was delayed by 4 days compared with the control group (pCLBV202). On the 24th day past inoculation (24 dpi), the incidence rate of the treatment group was 14.3%, and that of the control group was 100%. Compared with the control group, the resistance index of the treatment group was -2.66, and the resistance evaluation was high, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to tobacco bacterial wilt. When C. limon leaves was inoculated by Xcc, the number of detriment in the treatment group at 7 dpi was 47, and the incidence rate was 43.5%, while that of the control group was 73, and 67.6%. The detection of colony concentration changes of Xcc showed that the number of Xcc in the treatment group was less than that in the control group, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to citrus canker.【Conclusion】 The CLBV-based viral expression vector pCLBV202 was constructed. Using pCLBV202 to systematically express CB in N. benthamiana and citrus can improve the resistance of plants to bacterial diseases, which provided a new technology for the prevention and control of citrus bacterial diseases.

Complete Genome Sequence Characteristics and TC-RT-PCR Detection of East Asian Passiflora Virus Infecting Passiflora edulis

XIE LiXue,ZHANG XiaoYan,ZHANG LiJie,ZHENG Shan,LI Tao

Scientia Agricultura Sinica. 2022, 55(22):  4408-4418.  doi:10.3864/j.issn.0578-1752.2022.22.007

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【Objective】 East Asian Passiflora virus (EAPV) is an important virus in Passiflora edulis. The objective of this study is to determine the complete genome sequence of EAPV Fujian isolate (EAPV-FJ) in mainland China, define the genome sequence characteristics of EAPV-FJ, establish the TC-RT-PCR (tube capture RT-PCR) assay for the specific detection, and to provide theoretical basis and technical support for the detection, monitoring and control of the virus.【Method】The complete genome sequence of EAPV-FJ was determined by using small RNA deep sequencing technology, combined with segmental cloning and RACE technology. The obtained EAPV-FJ sequence was analyzed for sequence characteristics, phylogenetic relationship and recombination. The TC-RT-PCR technology for rapid detection of EAPV was established through optimization of reaction conditions and reaction system. The specificity and sensitivity of the TC-RT-PCR were determined. The established TC-RT-PCR technology was used to detect samples collected from P. edulis orchards in Fujian Province, and the conventional RT-PCR was used for verification.【Result】The full length of the obtained EAPV-FJ was 10 065 nt (excluding polyA tail), containing an open reading frame of 9 663 nt in length that encodes a polyprotein of 3 220 aa. The polyprotein was cleavaged into 10 proteins, which are P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb and CP, respectively. The results of genome sequence identity analysis showed that the nucleotide sequence identity between EAPV-FJ genome and the four EAPV representative isolates registered in GenBank was 80%-99%, among which Vietnamese isolate EAPV-GL1 (GenBank accession number MT450870) of AO strain had the highest identity (99%). The nucleotide and amino acid sequences of the polyprotein were 79%-99% and 82%-98% identical to the four EAPV representative isolates registered in GenBank, respectively. Phylogenetic analysis based on the nucleotide sequence of EAPV-FJ polyprotein showed that EAPV isolates were divided into two groups (Group I was AO strain and Group Ⅱ was IB strain), which did not show obvious geographical correlation. EAPV-FJ belonged to Group I and was most closely related to the reported Vietnamese isolate EAPV-GL1. The results of recombination analysis revealed that EAPV-FJ is not a recombinant of EAPV. The established TC-RT-PCR detection technology showed good specificity and sensitivity, and can only detect EAPV-infected samples of P. edulis, while other viruses including cucumber mosaic virus (CMV), Passiflora latent virus (PLV), hibiscus latent Fort Pierce virus (HLFPV), turnip mosaic virus (TuMV), soybean mosaic virus (SMV), telosma mosaic virus (TeMV) as well as healthy samples cannot be detected. The lowest sensitivity of the TC-RT-PCR could detect the crude leaf extract of EAPV-infected samples of P. edulis diluted by 10 times, which was similar to the sensitivity of conventional RT-PCR. A total of 13 EAPV-positive samples were detected from 60 suspected virus disease samples of P. edulis collected from P. edulis orchards in Fujian Province by using the established TC-RT-PCR assay, and the results were in good agreement with conventional RT-PCR.【Conclusion】 This is the first report of complete genome sequence of EAPV in mainland China. The genome structure of this isolate (EAPV-FJ) is consistent with other reported isolates. EAPV-FJ has the closest relative to the Vietnamese isolate EAPV-GL1 in phylogenetic relationship, and no recombination sites were detected. The established TC-RT-PCR assay has the advantages of convenient operation, strong specificity, high sensitivity and low cost, and can be effectively used for the actual detection of EAPV in P. edulis orchard samples.

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

Characteristics of Phosphorus Fractions and Its Response to Soil Chemical Properties Under the Threshold Region of Olsen P in Black Soil

QIN ZhenHan,WANG Qiong,ZHANG NaiYu,JIN YuWen,ZHANG ShuXiang

Scientia Agricultura Sinica. 2022, 55(22):  4419-4432.  doi:10.3864/j.issn.0578-1752.2022.22.008

