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    Breeding and Application of a New Thermo-Tolerance Rice Germplasm R203
    LIU Gang, XIA KuaiFei, WU Yan, ZHANG MingYong, ZHANG ZaiJun, YANG JinSong, QIU DongFeng
    Scientia Agricultura Sinica    2023, 56 (3): 405-415.   DOI: 10.3864/j.issn.0578-1752.2023.03.001
    Abstract449)   HTML69)    PDF (2812KB)(276)       Save

    【Objective】 The global warming has led to the increasingly serious heat damage on the heading and flowering stage of rice. To reduce the impact of heat damage on rice production and to ensure food security in China and even the world, new rice germplasms with thermo-tolerance on heading stage should be identified and new thermo-tolerance varieties need be bred. 【Method】Guanghui 128 (Qiguizao/Ce64//Minghui 63) was used as the heat resistant parent, through hybridization, multiple crossing and pedigree selection, the lines with high seed setting rate and small variation on heading and flowering stage during the high temperature were screened out for several generations’ breeding process. Then the selected higher generation lines were identified to create new thermos-tolerance rice germplasms in artificial climate chamber (The treated plants will be moved into the chamber on the flowering day, high temperature treatment is 9:00-15:00, 38℃, 15:01-8:59 28℃, the relative humidity is 75%, and the treatment lasts for 7 days), with analysis of agronomic trait. 【Result】The new germplasm R203 has stronger thermo-tolerance and higher seed setting rates under both normal and high temperature conditions (94.5% at normal temperature, 81.9% at high temperature, and 86.7% at relative). Its agronomic traits, quality and comprehensive resistance all meet the production standards. Above all, R203 has the potential to breed new thermos-tolerance hybrid rice varieties. The seed setting rates of 7 hybrid combinations with R203 as the male parent and seven three-line male sterile lines as the female parent were between 83.4%-99.4% under natural high temperature conditions. Among them, Taiyou 203, a new three-line medium indica hybrid rice has good qualities, the seed setting rate was 87.9%, the comprehensive relative heat resistance coefficient was 1.11, and the heat resistance reached level 1. In the production test, the yield increased by 5.36% compared with the control, and the yield increase point accounted for 85.71%. It has good high and stable yield, and the rice quality reached the second level of the ministerial standard. Thus Taiyou 203 has good promotion and application value. 【Conclusion】Currently, basic research on heat resistance is not enough to support the breeding of new practical heat resistant varieties, the rice resources in areas prone to high temperature and humidity are preferred as materials for breeding new heat tolerance lines, a new heat-resistant rice variety R203 was created by phenotypic selection, and a practical heat-resistant rice variety Taiyou 203 was developed by using heterosis.

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    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
    Abstract456)   HTML76)    PDF (3232KB)(387)       Save

    【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.

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    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
    Abstract407)   HTML56)    PDF (4862KB)(204)       Save

    【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.

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    Phenotypic Analysis and Gene Cloning of Rice Panicle Apical Abortion Mutant paa21
    HE Lei,LU Kai,ZHAO ChunFang,YAO Shu,ZHOU LiHui,ZHAO Ling,CHEN Tao,ZHU Zhen,ZHAO QingYong,LIANG WenHua,WANG CaiLin,ZHU Li,ZHANG YaDong
    Scientia Agricultura Sinica    2022, 55 (24): 4781-4792.   DOI: 10.3864/j.issn.0578-1752.2022.24.001
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    【Objective】Rice panicle apical abortion affects yield. Identification and cloning of genes related to rice panicle apical abortion can enrich the molecular mechanism of rice panicle development regulation, and provide theoretical basis and genetic resources for rice high-yield molecular design breeding. 【Method】Here, a stably inherited panicle apical abortion 21 (paa21) mutant was screened from EMS mutant library of the japonica rice variety "Wuyunjing 30". Agronomic traits, such as ratio of degraded primary branches, degraded apical spikelets, grains per panicle, plant height, panicle length, and grain yield per plant, were statistically analyzed. Trypan blue and Evans blue staining were used to detect whether programmed cell death occurred in the apical spikelets. H2O2 content in young panicles at different development stages and different panicle parts of WT and paa21 was determined. Genetic analysis was carried out by reciprocal cross of paa21 with indica rice II-32B and 9311 respectively. The F2 population constructed by crossing paa21 with indica rice II-32B was used for gene mapping and cloning. The three-dimensional structure of wild-type and paa21 proteins were predicted using SWISS-MODEL website. The expression levels of ROS response marker genes, programmed cell death related genes and catalase related genes were analyzed by RT-qPCR. 【Result】paa21 produced panicle apical abortion phenotype and the degenerated spikelets were mainly located on the primary branches at the apical panicle. The plant height, grain number per panicle, panicle length and grain yield per plant of paa21 were lower than those of WT. After observing the young panicles at different development stages, we found that the paa21 mutant had a panicle apical abortion phenotype when panicle developed to 12 cm. Trypan blue and Evans blue staining results showed that the apical spikelets of the paa21 mutant had programmed cell death. Stronger DAB staining was observed in the degenerated apical spikelets of paa21 than WT. The results of H2O2 content determination showed that higher level of ROS was accumulated in panicle of paa21 compared with WT. Genetic analysis suggested that paa21 mutant phenotype is controlled by a pair of recessive nuclear genes. The results of map-based cloning showed that a C to T mutation occurred in the second exon of Os02g0673100 in paa21, resulting in the mutation of alanine to valine. This gene encodes an aluminum activated malate transporter, ALMT7. The mutation site was located at the fourth transmembrane helix. SWISS-MODEL prediction results showed that the mutation site did not significantly affect the three-dimensional structure of the mutant protein. The expression level of ROS response marker genes Os01g0826400, Os05g0474800 and Os02g0181300 in paa21 was significantly higher than that in WT when the young spike developed to 10 cm. Compared with WT, the expression level of programmed cell death related genes VPE2 and VPE3 increased significantly in paa21. The expression level of CATA, CATB and CATC which encode catalase in 10 cm young panicle of paa21 was significantly higher than that of WT. The activity of CAT in paa21 10 cm young spikelet was significantly lower than that of WT. 【Conclusion】paa21 accumulate excess ROS in the apical spikelet at late stage of panicle development, resulting in programmed cell death, which eventually leads to the degeneration of the apical spikelet. These results lay a good foundation for further enriching the genetic regulatory network of panicle development.