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【Objective】 Agronomic and environmental thresholds of Olsen phosphorus (P) are the most important parameters for soil P management, but the characteristics of phosphorus fractions under the different threshold regions are not clear. This research evaluated the characteristics of the P fraction under the different threshold regions of Olsen P and its influencing factors in black soils for enabling to understand the transformation process of soil P, so as to provide a reference for optimizing the Olsen-P management strategy and improving the efficiency of P resource utilization.【Method】9 Olsen P levels (11, 21, 31, 40, 57, 69, 128, 331, and 490 mg·kg^-1) of agricultural fields plow layer (0-20 cm) soil samples were collected in Gongzhuling, Jilin Province. TIESSEN-Moir modified HEDLEY phosphorus classification method was used to classify soil inorganic phosphorus and organic phosphorus. The relationship between the phosphorus fractions and soil chemical properties, such as soil organic matter (SOM), C/P, Fe, and Al oxides, was also analyzed to clarify the characteristics of phosphorus fractions and the main controlling factors under the different threshold regions of soil Olsen P.【Result】The P pool was dominated by Pi, accounting for 71.25%-96.19%, with Po accounting for 3.81%-28.75%. When the Olsen P level was below the agronomic threshold (7.4-13 mg·kg^-1), the proportion of liable P (LP) of 19.89% was the lowest in comparation with other P fractions. When the Olsen P level was below the environmental threshold (51.0-56.4 mg·kg^-1), the proportion of medium active phosphorus (ML-P) and stable phosphorus (OP) is close, 36.03% and 35.49% respectively, both higher than the proportion of LP (28.48%). The highest proportion of LP (42.86%) was observed when the Olsen P level was above the environmental threshold. When the Olsen P level is higher than the environmental threshold, the content of LP and ML-P in the soil is significantly higher than that in the soil where the Olsen P level is lower than the environmental threshold, and the resin-P showed the greatest variation with Olsen P above and below the environmental threshold. The value of P activation coefficient (PAC), and the concentration of Mehlich-3 extractable aluminum (M3-Al), free Al oxide (Ald), organic-bound Fe, Al oxide (Fep, Alp), and amorphous Fe, Al oxide (Feo, Alo) increased significantly, while a significant decrease in C/P was observed with increasing Olsen P levels. The correlation analysis shows that when the Olsen P level was below the environmental threshold, the soil organic matter was positively and significantly correlated with the highly active inorganic P fractions (Resin-P, NaHCO₃-Pi, NaOH-Pi) above the environmental threshold; when the Olsen P level was above the environmental threshold, Fep+Alp showed a strong positive correlation with each inorganic P fraction blow the environmental threshold. The redundancy analysis results showed that when the Olsen P level was below the environmental threshold, SOM and M3-Fe were the key factors for affecting the change of P fractions in black soils, explaining 50.2% and 24.1% of the total variation, respectively; when the Olsen P level was above the environmental threshold, Fep+Alp was the main factor influencing the change of P fractions, explaining 68.1% of the total variation.【Conclusion】 When the Olsen P level was below the agronomic threshold, the liable P accounted for the lowest proportion; however, which was the greatest proportion when the Olsen P level was above the environmental threshold. In addition, the Resin-P is the phosphorus fraction with the largest variation below and above the environmental threshold. SOM, M3-Fe, and Fep+Alp were the key factors affecting the change of P fractions below and above the environmental threshold, respectively.

Effects of Foliar-Spraying Selenium Coupled with Soil Moisture on the Yield and Quality of Tomato

LIU Hao,PANG Jie,LI HuanHuan,QIANG XiaoMan,ZHANG YingYing,SONG JiaWen

Scientia Agricultura Sinica. 2022, 55(22):  4433-4444.  doi:10.3864/j.issn.0578-1752.2022.22.009

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【Objective】 Both exogenous selenium and soil moisture can affect crop growth and quality properties. It is very important to clarify the influences of exogenous selenium coupled with soil moisture on the tomato yield and quality, which maybe provide a theoretical basis on the water use for production of selenium-enriched tomato. 【Method】Three foliar-spraying selenium concentrations, such as 0 (S₀), 5 (S₅) and 10 mg·L^-1 (S₁₀), were considered with sodium selenite (Na₂SeO₃) as the selenium source by using pot experiment in this paper. Each selenium concentration was associated with two irrigation levels, which were scheduled to irrigate the crop as soon as the soil water moisture decreased to 50% (W₁) and 75% (W₂) of the field capacity, respectively. The effects of the different treatments on the selenium content, plant growth, yield and quality of tomato were studied.【Result】The different soil moistures had no significant impact on the selenium contents in soil, leaf and fruit (P>0.05). Although the different selenium concentrations had no marked influence on the soil selenium content for each irrigation level, the increasing selenium concentration led to an significant (P<0.01) increase in selenium content of leaves and fruits. Compared with S₀ over the two irrigation levels, selenium-treated fruits increased the average selenium content with 2-4 fold. Drought stress significantly reduced plant height and stem diameter. The foliar-spraying selenium moderately alleviated the inhibition effect on plant height when crop suffered drought stress, whlie no significant effect was found on stem diameter. The drought stress gave a significant reduction in yield by 39.5% compared with the sufficient soil water applied. The foliar-spraying selenium had an increase in fruit number, but tended to decrease fruit weight under the drought stress conditions, thus no noticeable difference in yield was investigated between different foliar-spraying selenium. Compared with the sufficient soil water applied, the drought stress gave a significant increase in soluble sugar (SSC), organic acid (OA), vitamin C, and total soluble solid content (TSS) by 28.7%, 24.3%, 18.7%, and 24.0%, respectively. The foliar-spraying selenium improved SSC, whereas there was no noticeable difference in SSC between different selenium concentrations when the soil moisture was the same. Foliar-spraying selenium significantly increased OA except for S₅W₂ treatment compared with the control, thus S₅W₂ had the highest sugar-acid ratio (SAR), while the lowest SAR was observed in S₀W₂ treatment. There was no marked effect of foliar-spraying selenium on TSS under sufficient soil water applied conditions, however, the TSS was increased firstly and then decreased with selenium concentration increasing under drought stress, indicating that a further increase in selenium concentration from 5 mg·L^-1 did not give a noticeable increase in fruit quality. 【Conclusion】 The coupling effect of foliar-spraying selenium and soil moisture on tomato quality was obvious. The spraying sodium selenite at 5 mg·L^-1 significantly increased SSC and TSS and thereby improved nutritional quality of fruit when the plant suffered drought stress, but promoted flavor quality of fruits with no yield decreasing and thereby achieved stable yield and high quality when the soil moisture applied was sufficient.

Effect of CO₂ Like Fertilization on Rice Growth by HCO₃^- in Biogas Slurry

WAN HuaQin,GU Xu,HE HongMei,TANG YiFan,SHEN JianHua,HAN JianGang,ZHU YongLi

Scientia Agricultura Sinica. 2022, 55(22):  4445-4457.  doi:10.3864/j.issn.0578-1752.2022.22.010

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【Objective】 Biogas slurry is rich in nitrogen (N), which is the basis of its farmland utilization. However, it also contains a large amount of HCO₃^-. In this study, the conversion of HCO₃^- in biogas slurry and its effect on rice growth were investigated, in order to provide a new theoretical basis for replacing chemical fertilizer with biogas slurry, reducing the amount of chemical fertilizer, and whether biogas slurry could be utilized as resources. 【Method】Four treatments, including biogas slurry (BS), biogas slurry (BS-B), deionized water plus HCO₃^- (W+B) and deionized water (W), were set up in this paper. Using ¹³C labeling technology, the characteristics of CO₂ release were observed through pot experiment at Rice Seedling Stage, and the photosynthetic rate, intercellular CO₂ concentration, dry and fresh weight, plant height, overlying water and soil pH, and HCO₃^- and NH₄⁺ contents were analyzed. 【Result】(1) Under BS treatment, CO₂ release rates ranged from 9.55 to 38.07 mg·kg^-1·h^-1, with a net cumulative release of 4 654.06 mg·kg^-1. Under BS-B treatment, the CO₂ release rates ranged from 4.55 to 17.25 mg·kg^-1·h^-1, and the net cumulative release was 780.68 mg·kg^-1. Under W+B treatment, the CO₂ release rate was 3.93-26.33 mg·kg^-1·h^-1, and the net cumulative release was 1 274.07 mg·kg^-1. Under W treatment, the CO₂ release rate was 3.22-11.90 mg·kg^-1·h^-1, and the cumulative release amount was 2 265.20 mg·kg^-1. Under BS treatment, the average CO₂ release rate was 4.18 times and 2.44 times of BS?B and W+B, respectively, and the net cumulative CO₂ release was 5.96 times and 3.65 times of BS-B and W+B, respectively, which were significantly higher than those under BS?B and W+B treatments. At the same time, the net cumulative release under BS treatment was greater than the sum of the two treatments (BS-B) + (W+B), and HCO₃^- had a synergistic effect with other components in biogas slurry on CO₂ release. (2) The net cumulative release of ¹³CO₂ under BS treatment was 32.87 mg·kg^-1, accounted for 0.71% of the net cumulative release of CO₂ in soil-rice system. The net cumulative release of ¹³CO₂ under W+B treatment was 13.18 mg·kg^-1. In comparison, the net cumulative release amount of ¹³CO₂ under BS treatment was significantly higher than that under W+B treatment (P<0.05), indicated that other components in biogas slurry promoted the conversion of HCO₃^- to CO₂. (3) The net photosynthetic rate of BS and BS?B treatments in the first 12 h was significantly higher than that under W+B and W treatments. After the addition of culture medium, the net photosynthetic rate under BS treatment was significantly higher than that under BS?B treatment at 2-7 d, and was significantly higher than that under W+B treatment during the whole culture period (P<0.05). HCO₃^- in biogas slurry significantly improved the photosynthesis of rice leaves. In comparison, the intercellular CO₂ concentration was significantly lower than that of the other three treatments 5 days before BS treatment. Rice plant height and fresh weight under BS and BS-B treatments were significantly higher than those under W+B and W treatments (P<0.05), and there was no significant difference in dry weight among the four treatments. (4) The fixed ¹³CO₂ content of rice seedlings treated by BS was 4.05 g·kg^-1, and the utilization rate of marker HCO₃^- was 18.54%. The fixed amount of ¹³CO₂ in W+B treatment was 3.29 g·kg^-1 and the H¹³CO₃^- utilization rate was 14.20%. The ¹³CO₂ of H¹³CO₃^- promoted the photosynthesis of rice and was beneficial to the growth of rice. (5) The release rates of CO₂ and ¹³CO₂ under BS and W+B treatments were significantly correlated with overlying water and soil HCO₃^- content and pH. At the same time, the photosynthetic rate of rice under BS and W+B treatments was significantly positively correlated with HCO₃^- content in soil. 【Conclusion】 When biogas slurry was returned to the field, a large amount of HCO₃^- transformation significantly promoted the release of CO₂, which was beneficial to rice photosynthesis. Soil HCO₃^- content and soil pH value were important factors for affecting CO₂ release and rice photosynthesis. At the same time, the rice had a higher utilization rate of HCO₃^- in biogas slurry, and HCO₃^- in biogas slurry had obvious CO₂-like fertilization effect.