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    Dynamic Changes of Gibberellin Content During the Development and Its Relationship with Yield of Brassica napus L.
    XIE LingLi,WEI DingYi,ZHANG ZiShuang,XU JinSong,ZHANG XueKun,XU BenBo
    Scientia Agricultura Sinica    2022, 55 (24): 4793-4807.   DOI: 10.3864/j.issn.0578-1752.2022.24.002
    Abstract255)   HTML31)    PDF (765KB)(123)       Save

    【Objective】It was found that there were significant genetic differences between Chinese and European Brassica napus lines, and the yield advantage of hybrid F1 was obvious. To explore the effect of gibberellin (GA) on the growth and development of B. napus, the dynamic changes of GA during the developmental process of B. napus from different lines were measured. For explaining the role of GA in the formation of yield, the transcriptional levels of key enzyme genes involved in GA synthesis were analyzed, and the relationship between GA content and yield in parents and F1 was clarified. 【Method】The dynamic changes of GA content in B. napus lines (15 Chinese lines and 15 European lines) with different sources from November 2020 to May 2021were determined using high performance liquid chromatography. Two F1hybrid lines (YG2009×YC4, ZS11×YC4) with strong heterosis and their parents were used as materials to investigate the changes of GA content in different periods (D1, 15 January 2022; D2, 15 February 2022; D3, 15 March 2022)and measure growth indicators (plant height, root length, fresh weight, etc.), yield and component factors (silique number per plant, seed number per silique and thousand-seed weight), as well as photosynthetic indicators (photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration).The change trend of GA content with temperature and the relationship between GA content and agronomic characters, photosynthetic characteristics and yield of parents and F1hybrid from November 2021 to May 2022 were analyzed. Besides, the transcriptional levels of key enzyme genes involved in GA synthesis of the parents and F1 at different stages (D1, D2, D3) also examined by quantitative real-time PCR. 【Result】The content of GA was closely related to the ambient temperature, and the GA content in the tested B. napus lines decreased first and then increased with the change of temperature. However, it was found that the average content of GA in B. napus lines from Europe was higher than that of Chinese lines. F1 showed significant yield heterosis. Compared with the male and female parents, the yield of YG2009×YC4 and ZS11×YC4 increased by 18.06% and 10.35%, and 29.92% and 28.6%, respectively. There were significant differences in agronomic traits between parents and F1, and the yield was correlated with GA content. The results also showed that the transcriptional levels of GA20ox4, GA3ox2 and SLR1 in parents and hybrid F1 varied greatly. 【Conclusion】The GA content of B. napus lines from Europe was more sensitive to temperature changes, but its average content of GA was higher than that from China. The yield of F1 between European rape and Chinese rape lines showed strong heterosis, which was correlated with GA content. The transcriptional levels of GA20ox4 and GA3ox2 regulated the GA content.

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    Screening of Core Markers and Construction of DNA Fingerprints of Semi-Waxy Japonica Rice Varieties
    ZHAO ChunFang,ZHAO QingYong,LÜ YuanDa,CHEN Tao,YAO Shu,ZHAO Ling,ZHOU LiHui,LIANG WenHua,ZHU Zhen,WANG CaiLin,ZHANG YaDong
    Scientia Agricultura Sinica    2022, 55 (23): 4567-4582.   DOI: 10.3864/j.issn.0578-1752.2022.23.001
    Abstract392)   HTML67)    PDF (2482KB)(218)       Save

    【Objective】 A set of variety DNA fingerprint identification system based on the core markers of genes regulating rice important traits was constructed, which will establish a foundation for strengthening the germplasm management and protection of the mainly promoted semi-waxy japonica rice varieties with high eating quality.【Method】 34 semi-waxy japonica rice varieties mainly cultivated in Jiangsu, Zhejiang and Shanghai were used as the test materials. The key differential sequence sites in genes regulating rice important traits were screened and core SNP or InDel markers were developed through multiple methods such as polymorphism testing of existing markers, gene sequence alignment from public databases and genome resequencing. SNP markers were developed into simple PCR markers based on electrophoretic bands by As-PCR technology. Genotype information was obtained by electrophoretic band characterization and type analysis, and the DNA fingerprint database of the semi-waxy japonica rice varieties was constructed..【Result】 54 core markers derived from 40 key genes regulating rice important traits were obtained, including 18 SNP and 36 InDel markers; 155 characteristic and effective bands were identified by 54 markers in the tested rice varieties, which were transformed into 155 0/1 data sites. The DNA fingerprint database of each variety was established and could distinguish it from all tested varieties. Genetic diversity analysis showed that the variation range of genetic similarity among varieties was 0.47-0.90, among which the lowest similarity coefficient was detected between Nanjing 7718 and Suxiangjing 100, while the highest similarity coefficient was detected between Nanjing 9308 and Nanjing 9036, among which there were 8 differential data sites. Genetic relationship analysis indicated that 34 varieties were divided into 6 branches, of which Nanjing 7718 was an independent branch, suggesting it has a distant relationship from other varieties. Further verification of the identification effect of core markers showed that the set of markers could effectively distinguish 14 new semi-waxy japonica rice varieties. The cluster diagram showed that they were distributed in three groups of Ⅱ, Ⅲ and Ⅳ, confirming the differences of genotype information among varieties; using this set of markers, the authenticity of an unknown semi-waxy rice variety was also identified. According to genotype and cluster analysis, it was determined as Nanjing 9108.【Conclusion】 After optimization and screening, 54 core markers that could accurately distinguish all the tested semi-waxy japonica varieties were obtained, and developed into simple PCR markers detected by electrophoresis. Using this set of marker combinations, the DNA fingerprints of 34 semi-waxy japonica varieties in Jiangsu, Zhejiang and Shanghai were constructed.

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    Identification of Wheat Circular RNAs Responsive to Drought Stress
    LI Ning,LIU Kun,LIU TongTong,SHI YuGang,WANG ShuGuang,YANG JinWen,SUN DaiZhen
    Scientia Agricultura Sinica    2022, 55 (23): 4583-4599.   DOI: 10.3864/j.issn.0578-1752.2022.23.002
    Abstract347)   HTML50)    PDF (2182KB)(171)       Save

    【Objective】 Drought is one of the foremost abiotic stress limiting global wheat production. Exploring the molecular mechanism of wheat response to drought stress have great significance in wheat molecular breeding. Circular RNAs (circRNAs) have been proved to play an important role in the process of plants tolerance to environmental stresses. Therefore, identifying circRNAs involved in drought stress response will help to construct a regulatory network of wheat drought tolerance, and lay a foundation for analyzing the drought resistance mechanism in wheat. 【Method】 In this study, two wheat varieties (Zhoumai13 and Jimai38) with significant differences in drought resistance were used and circRNA-seq was performed on their root samples under well-watered and drought conditions. Differentially expressed circRNAs related to drought stress response were screened based on the identified circRNAs and their microRNAs (miRNAs) targets were predicted. Further, potential circRNA-miRNA-mRNA regulatory modules related to wheat drought stress response were constructed according to the expression patterns of miRNAs and their target genes under drought stress..【Result】 A total of 1 409 wheat circRNAs were identified, most of which (68.91%) were exonic circRNAs. Only 133 circRNAs were simultaneously identified in both varieties. A total of 239 differentially expressed circRNAs were identified under drought stress, of which 138 circRNAs were specifically differentially expressed in the drought-resistant variety Zhoumai 13 (ZM13), and 19 circRNAs were differentially expressed simultaneously in both varieties. Besides, 34 targeted miRNAs and 1 408 miRNA target genes were predicted. Based on the expression patterns of these differentially expressed circRNAs, targeted miRNAs, and miRNA target genes, five potential circRNA-miRNA-mRNA regulatory modules centered on tae-miR9664-3p, tae-miR1122b-3p, tae-miR9662a-3p, tae-miR6197-5p and tae-miR1120c-5p in response to drought stress were screened..【Conclusion】 Wheat circRNAs have obvious specificity in different cultivars and different expression patterns among different drought-tolerant wheat cultivars. A total of 239 wheat circRNAs and 5 potential circRNA-miRNA-mRNA regulatory modules in response to drought stress were identified in the present study.

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    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
    Abstract531)   HTML107)    PDF (5241KB)(252)       Save

    【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.

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    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
    Abstract313)   HTML58)    PDF (2550KB)(177)       Save

    【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.

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    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.

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    PSORA: A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites
    MA XueMeng,YU ChengMin,SAI XiaoLing,LIU Zhen,SANG HaiYang,CUI BaiMing
    Scientia Agricultura Sinica    2022, 55 (15): 2875-2882.   DOI: 10.3864/j.issn.0578-1752.2022.15.001
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    【Objective】The purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites. 【Method】A T-DNA insertion sites analysis approach based on high-throughput sequence technologies was developed, called PSORA: Parallel sequencing of one round amplicons. The process involves high-throughput amplicon sequencing of a round of thermal asymmetric PCR (TAIL-PCR) and bioinformatics analysis of T-DNA insertion sites, which reduces concerns about the specificity of TAIL-PCR. In PSORA, only two primers are required, a degenerate primer and a T-DNA specific primer. A 6-nt Barcode was designed at the 5’ end of the specific primers for labeling different transgenic events. All five transgenic events (L1, L6, L9, L15 and L19) of tobacco used in this study were produced via Agrobacterium mediated transformation with plasmids pBI121. In addition, the results of PSORA are confirmed by standard PCR. 【Result】The T-DNA insertion sites of five transgenic events were analyzed by PSORA. The results showed that L6 contained two insertion sites (36 316 bp on NW_015801367 and 42 202 bp on NW_015950898), the lines of L9, L15 and L19 each contained one insertion site (The insertion site of L9 was located at 235 969 bp on NW_015943682. The insertion site of L15 was located at 60 529 bp on NW_015802951 and the insertion site of L19 was located at 12 188 bp on NW_015863435), but the insertion site of L1 could not be detected. PCR was performed to validate the results from bioinformatics analysis, transgenic events with different insertion sites were used as negative controls for each other, and the wild type (WT) was used as a blank control. The results showed that specific amplification consistent with expectations was obtained in each transgenic event. The effectiveness of PSORA was successfully confirmed. 【Conclusion】PSORA is an effective strategy to analyze T-DNA insertion sites. PSORA can parse the comprehensive molecular characteristics of all T-DNA insertion events simultaneously, making it simpler and faster than the traditional methods of genome walking.