HORTICULTURE

Identification of 60 Citrus Accessions Using Target SSR-seq Technology

ZHU YanSong,ZHANG YaFei,CHENG Li,YANG ShengNan,ZHAO WanTong,JIANG Dong

Scientia Agricultura Sinica. 2022, 55(22):  4458-4472.  doi:10.3864/j.issn.0578-1752.2022.22.011

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【Background】 Bud sports mutation is a DNA mutation occurred in somatic meristem, and it often displays visible morphological and other characteristic changes different from its mother plants in branches, leaves, flowers and fruits. However, the discriminating bud sports mutation from the epigenetic variation caused by environmental conditions and cultivation measures etc. external factors was still mostly depended on the molecular fingerprint detection. 【Objective】 This study objective was to identify citrus bud sports mutant through Target-SSR sequencing technology.【Method】Firstly, the genome of clementine mandarin (Citrus reticulata Blanco) and satsuma (Citrus unshiu Macf.), as well as GSS and EST sequences of satsuma were used to scan SSRs loci with GMATA, and the highly polymorphic SSRs loci were screened out to design primers. The multiplex PCR with optimized primers were ampllified on 60 citrus bud sports mutants to construct high-thoughout sequencing library, and the amplification products were then sequenced on illumina Minseq platform. The clean sequencing short reads were mapped to reference target sequences to find differentiated SSRs loci presented in citrus bud sports mutants.【Result】 A total of 77 pairs of SSR primers were designed from highly polymorphic SSRs loci. The primer pair combinations were optimized and 18 multiplex PCR amplification products were sequenced. The target SSR-seq analysis showed that the genotyping data of SSRs could divided 60 citrus accessions into two groups corresponding to sweet orange and mandarin, and the mandarin group could be further subdivided into different citrus cultivars, such as Orah, ponkan etc. 11 SSR loci containing mostly ATT motif were found in 7 Tarroco blood orange mutants, 8 SSR loci containing mostly TAA motif were found in 2 ‘Wu Yue Hong’ mutants and 5 navel orange, 16 SSR loci containing mostly GA motif were found in 9 Bing Tang Cheng mutants, 9 SSR loci containing mostly AAT motif were found in 2 Sha Tang Ju mutants, and 15 SSR loci with mostly AAT motif were found in 4 satsuma mutants. This study showed that Target-SSR sequencing technology provided an excellent resolving approach to discriminate citrus bud mutations.【Conclusion】 In this study, an effective method for citrus bud mutant identification by using Target SSR-seq technology were established, and 60 citrus germplasm accessions could be discriminated. The precision and reliability of SSR genotyping information could be utilized in citrus germplasm resources management and variety intellectual property protection.

Difference in Flavonoid Composition and Content Between Summer and Winter Grape Berries of Shine Muscat Under Two-Crop-a-Year Cultivation

WANG Bo,QIN FuQiang,DENG FengYing,LUO HuiGe,CHEN XiangFei,CHENG Guo,BAI Yang,HUANG XiaoYun,HAN JiaYu,CAO XiongJun,BAI XianJin

Scientia Agricultura Sinica. 2022, 55(22):  4473-4486.  doi:10.3864/j.issn.0578-1752.2022.22.012

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【Objective】 3-year-old Shine Muscat grape under two-crop-a-year cultivation was used as the material to investigate the differences of physical and chemical indexes of basic quality, flavonoid components and contents between summer grape and winter grape, which would provide the theoretical basis for the quality control of Shine Muscat grape under two-crop-a-year cultivation.【Method】The climatic data, such as the sunshine duration, light intensity, temperature and rainfall during the whole growth period of Shine Muscat grape, were recorded. The physical and chemical indexes of basic quality in berries of summer and winter grapes of Shine Muscat were determined at the young fruit stage, expansion stage, softening stage, beginning maturity stage, and maturity stage, respectively. Meanwhile, the components and contents of flavonols and flavanols in the peel of summer and winter grapes of Shine Muscat grape were detected by LC-MS/MS.【Result】In terms of the climate factors, the summer grape of Shine Muscat displayed weak illumination and low temperature at early growth stage but strong illumination and high temperature at late growth stage of the whole developing period, while the winter grape was opposite. The average sunshine duration, average temperature and effective accumulated temperature in the growth period of summer grape were higher than those of winter grape, but the rainfall was lower than that of winter grape. The hydrothermal coefficient of summer grape was higher than that of winter grape from the beginning of maturity stage to maturity stage. In terms of the basic quality, the content of soluble solid of summer grape was significantly higher than that of winter grape at maturity stage, and the peel thickness of summer grape was significantly lower than that of winter grape. There was no significant difference in the single berry weight, fruit equatorial and longitudinal diameter and titratable acid content between summer and winter grape. In terms of the components and contents of flavonols, the content of total flavonols in peels of summer and winter grape showed a downward trend during the fruit developing stage. The content of total flavonols in different periods of summer grape was significantly higher than that of winter grape. The main flavonol in summer grape was quercetin-3-O-glucoside, while the main flavonol in winter grape was kaempferol-3-O-galactoside. In terms of the components and contents of flavanols, the content of total flavanols in summer and winter grape also showed a downward trend. The eight identical flavanols were detected in the peels of both summer and winter grape, and the main flavanols were catechin, epicatechin and procyanidin B₁. The contents of total flavanols, catechin, epicatechin and procyanidin B₁ in summer grape were significantly lower than those in winter grape during fruit development. The contents of gallic catechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and procyanidin B₂ at fruit maturation stage of summer grape were significantly higher than those of winter grape. The principal component analysis showed that there were differences in flavonols between summer and winter fruits. The regression analysis showed that catechin, quercetin-3-O-Glucoside and procyanidin B₁ were the main components for distinguishing the flavonoids from summer and winter grapes.【Conclusion】 In the study conducting year, the quality of summer grape of Shine Muscat was better than that of winter grape. During the whole grape developing stage, the content of total flavonols in summer grape was significantly higher than that in winter grape, while the content of total flavanols in summer grape was significantly lower than that in winter grape. The main components of flavonols were different between summer and winter grape of Shine Muscat, while the main components of flavanols were the same, namely catechin, epicatechin and procyanidin B₁. The content of the main components of flavanols in winter grapes was significantly higher than that in summer grape, which might probably explained why the astringency taste of winter grape was stronger than that of summer grape. The differences in light and temperature during the growth period may be an important factor that caused the difference in the components and contents of summer and winter grapes of Shine Muscat.