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    QTL Analysis for Growth Period and Panicle-Related Traits in Foxtail Millet
    GUO ShuQing,SONG Hui,CHAI ShaoHua,GUO Yan,SHI Xing,DU LiHong,XING Lu,XIE HuiFang,ZHANG Yang,LI Long,FENG BaiLi,LIU JinRong,YANG Pu
    Scientia Agricultura Sinica    2022, 55 (15): 2883-2898.   DOI: 10.3864/j.issn.0578-1752.2022.15.002
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    【Objective】Growth period and panicle-related traits are the main factors affecting foxtail millet yield and adaptability. To analyze the molecular genetic mechanisms of the growth period and panicle-related traits, it is necessary to map the related genes. 【Method】A recombinant inbred line (RIL) population of foxtail millet consisting of 400 lines derived from a cross between the elite varieties, Yugu18 and Jigu19, were used in this study. Phenotype surveys were carried out across four independent environments in 2018—2019 to study the days from emergence to heading and heading to maturity, the growth period, panicle length, panicle diameter, and single panicle weight. Based on a high-density genetic map that spanned 2 196 cM of the foxtail millet genome with an average of 1.68 cM/bin, composed of 1 304 bin markers, QTL for growth period and panicle-related traits were mapped using composite interval mapping (CIM), and the candidate genes of the confidence interval were predicted. 【Result】The growth period and panicle-related traits of RIL population exhibited continuous distribution with apparent transgressive segregation in the 4 environments, which accorded with the genetic characteristics of typical quantitative traits and were suitable for QTL genetic analysis. Correlation analysis showed that the days from emergence to heading were significantly positively correlated with the growth period, and negatively correlated with those heading to maturity. A positive correlation between panicle length and diameter was also observed. A total of 88 QTL for the growth period and panicle-related traits were detected on chromosomes 1, 3, 5, 6, 8 and 9, respectively. Among them, 45 QTL were significantly associated with the days from emergence to heading and explained 1.61%-27.60% of the phenotypic variance; seven QTL were significantly associated with the days from heading to maturity and explained 2.65%-12.14% of the phenotypic variance; 20 QTL were significantly associated with the growth period and explained 1.98%-16.97% of the phenotypic variance; nine QTL was significantly associated with the panicle length and explained 3.51%-11.65% of the phenotypic variance; five QTL were significantly associated with the panicle diameter and explained 3.74%-8.34% of the phenotypic variance; two QTL was significantly associated with the single panicle weight and explained 5.16%-5.20% of the phenotypic variance. A total of 12 major QTL were detected in this study, of which five major QTL, including qEHD-9-1, qEHD-9-2, qHMD-9-2, qGRP-9-2 and qPL-5-1, were repeatedly detected in at least two environments and BLUP value. The genomic regions of qEHD-9-1, qHMD-9-1, qGRP-9-1 and qPL-9-1 overlapped on chromosome 9, the genomic regions of qEHD-9-2, qHMD-9-3, qGRP-9-2 and qPL-9-3 overlapped on chromosome 9, and the genomic regions of qPL-5-1 overlapped with qPD-5-1 on chromosome 5. Five candidate genes related to the growth period and panicle-related traits were identified from the confidence interval of the 3 QTL clusters, of which two candidate genes played an important role both in the growth period and panicle-related traits.【Conclusion】A total of 88 QTL for the growth period and panicle-related traits were detected, and 12 were major QTL, of which 5 major QTL were repeatedly detected in multiple environments by clustering distribution. Five candidate genes related to the growth period and panicle-related traits were identified via gene annotation.

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    Cloning of the Soybean Single Zinc Finger Protein Gene GmSZFP and Its Functional Analysis in SMV-Host Interactions
    ZHAO DingLing,WANG MengXuan,SUN TianJie,SU WeiHua,ZHAO ZhiHua,XIAO FuMing,ZHAO QingSong,YAN Long,ZHANG Jie,WANG DongMei
    Scientia Agricultura Sinica    2022, 55 (14): 2685-2695.   DOI: 10.3864/j.issn.0578-1752.2022.14.001
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    【Objective】The molecular mechanism underlying the resistance to soybean mosaic virus (SMV) infection in soybean is of great importance as soybean mosaic caused by SMV has become one of the major soybean disease worldwide. We have previously performed transcriptome analysis of SMV-inoculated soybean after inhibition of H2O2 production and have identified a differentially expressed C2H2-type single zinc finger protein gene, Glyma.18G003600.1, named GmSZFP. In this study, we use virus induced gene silencing (VIGS) technique to investigate the function of GmSZFP in soybean-SMV interaction, providing a foundation for further investigation of the molecular mechanism of GmSZFP in soybean-SMV interaction. 【Method】Soybean cultivar Jidou 7 and SMV strains SC-8 (susceptive) and N3 (resistance) were used as the materials in this study. Bioinformatic analysis was conducted to predict the protein domains of GmSZFP; its transcription factor activity was measured by transcriptional activation assay in yeast; the expression characteristics of GmSZFP at the transcriptional level in soybean-SMV interaction were verified by real-time quantitative PCR (qPCR); and the function of GmSZFP in soybean-SMV interaction was investigated by VIGS technique. 【Result】The CDS region of GmSZFP gene was cloned; amino acid sequence analysis and transcriptional activation assay in yeast revealed that GmSZFP is a C2H2-type zinc finger protein transcription factor with transcriptional activation activity; qPCR results showed that GmSZFP was strongly induced by SMV inoculation, and the expression pattern was different between the compatible and the incompatible combinations. The expression of GmSZFP was elevated after SMV inoculation, and then decreased in the incompatible combination, and the expression level of GmSZFP was significantly lower in the compatible combination than that in the former. Moreover, the expression level of GmSZFP was found to be reduced to the level that is similar to the level in the compatible combination that was pre-inoculated with imidazole, indicating that GmSZFP responds to SMV infestation at the transcriptional level and is regulated by H2O2; After silencing GmSZFP, we found that callose at the SMV inoculation site was greatly reduced compared to the control, and the expression of callose synthase genes GmGSL7c and GmGSL12b was reduced compared to the control, and the expression of callose hydrolase gene BG was elevated compared to the control; In addition, after GmSZFP was silenced, the virus spread outward to a distance of 2 mm at 72 h and to a distance of 3 mm at 96 h from the central source after SMV was inoculated in a small area, while the expression of SMV capsid protein (CP) gene was not detectable outside the inoculation site in control leaves; 10 d after SMV inoculation, the upper leaves (of the SMV inoculated leaves) in the GmSZFP-silenced plants showed mosaic, greening and curling symptoms, and CP gene was expressed, indicating that silencing of GmSZFP enabled SMV to transport in an unrestricted manner. 【Conclusion】GmSZFP is a canonical C2H2-type mono-zinc finger protein, and the GmSZFP gene plays a positive regulatory role in soybean resistance to SMV infection.

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    Effect of Salicylic Acid Priming on Salt Tolerance of Kenaf Seedlings
    HU YaLi,NIE JingZhi,WU Xia,PAN Jiao,CAO Shan,YUE Jiao,LUO DengJie,WANG CaiJin,LI ZengQiang,ZHANG Hui,WU QiJing,CHEN Peng
    Scientia Agricultura Sinica    2022, 55 (14): 2696-2708.   DOI: 10.3864/j.issn.0578-1752.2022.14.002
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    【Objective】To study the growth and physiological response of salicylic acid (SA) priming in kenaf under salt stress, and further reveal the induction pattern of SA priming on the stress-related genes in kenaf, thus provide a theoretical basis for salt tolerance study in kenaf. 【Method】Two different salt-tolerant kenaf cultivars (resistant and sensitive cultivars codenamed CP018 and CP047, respectively) were used as materials. The seeds were tested by SA priming and then subjected to hydroponics experiments to analyze the effect of SA priming on kenaf seed germination and the agronomic and physiological aspects of seedling under 150 mmol·L-1 NaCl stress, and the expression patterns of SA priming stress-related genes were analyzed by qRT-PCR. 【Result】The germination rate, germination potential and germination index of the salt-resistant cultivar CP018 were significantly improved after 0.2 mmol·L-1 SA priming, by 34.78%, 31.30% and 58.07%, respectively; the salt-susceptible cultivar CP047 also showed some improvement, by 7.50%, 10.56% and 6.23%, respectively, but did not reached the significant level. Under salt stress conditions, plant height inhibition was significantly reduced by 4.07% (CP018) and 3.91% (CP047) in the 2 cultivars by SA priming (S1) compared with un-priming (N1), and dry weight inhibition was significantly reduced by 15.50% (CP018) and 15.68% (CP047), in the 2 cultivars, respectively; fresh weight inhibition was significantly reduced by 4.46% in CP047, but not in CP018. Analysis of the root systems showed that root length inhibition was significantly reduced by 10.74% (CP018) and 10.77% (CP047) in the two cultivars, respectively, root surface area inhibition decreased by 5.09% (CP018) and 2.95% (CP047) in the two cultivars, reaching a significant level only in the salt-resistant cultivar CP018, while root activity inhibition was significantly reduced by 46.21% in the salt-susceptible cultivar CP047 and 6.56% in the salt-resistant cultivar CP018, reaching a significant level only in the salt-susceptible cultivar CP047. A grey correlation analysis of the indicators revealed that root activity was the most relevant factor influencing plants dry weight. SA priming reduced the MDA content and increased the POD and SOD enzyme activities of kenaf leaves under salt stress. Expression analysis of 12 stress-related genes showed that ACCD, APX2, SOS1, ARR2, PAL, CHIT and TIFY11 genes expression levels were significantly up-regulated after SA priming, while ERF9, ERS1, ERF.C3 and MYC2 and XTH22 expression patterns differed between the two cultivars, with XTH22 being significantly up-regulated in salt sensitive cultivar CP047 but not in the salt resistant cultivar CP018, ERS1 and MYC2 were significantly up-regulated in the salt resistant cultivar CP018 but significantly down regulated in the salt sensitive cultivar CP047, while the trend of ERF9 was opposite in the two cultivars. 【Conclusion】SA priming at a suitable concentration could significantly alleviate the growth of kenaf under salt stress, and differed in the degree and patterns of effects on different kenaf germplasm resources. SA may regulate kenaf plant response to abiotic stresses by affecting physiological processes such as antioxidant enzyme systems and mediating the expression of specific genes.