Characteristics of Volatile Components in Peach Fruits of 21shiji and Jiucui and Their Hybrid Progenies

HAO Yan,LI XiaoYing,YE Mao,LIU YaTing,WANG TianYu,WANG HaiJing,ZHANG LiBin,XIAO Xiao,WU JunKai

Scientia Agricultura Sinica. 2022, 55(22):  4487-4499.  doi:10.3864/j.issn.0578-1752.2022.22.013

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【Objective】 The fruit aroma components and content as well as the expression of key genes between peach parents and hybrid progenies were studied to reveal the variation characteristics of peach fruit aroma components and to clear the main components and changes of peach fruit aroma components between parents and hybrid progenies, so as to provide data support for further exploring the genetic and regulatory mechanism of fruit aroma and breeding of peach parents and hybrid progeny.【Method】 In this study, the volatile components and content of peach fruit of 21shiji and Jiucui peach as well as their hybrid progenies were analyzed by SPME-GC-MS, and the expression levels of genes related to ester synthesis (PpAAT1) and ester hydrolysis (PpCXE1) were analyzed by real-time PCR. In addition, the effects on volatile components and content of peach fruits by 1-MCP and ethylene treatments which used to change fruit ripeness were analyzed.【Result】 A total of 125 volatile compounds were detected in hybrid parents and progenies, including alcohols, lactones, acids, terpenoids, ketones, alkanes, alkenes, and esters. The results showed that the number of volatile components of the hybrids was lower than that of the parent Jiucui except for Shijizhixing, but the volatile components of 55.56% progenies were higher than that of these two parents. Esters were the most abundant volatile components detected in different peach fruits, accounting for more than 50.98% of the total volatile content. The contents of esters of these two parents 21shiji and Jiucui accounted for 60.24% and 43.45% of the total volatiles, respectively. Except hy-7 (38.71%), the esters in other hybrids were higher than that in Jiucui peach. Alkanes were also the main volatile components detected in this experiment. The proportion of alkanes in the total volatile content of each hybrid was lower than that of the parent Jiucui, and 66.67% of the hybrid was lower than that of the parent 21shiji. Lactones were not detected in the two parents, but were detected in some progenies, such as Shijizhixing, hy-1, hy-9, hy-16, and hy-18, and the content was significantly higher than that of the two parents. Trans-3-hexene acetate, hexyl acetate, ethyl caprylate, linalool, and dihydrogen -β-ionone were the main components of peach fruit aroma (OAVs > 1). The trans-3-hexene acetate, one of the main components contributing to peach aroma, was not detected in parents, but was detected in 77.78% progenies, and the aroma intensity value was above 1.29, and the highest was 5.04 of Shijizhixing. And γ -decalactone, the main volatile compound of peach fruit, was detected only in 3 progenies. According to the OAVs summing distribution, 21shiji and most of the hybrid progenies were mainly fruity type, while only Jiucui and hy-7 were floral type. The hybrid progeny Shijizhixing owned very strong aroma with both fruity and floral type characteristics, and its OAVs values were significantly higher than both parents and other hybrids. Furthermore, the results showed that 1-MCP inhibited the synthesis of esters and lactones in fruit, while the exogenous ethylene accelerated fruit ripening. And the exogenous ethylene promoted the release of esters in advance, and the content of esters reached the highest on the second day. Moreover, lactones were not detected on the 1-3 days after treatment, and the contents of lactones in the three groups showed an increasing from 4 to 10 days. On the 10th day, the contents of lactones in the ethylene treatment group and 1-MCP treatment group were significantly lower than those in the control group.【Conclusion】 The type, quantity, and content of volatile components were significantly different between 21shiji, Jiucui, and their hybrid progenies. The content of esters and lactones in hybrid progenies were significantly higher than that of their parents. The fruit aroma characteristics of most hybrid progenies were consistent with that of the parent 21shiji, which was fruity type. Some of them had the same floral type as their parent Jiucui, while some of their hybrid progenies was both floral and fruity type. Compared with the control group, 1-MCP treatment inhibited the synthesis of esters and lactones, and weakened the fruit aroma. However, exogenous ethylene promoted the early release of esters and the highest content of esters was not significantly different from that in the control group, but significantly reduced the content of lactones in fruits.

FOOD SCIENCE AND ENGINEERING

Differentiation of Porcine Muscle Stem Cells in Three-Dimensional Hydrogels

CHEN Yu,ZHU HaoZhe,CHEN YiChun,LIU Zheng,DING Xi,GUO Yun,DING ShiJie,ZHOU GuangHong

Scientia Agricultura Sinica. 2022, 55(22):  4500-4512.  doi:10.3864/j.issn.0578-1752.2022.22.014