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    Screening of Sweetpotato Varieties Tolerant to Low Nitrogen at Seedling Stage and Evaluation of Different Nitrogen Efficiencies
    FAN WenJing,LIU Ming,ZHAO Peng,ZHANG QiangQiang,WU DeXiang,GUO PengYu,ZHU XiaoYa,JIN Rong,ZHANG AiJun,TANG ZhongHou
    Scientia Agricultura Sinica    2022, 55 (10): 1891-1902.   DOI: 10.3864/j.issn.0578-1752.2022.10.002
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    【Objective】The objective of this article was to establish a low-N tolerance evaluation system for sweetpotato varieties, to screen low-N-tolerant genotypes and evaluate different N efficiency categories and to provide a theoretical basis for studying the low-N-tolerant physiological mechanism of sweetpotato varieties and mining N-efficient genes.【Method】Under the treatment of low N stress (0 mmol·L-1) and normal N application (14 mmol·L-1) of hydroponic experiment. Selected 126 sweetpotato varieties from different sweetpotato areas at home and abroad, we collected eleven indicators, including the shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, root-to-shoot ratio, vine length, root length, leaf number, CCI, nitrogen accumulation, nitrogen physiological utilization efficiency, and calculated the low-N tolerance index of all indicators. The study carried out principal component analysis by using the comprehensive membership function method, regression analysis and cluster analysis to comprehensively evaluate the low-N-tolerant sweetpotato varieties and N efficiency types.【Result】1) Under low N level, the average of the shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, vine length, root length, leaf number, CCI, and N accumulation of 126 tested sweetpotato varieties was lower than that under normal N level, while the average of root-to-shoot ratio and nitrogen physiological utilization efficiency were higher than that under normal N level; 2) The variation coefficient of shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, root-to-shoot ratio, vine length, root length, leaf number, nitrogen accumulation, nitrogen physiological utilization efficiency at low-N stress was higher than that at normal N level, and the amplification of them were ranked as shoot biomass increase>plant biomass increase ratio>root biomass increase>leaf number>shoot biomass>nitrogen physiological utilization efficiency>nitrogen accumulation>root length> root-to-shoot ratio>Vine length; 3) The principal component analysis was carried out on the low-N-tolerant index of eleven indicators, extracted three principal components, the cumulative variance contribution rate of which was 72.67%, and calculated the comprehensive evaluation Y-value; 4) The correlation between the low-N-tolerant index of shoot biomass, shoot biomass increase, root biomass increase, plant biomass increase ratio, leaf number, vine length, root length, root-to-shoot ratio, nitrogen accumulation, nitrogen physiological utilization efficiency and the Y-value were highly significant(P<0.01), among them, the correlation of shoot biomass increase, root biomass increase, plant biomass increase ratio, nitrogen accumulation and shoot biomass was higher, with the correlation coefficients of 0.85, 0.86, 0.81, 0.79 and 0.73, respectively; 5) The regression equation of the Y-value screened eight indicators to evaluate low-N tolerance of sweetpotato varieties, and the cluster analysis on eight indicators showed that the sweetpotato genotypes were classified into three types, low-N-tolerant, intermediate, and low-N-sensitive. The agronomic traits and N efficiency traits of three sweetpotato categories were analyzed by variance analysis.【Conclusion】Shoot biomass, shoot biomass increase, root biomass increase, root length, vine length, leaf number, nitrogen accumulation and nitrogen physiological utilization efficiency were selected as the low-N tolerance evaluation indicators of sweetpotato; The study screened 7 low-N-tolerant sweetpotato varieties: 13104-2/Zishu1, Yibinhongxinshu, Zhezishu 2, Yuzi 3, Yuzi 6, Luozi 1 and Yuzixiang 10; The results of variance analysis showed that the low-N-tolerant varieties performed better than the intermediate and the low-N sensitive varieties, and there are significant difference in five traits: shoot biomass, shoot biomass increase, root biomass increase, vine length and nitrogen accumulation.

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    Precise Characterization of Maize Germplasm for Resistance to Pythium Stalk Rot and Gibberella Stalk Rot
    DUAN CanXing,CAO YanYong,DONG HuaiYu,XIA YuSheng,LI Hong,HU QingYu,YANG ZhiHuan,WANG XiaoMing
    Scientia Agricultura Sinica    2022, 55 (2): 265-279.   DOI: 10.3864/j.issn.0578-1752.2022.02.003
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    【Objective】The objective of this study is to establish and perfect the method for precise characterization of maize germplasm resistance to Pythium stalk rot (PSR) and Gibberella stalk rot (GSR), and then precisely evaluate maize accessions with rich genetic background for resistance to stalk rot, and to screen out resistant germplasm and provide valuable resources to the maize community for enhancing disease resistance. 【Method】The total of 2 004 maize accessions with extensive genetic background were identified for resistance to stalk rot in multi-plot demonstration for three years under natural condition in order to preliminarily screen out germplasm with resistance or other excellent traits. Using drilling inoculation, precise evaluation of 690 maize germplasm for resistance to PSR and GSR was conducted at 6 different sites from 2018 to 2020. 【Result】During 2016-2018, 508 accessions with different resistance to stalk rot were identified at multiple sites in non-inoculated fields, including 79, 106, and 323 highly resistant, resistant, and moderately resistant germplasm, respectively. The method of drilling inoculation at the base of stem for precise characterization of maize resistance to stalk rot was established and perfected. Correlation coefficient (r) between soil inoculation and drilling inoculation for screening of maize resistance to PSR was 0.87, with 0.84 for GSR, indicating strong correlation between maize resistance to stalk rot identified by drilling inoculation and soil inoculation. Compared with soil inoculation, the severity of stalk rot on maize treated with drilling inoculation is higher. The precise resistance evaluation of 690 maize accessions to PSR was conducted with drilling inoculation at 6 different environments in Changping of Beijing, Changge of Henan, and Yuanyang of Henan from 2018 to 2020. The incidence of PSR was considerable and the majority of accessions were susceptible or highly susceptible, merely 3.5% of maize exhibited stable PSR resistance. Correlation coefficient of maize resistance to PSR between any two sites among six different environments varied from 0.46 to 0.72. The r values between two sites in 2018, 2019, and 2020 were 0.66, 0.60, and 0.65, respectively. The r values between pairwise annual comprehensive resistance to PSR among 3 years were 0.67, 0.84, and 0.87, indicating the resistance reaction of 690 maize accessions to PSR exhibited good consistency among different environments. A total of 24 maize germplasm expressed stable resistance to PSR, such as Jizi H676, Liao 2235, Jizi 14L88, Jizi 14L101, Dan 337, M02N-23, Y1747, HRB16232, and T628358, and so on. During 2018-2020, 690 maize resources were precisely identified for resistance to GSR with drilling inoculation at 6 different environments in Shenyang of Liaoning, Lishu of Jilin, and Gongzhuling of Jilin. The results indicated GSR on maize accessions in Shenyang was severe, with the majority of maize expressing susceptibility or high susceptibility, while it was significantly slight in Lishu and Gongzhuling. Correlation coefficient of GSR resistance in 690 maize germplasm between any two sites among six environments ranged from 0.00 to 0.76. The r values between two sites in 2018, 2019, and 2020 were 0.12, 0.04, and 0.05, respectively, indicating considerable differences in GSR resistance at two sites. The r values between pairwise annual comprehensive resistance to GSR among 3 years were 0.40, 0.74, and 0.72, showing annual comprehensive GSR resistance in 690 maize accessions were relatively consistent. The six-point experimental data showed that 5 accessions (Jizi C32, Liao 785, Liao 2235, Jizi 1034, and 16SD088) exhibited stable resistance to GSR. The r value of 0.44 between PSR and GSR resistance in 690 maize germplasm indicated moderate correlation between two stalk rot resistances. 【Conclusion】The resistance level of 2004 maize accessions to stalk rot was determined at multiple environments under natural conditions. The method of drilling inoculation at the base of stem for precise characterization of maize germplasm resistance to stalk rot was established and validated. The precise resistance characterization of 690 maize accessions to PSR and GSR was performed with drilling inoculation at 6 different environments among 3 years. Twenty-four and five maize germplasm expressed stable resistance to PSR and GSR at diverse sites among 3 years, respectively, which were important sources for breeding stalk rot resistant varieties or improvement of cultivar resistance.

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    Genome-Wide Association Analysis for Yield and Nitrogen Efficiency Related Traits of Wheat at Seedling Stage
    ZHANG PengXia,ZHOU XiuWen,LIANG Xue,GUO Ying,ZHAO Yan,LI SiShen,KONG FanMei
    Scientia Agricultura Sinica    2021, 54 (21): 4487-4499.   DOI: 10.3864/j.issn.0578-1752.2021.21.001
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    【Objective】 To identify and locate the molecular markers which were stable and significantly correlated to the traits of biomass and N efficiency under different N nutrition levels will help to provide reference for cloning and characterization of the related genes.【Method】A group of 134 wheat varieties (or lines) were used in a two-years (2013 and 2014) hydroponic experiments, in which three treatments applying normal level N, low level N and high level N were set up. Fourteen traits related to biomass and N efficiency were measured, as the respective average values of each treatment in one year and two years. Genome-wide association analysis using 90K SNP molecular markers was carried out for the tested traits by MLM+K+Q mixed linear model. 【Result】 Compared with normal nitrogen treatments, roots, shoots, and plant nitrogen content and nitrogen accumulation were significantly reduced in low nitrogen treatments, while root biomass and root and plant nitrogen efficiency were significantly increased. In high nitrogen treatments, almost all traits are significantly increased. The heritability of all the tested traits were above 40%. According to genome-wide association analysis on the 9 329 SNPs, a total of 838 molecular marker sites were identified associating with 14 traits significantly (P≤0.001). These markers located on 21 chromosomes, among which 435 (51.91%) molecular marker sites were detected in only one environment, 403 and 8 environment stable sites were identified in at least two or three environments, two environmental stable SNP marker sites were identified in at least four environments. The two stable markers (Kukri_c65481_121 and tplb0025f09_1052) were significantly related to total nitrogen use efficiency of plant (TNUE) and root nitrogen use efficiency (RNUE), respectively. Five multi-trait co-location SNP marker sites which simultaneously associated with at least six traits were located on chromosomes 1A, 1B(2), and 2A(2). Furtherly, candidate gene prediction was conducted in the 214 kb genomic region of 5 SNP sites co-located with 6 traits (biomass and N efficiency traits) and 2 SNP sites associated with multiple environments (4 environments). According to the genome annotation and LD attenuation level, a total of 84 candidate genes were determined. Gene function annotations of these genes were performed using the coding protein types of known cloned nitrogen efficiency genes, candidate gene function annotation information and the use of plant comparative genomics resource library protein sequence homology analysis, 3 candidate genes were initially determined. 【Conclusion】 Different N treatments significantly affected the phenotypic traits of biomass, N efficiency and the expression of related QTLs at seedling stage of wheat. Most SNPs were detected in only one N environment, but there were some locations with relatively strong environmental stability. There was a significant correlation between biomass and N efficiency related traits, and they might be partly controlled by the same QTL/gene. The functions of related candidate genes related to N efficiency and biomass of wheat selected in this paper needed to be further verified.