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【Objective】 The objective of this study was to explore the differentiation effect of porcine muscle stem cells in three-dimensional hydrogels, and to provide a guidance for inducing muscle stem cells to differentiate into muscle tissue in vitro.【Method】 Some porcine muscle stem cells were respectively induced to differentiate under the conditions of 2D and 3D (2D condition means culturing cells in culture dishes; 3D condition means culturing cells in hydrogels). The RNA and protein samples of porcine muscle stem cells cultured in 2D were collected at proliferation, pre-differentiation, early differentiation, mature differentiation, and late differentiation, respectively, and those in 3D were also collected at day 7 and day 14 of differentiation, respectively. Then, RT-qPCR was used to compare the expression levels of the myogenic-related genes, including the genes of MYOG, CAV-3, MyHC-slow, and MyHC-2a, under 2D and 3D differentiation conditions. Correspondingly, the Western Blot was used to detect the expression levels of MyHC protein and MYOG protein in the two conditions. Moreover, the immunofluorescence staining was used to observe the myotubes formed in cell culture dishes and hydrogels. Further, the amino acid content and composition of the cultured muscle tissue were analyzed by an amino acid automatic analyzer at day 14 of differentiation. 【Result】 The porcine muscle stem cells started to fuse to form myotubes at day 3 of differentiation in 2D. The myotubes formed in 2D matured at day 7 and divorced from culture dish afterwards. The porcine muscle stem cells were still globe and had low expression of MYOG and CAV-3 at day 7 of differentiation in 3D. Multinucleated myotubes formed at day 14 and the expression of MYOG and CAV-3 reached levels of 2D differentiation. The cells in hydrogels had higher expression of terminal differentiation genes MyHC-slow and MyHC-2a than the cells in culture dishes. The expression of MyHC-slow was 12 times that at day 7 in 2D and the expression of MyHC-2a was 4 times that at day7 in 2D, but the expression of MyHC protein was only 1/6 that at day 7 in 2D. Amino acid analysis results showed that the contents of 17 hydrolyzed amino acids in cultured muscle tissue were all lower than those in pork, and the ratio of essential amino acids was also lower in cultured muscle tissue, but the ratio of flavor amino acids was higher. 【Conclusion】 The porcine muscle stem cells could differentiate into myotubes in 3D collagen hydrogels in vitro, and the 3D condition was positive to the expression of myogenic differentiation related genes, but further research was needed to achieve high expression of MyHC protein. The flavor amino acid content of the muscle tissue cultured in this way was high, which might mean good flavor.

ANIMAL SCIENCE·VETERINARY SCIENCE

Risk Zoning of Heat Stress Risk Zoning of Dairy Cows in Jiangsu Province and Its Characteristics Affected by Climate Change

REN Yifang,YANG ZhangPing,LING Fenghua,XIAO LiangWen

Scientia Agricultura Sinica. 2022, 55(22):  4513-4525.  doi:10.3864/j.issn.0578-1752.2022.22.015

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【Objective】 The aim of this study was to master the occurrence law of cow heat stress under the background of climate change to optimize the production management of pasture and to promote ecological and healthy breeding level of dairy cows, which could provide the reference for optimizing the layout of animal husbandry, intelligent management and control of pasture, site selection and transformation, benefit improvement and so on. 【Method】Taking Jiangsu Province as an example, the risk index (RI) was constructed based on the temperature humidity index (THI) by using the Era5 data set of global atmospheric reanalysis data from 1980 to 2020, which represented the degree of cow heat stress. The K-means clustering algorithm was selected to realize the risk zoning of heat stress of dairy cows, and the regional evaluation was carried out in combination with the characteristics of occurrence intensity, frequency, start and end time, as well as duration of heat stress. The climate tendency rate was calculated to analyze the change trend of the characteristics of cow heat stress in the different risk areas. Based on the cumulative temperature humidity index (CTHI), Mann-Kendall test was used to determine the climate mutation points in the different risk areas. Furthermore, the impacts of climate change on the occurrence characteristics of cow heat stress in different risk areas were analyzed from the daily and hourly time scales, respectively. 【Result】The risk of heat stress of dairy cows in Jiangsu Province presented the distribution features of "high in the southwest and low in the northeast". The low-risk areas mainly included Huaibei and the middle-eastern area of Yangtze River and Huai River valley. The regional averaged value of THI was 73.63, and the mild heat stress mainly occurred. The high-risk areas mainly included the areas along the southern Jiangsu and the west area of Yangtze River and Huai River valley. The regional averaged value of THI was 75.12, and the occurrence frequency of mild and moderate thermal stress was nearly the same. In the low-risk and high-risk areas, the start and end time of heat stress showed an advanced and delay trend, and the duration days showed an extended trend of 4.0 d/(10a) and 4.2 d/(10a), respectively, the values of THI all showed an increasing trend of 0.2/(10a), while the value of CTHI showed an increasing trend of 301.2/(10a) and 256.1/(10a), respectively. The frequencies of mild thermal stress were bimodal, which mainly occurred from the early-June to the mid-July, and from the early-August to the middle-September, while the frequency of moderate thermal stress was unimodal, which mainly occurred from mid-July to mid-August. The change of daily heat stress intensity basically presented a distribution of "sinusoidal", and the high incidence period was concentrated in 11:00-17:00. Affected by climate change, the heat stress of dairy cows in Jiangsu Province showed an obvious increasing trend, reaching a small peak in 2010. Then after a decline, it showed a steady strengthening trend, which exceeded the threshold of 0.05 significance level. In the low and high risk areas, the highly impacted periods of cow heat stress were prolonged, the occurrence frequency increased, the coverage increased, and the starting-time moved forward. The daily beginning time of the high incidence period of cow heat stress moved forward for about 1 hour, and the intensity of heat stress in high-risk areas was basically increased close to the medium level. 【Conclusion】 Based on THI, RI and CTHI, the risk zoning assessment and climate impact analysis of cow heat stress could be realized, the high-risk areas as well as key prevention and control periods of cow heat stress could be determined, and the climate change trend could be grasped. With the climate change, the heat stress of dairy cows in Jiangsu Province showed the characteristics of "earlier, stronger, longer and more", which should be actively dealt with.

Genetic Diversity of Indigenous Yellow-Feathered Chickens in Southern China Inferred from Mitochondrial DNA D-Loop Region

HUANG XunHe,WENG ZhuoXian,LI WeiNa,WANG Qing,HE DanLin,LUO Wei,ZHANG XiQuan,DU BingWang

Scientia Agricultura Sinica. 2022, 55(22):  4526-4538.  doi:10.3864/j.issn.0578-1752.2022.22.016

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【Objective】 Indigenous yellow-feathered chicken is an important poultry genetic resource in China. The genetic diversity of these chickens was evaluated to facilitate scientific protection policies and to improve their use.【Method】 A total of 1283 mitochondrial DNA fragments (mtDNA D-loop, 519 bp) were investigated including 694 de novo and 589 previously published units from 28 indigenous yellow-feathered chicken breeds from southern China. These data were subsequently used to calculate the genetic diversity, to construct median-joining networks of haplotypes, and to investigate geographical distribution patterns of haplotypes by using principal coordinate analysis, analysis of molecular variance, and neutral test. The origin of rare haplogroups was inferred from the geographical distribution pattern of mitochondrial DNA haplogroups of Asian and Pacific indigenous chickens.【Result】One hundred and one mutation sites were detected in 1 283 samples, of which 92 were polymorphic. One hundred and sixty-nine haplotypes, belonging to haplogroups A-E and G, were defined. The predominant haplogroups were A (15.43%), B (49.26%), C (18.55%), and E (16.37%), while D (0.31%) and G (0.08%) were the rare haplogroups within the total samples. Chickens from Henan Province covered all six haplogroups; Chickens from Guangdong Province had the largest number of haplotypes (64), whereas those from Zhejiang (19) and Hainan (12) Provinces had the lowest. Haplogroups A and B occurred in all 28 breeds. Haplogroup D existed only in Huainan yellow, Gushi, Ningdu yellow and Xiayan chickens, whereas haplogroup G only occurred in Gushi chickens. The distribution frequency of haplogroup D and the number of haplotypes indicated that haplogroup D of indigenous yellow-feathered chickens in southern China could originated from Southeast Asia. The evidence from geographical distribution and median-joining networks of haplogroup G indicated that haplogroup G of chickens from Henan Province and South Asia could originated from Southwest China. The indigenous yellow-feathered chickens in southern China had a total haplotype diversity of 0.903±0.005 and nucleotide diversity of 0.01269±n.d. Indigenous yellow-feathered chickens from Henan and Hunan Provinces had the highest level of haplotype diversity of 0.916±0.011 and 0.01358±0.00039, respectively, and the nucleotide diversity of 0.913±0.012 and 0.01345±0.00042, respectively. The lowest level of haplotype diversity was found in Hainan Province (0.736±0.076), and the lowest level of nucleotide diversity was found in Jiangxi Province (0.00981±0.00072). Huainan yellow, Gushi and Yunyang da chickens retained the highest level of haplotype diversity; Jianghan, Huainan yellow and Huanglang chickens had the highest level of nucleotide diversity, whereas the haplotype diversity and nucleotide diversity in Hongshan, Guangxi yellow and Xiaoshan chickens were the lowest. Wenchang chickens were genetically close to Hentian chickens, which was consistent with their breeding history. The population genetic structure of yellow-feathered chickens was less clear, and the molecular variance component of within-population was significantly higher than others. The neutral test indicated that the yellow-feathered chickens in southern China did not sustain obvious population expansion at the breed level, with the exception of haplogroups A, B and E.【Conclusion】 The results suggested that yellow-feathered chickens had a good conservation status with high level of genetic diversity, although Hongshan, Xiaoshan and Guangxi yellow chickens required further protection. Hybridization between chickens was common in yellow-feathered chickens, which had experienced population expansion. Indigenous chickens from Southeast Asia and Southwest China had substantial genetic contributions to indigenous yellow-feathered chickens in southern China.

Association of Levels of Cholecystokinin A Receptor Expression and Sequence Variants with Feed Conversion Efficiency of Lueyang Black-Boned Chicken

WANG ZhePeng,ZHOU WenXin,HE JunXi,HU QiaoYan,ZHAO JiaYue

Scientia Agricultura Sinica. 2022, 55(22):  4539-4549.  doi:10.3864/j.issn.0578-1752.2022.22.017