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    Over-expression of ZmIBH1-1 to Improve Drought Resistance in Maize Seedlings
    ZHU FangFang,DONG YaHui,REN ZhenZhen,WANG ZhiYong,SU HuiHui,KU LiXia,CHEN YanHui
    Scientia Agricultura Sinica    2021, 54 (21): 4500-4513.   DOI: 10.3864/j.issn.0578-1752.2021.21.002
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    【Objective】Drought is an important factor that affects the growth and development of maize seriously. Through the mining of genes related to drought resistance in maize, transgene function verification and transcriptome analysis, analyzing the molecular regulation mechanism of key genes in response to drought stress, this paper provides the theoretical basis for drought resistance molecular breeding and genetic improvement. 【Method】In this study, the maize inbred line B104 (wild-type, WT) was used as the background to construct the ZmIBH1-1 overexpression (ZmIBH1-1-OE) transgenic line by Agrobacterium-mediated method. The transgenic plants and lines were identified via screening transgenic plants for glufosinate-ammonium resistance, PCR detection of marker gene and target gene and expression analysis of target gene by qRT-PCR. We used the ZmIBH1-1-OE and WT transgenic lines as materials. Through drought treatment (20% PEG6000), phenotype identification and drought tolerance physiological and biochemical index determination were carried out to verify the drought resistance function of ZmIBH1-1; RNA-Seq was used to identify differentially expressed genes (DEGs) under drought stress at the 4-leaf stage; Combined with DAP-seq (DNA affinity purification sequencing) analysis, it is preliminarily determined that ZmIBH1-1 protein directly regulates downstream target genes related to drought resistance, and IGV (Integrative Genomics Viewer) was used to analyze the position of the ZmIBH1-1 protein binding candidate target gene, and then the Dual-Luciferase assay was used to verify the regulatory relationship between ZmIBH1-1 protein and target genes. 【Result】12 transformation events were obtained by genetic transformation of maize. In the T3 generation, there were 458 plants in which the marker gene Bar and the target gene ZmIBH1-1 were simultaneously detected. The results of qRT-PCR showed that the expression level of ZmIBH1-1 in ZmIBH1-1-OE lines was significantly higher than that of WT and the expression levels of transformation events 3 and 8 were the highest, which were self-crossed to obtain T4 generation for subsequent experiments. Under drought stress, the survival rate, the relative water content, the chlorophyll content, soluble protein content and the physiological and biochemical indicators (superoxide dismutase, peroxidase, catalase activity) of ZmIBH1-1-OE were higher than those of WT significantly, which indicating that the overexpression of ZmIBH1-1 in maize confers higher drought tolerance. The RNA-Seq results showed that there were 1 214 DEGs between WT and ZmIBH1-1-OE lines. Gene Ontology (GO) analysis showed that DEGs were mainly involved in biological processes, cell components and molecular functions, such as photosynthesis, stress response, dehydration response, etc. in biological processes; KEGG enrichment analysis showed that DEGs were mainly involved in the signal transduction of plant hormones, the metabolism and other processes. Combining the significantly DEGs of RNA-Seq and the target genes of ZmIBH1-1 obtained from DAP-seq analysis, it is preliminarily identified 11 candidate target genes related to drought resistance that may be directly regulated by ZmIBH1-1, including 2 calcium signal related genes, 3 cysteine metabolism related genes, 1 bHLH transcription factor, 1 stress response protein, 1 glutathione transferase, 1 redox process protein and 2 ethylene response factor; Integrative genomics viewer showed that ZmIBH1-1 protein could bind to the promoters of the target genes; Subsequent Dual-Luciferase assay further showed that ZmIBH1-1 protein can directly act on 11 candidate target genes, of which, ZmIBH1-1 directly binds to the promoters of ZmCa-M, ZmSYCO, ZmbHLH54, ZmGlu-r1, ZmCLPB3 and ZmP450-99A2 to promote their expression, and directly binds to the promoters of ZmAGD12, ZmCYS, ZmCYSB, ZmERF-107 and ZmEIN3 to repress their expression. In addition, transcription factors such as NAC, WRKY and MYB also differentially expressed between WT and ZmIBH1-1-OE under drought stress. 【Conclusion】The overexpression of ZmIBH1-1 can enhance the drought tolerance of maize; ZmIBH1-1 improves the drought tolerance of maize by directly regulating the expression of genes ZmERF-107 and ZmEIN3 in the ethylene signaling pathway; ZmIBH1-1 enhances the drought tolerance of maize by directly regulating the calcium signal-related genes ZmCa-M and ZmAGD12; ZmIBH1-1 may indirectly regulate NAC, WRKY, MYB and other transcription factors in response to drought stress.

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    Optimization of Cotton Mesophyll Protoplast Transient Expression System
    LI Qing,YU HaiPeng,ZHANG ZiHao,SUN ZhengWen,ZHANG Yan,ZHANG DongMei,WANG XingFen,MA ZhiYing,YAN YuanYuan
    Scientia Agricultura Sinica    2021, 54 (21): 4514-4524.   DOI: 10.3864/j.issn.0578-1752.2021.21.003
    Abstract640)   HTML33)    PDF (4819KB)(378)       Save

    【Objective】Cells of true leaves can well mimic the plant endogenous situation. It is an efficient way for expediting cotton functional study to establish an effective transient expression system using cotton protoplasts obtained from true leaves.【Method】The enzyme combination of cellulose and macerozyme were used to isolate protoplasts from true leaves of Gossypium hirsutum L. acc. TM-1. The effects of osmotic pressure, components of digestion buffer and digestion time on protoplast yield were studied and the validity of protoplasts were compared under different mannitol concentration and digestion time. To improve the transformation efficiency of cotton protoplast, the effects of mannitol and PEG concentration and buffers for protoplast culture were subsequently studied. In order to verify the optimized transient expression system, the vector 35S:LTP-GFP was constructed and transformed into protoplasts of Arabidopsis and cotton and tobacco epidermal cells followed by observation of fusion protein localization.【Result】High concentration of CaCl2 in the digestion buffer significantly inhibited the isolation of protoplast from cotton true leaves, which was opposite of that using cotyledon. 10 mmol·L -1 CaCl2 was employable for digestion buffer to isolate cotton protoplasts from true leaves. Mannitol concentration significantly affected protoplast yield that peaked under mannitol concentration of 0.5 mol·L-1, and protoplast validity decreased moiety under 0.4 mol·L-1 mannitol, suggesting that 0.5 mol·L-1 mannitol was most suitable to maintain the osmotic pressure of cotton protoplasts. Cotton protoplasts displayed suitable size when isolated from newly flattened true leaves, while protoplast enlarged and yield decreased when produced from young leaves flattened 5 days. The protoplasts dissociate slowly until being digested 9 h when the yield reached the peak. The transformation efficiency was greatly improved under isotonic condition of 40% PEG buffer. While hypotonic condition that is commonly applied to facilitate transformation was against the entrance of exogenous DNA into cotton protoplasts. After transformation, the protoplast ruptured abundantly in WI buffer,whereas the shape maintained well in W5 buffer adding 0.5 mol·L-1 mannitol. The transformation efficiency was improved to 90% using the optimized transient expression system. The subcellular location analysis results showed consistent GFP signal in protoplasts of cotton and Arabidopsis true leaf and epidermal cells of tobacco leaf.【Conclusion】Our study has optimized the cotton mesophyll protoplast transient expression system, which could produce 8.10×10 6·mL-1 fine protoplasts with validity above 95% and transformation efficiency reached to 90%. This system is applicable for analysis of subcellular location, protein interaction and research on metabolism and regulation network.

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