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【Background】 Increased appetite and feed conversion efficiency forms a physiological basis underlying high growth of modern commercial broiler chickens. Cholecystokinin A receptor (CCKAR) is a key receptor for mediating CCK signals and involving in regulation of appetite and digestion. In view of the important role of CCK signals in the control of energy balance, the levels of CCKAR expression are closely related to the growth of chickens. However, the mechanism by which the abundance of CCKAR affects the growth of chickens remains unclear. 【Objective】 The aim of this study was to elucidate the relationship of levels of CCKAR expression and sequence variants with feed conversion efficiency, which would promote the understanding of genetic basis of the growth traits and breeding for improvement of feed efficiency in the chicken. 【Method】The feed to meat conversion ratio of Lueyang black-boned chickens (rooster n=62, pullet n=67) from 12 to 16 weeks of age and the feed to egg conversion ratio from 26 to 30 weeks of age (n=150) were individually measured. Two synonymous mutations (g.73206714T>C and g.73209189C>T) in the CCKAR gene were genotyped by using PCR-RFLP. Association of two SNP with the feed to meat conversion ratio was tested by using two-way analysis of variance with sex treated as a fixed effect. Association of two SNP with the feed to egg conversion ratio was tested by using one-way analysis of variance. Disrupting effect of g.73206714T>C on transcription factor binding sites was predicted in silico by using JASPAR database. Ten birds that were heterozygotes (CT) at the g.73206714T>C locus were selected to analyze imbalance of CCKAR expression between T and C alleles. The difference of allele-specific expression was detected by using PCR-RFLP. The Levels of CCKAR expression were detected by qPCR in the hypothalamus, pituitary, pancreas, gallbladder and duodenum of Lueyang black-boned chickens (n=30). The correlation of levels of CCKAR expression with food intake and feed to meat conversion ratio was analyzed. 【Result】 Sex had a significant (F=29.44，P<0.0001) effect on the feed to meat conversion ratio, and the feed efficiency (5.41±0.76) of roosters was significantly superior to pullets (6.62± 1.00). Of two SNP, only g.73206714T>C was significantly associated with the feed to meat conversion ratio. The feed to meat conversion ratio (5.18±0.63) of the TT genotype was significantly lower than ones of CC (6.37±1.1) and CT (6.17±0.95) genotypes. The feed to meat conversion ratio had no significant difference between CC and CT. There was no significant interaction effect between genotypes of g.73206714T>C and sex. G.73206714T>C potentially disrupted binding sites of 28 transcription factors. Allele-specific expression assay showed that the abundance of transcripts with the T allele was higher than one of transcripts with the C allele, supporting that differential expression of CCKAR gene between alleles might likely contribute to genetic association of g.73206714 with the feed to meat conversion ratio. Average feed to egg conversion ratio of Lueyang black-boned chickens was 2.85±0.41 at the 26-30 weeks of age. Both g.73206714T>C and g.73209189C>T had no significant effect on the feed to egg conversion ratio. The levels of CCKAR expression in the pancreas were the highest among five tissues, which showed a negative correlation (r=-0.45, P=0.017) with feed intake. But there was no significant correlation between levels of CCKAR expression and feed intake in other tissues. In the pancreas (r=-0.41, P=0.03) and pituitary (r=-0.57, P=0.0018), the higher levels of CCKAR expression was, the lower the feed to meat conversion ratio was. But in other tissues, there was no the negative correlation.【Conclusion】 This study found that increased levels of CCKAR was associated with high feed efficiency. The T allele of g.73296714T>C was associated with high expression activity of CCKAR gene, and with high feed efficiency.

RESEARCH NOTES

Effect of Deep Tillage Under Continuous Rotary Tillage on Yield Formation of High-Quality Japonica Rice in Cold Regions

ZHAO LiMing,HUANG AnQi,WANG YaXin,JIANG WenXin,ZHOU Hang,SHEN XueFeng,FENG NaiJie,ZHENG DianFeng

Scientia Agricultura Sinica. 2022, 55(22):  4550-4566.  doi:10.3864/j.issn.0578-1752.2022.22.018

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【Objective】 The effects of deep tillage under continuous rotary tillage on growth dynamics, photosynthetic matter production and yield formation of different japonica rice were studied in order to provide the technical support for high yield and high efficiency cultivation of high quality japonica rice in cold regions. 【Method】 A field experiment was conducted in 2018-2019, under the condition of straw returning to the field, on the basis of rotary tillage in the previous stubble for two consecutive years. Here, two tillage practices, namely, deep tillage and rotary tillage, were adopted during the growth stages of three modern japonica varieties Suijing18, Kendao12, and Sanjiang6. The effects of deep tillage and rotary tillage on the growth dynamics and post-anthesis production characteristics of high-quality japonica rice were studied. 【Result】 There was no significant difference in yield between years, but tillage methods had significant effects on the growth and development, photosynthetic dry matter production characteristics and yield characters of high-quality japonica rice in cold regions. Compared with rotary tillage, the deep tillage significantly increased the number of tillers per square meter and the number of effective panicles, the flag leaf unfolded later and lasted longer, and the heading stage was late but the duration did not change; The biomass and dry matter transport capacity of stem and sheath under deep tillage increased, and the biomass and stem-sheath dry matter transport capacity were increased by 8.34% and 5.36% respectively at full heading stage; The output, output rate and transformation rate of stem-sheath matter under deep tillage increased by 13.19%, 6.70% and 9.17%, respectively (P<0.05); The deep tillage increased the leaf area index at full heading and mature stage, prolonged the duration of green leaf area and increased the population growth rate; The internode length, leaf length and width of the third and fourth nodes of the main stem under deep tillage were increased, and the plant height and ear length were increased too; The number of grains per ear and grain weight per ear under deep tillage increased by 7.05% and 3.37%, respectively, the harvest index increased by 1.90%, and the average yield increased by 12.78%. Under the same tillage method, Kendao12 was the best in tiller number, photosynthetic matter production capacity, stem and sheath dry matter accumulation and transport capacity, yield and its composition, followed by Suijing18; However, Sanjiang6 had higher leaf area index after anthesis, grain number per panicle and grain weight at mature stage, but which could not make up for its low dry matter transport capacity, effective panicle number and 1000-grain weight. In terms of interaction effect, the combination of deep tillage × Kendao12 treatment showed higher effective panicles per square meter, strong production and transport capacity of photosynthetic matter after anthesis, high grain-leaf ratio and population growth rate, high 1000-grain weight and harvest index, reasonable allocation of plant agronomic morphological characters and coordination of yield traits, which could achieve a yield increase of 9.15%-27.47%.【Conclusion】 Continuous rotary tillage combined with one-year deep tillage was the most effective and sustainable rice tillage system to improve the yield of high-quality japonica rice in this region.