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Function of FCS-Like Zinc-Finger Protein OsFLZ18 in Regulating Rice Flowering Time MA YaMei,ZHANG ShaoHong,ZHAO JunLiang,LIU Bin Scientia Agricultura Sinica    2022, 55 (20): 3875-3884.   DOI: 10.3864/j.issn.0578-1752.2022.20.001 Abstract （577）   HTML （115）    PDF （1565KB）（305）       Knowledge map    Save 【Objective】Flowering time is an important agronomic trait which determines the yield and regional adaptability of rice, but the underlining molecular regulatory mechanism need further study. FCS-like Zinc finger proteins (FLZs) are a class of plant specific regulatory proteins which play essential roles in plant growth and stress response, but their functions in regulating flowering time have not been reported. This study aims to investigate the potential function of FLZ proteins in rice flowering time control. The finding will broaden our understanding on the molecular regulatory mechanism of rice flowering time.and provide new theoretical basis and gene resource for rice breeding. 【Method】Based on the target sequences published in RGAP database, OsFLZ18 overexpression vector and CRISPR-Cas9 vector were generated and introduced into Japonica variety Nipponbare by Agrobacterium tumefaciens-mediated genetic transformation assay. Homozygous CRISPR knockout mutants were screened by PCR and sequencing analyses. The quantitative real-time PCR (qRT-PCR) assay was used to examine the spatial-temporal expression and diurnal rhythmic expression of OsFLZ18, as well as the effects of OsFLZ18 on the transcription of several known flowering time-related genes. Yeast two-hybrid assay (Y2H) was used to test the interaction between OsFLZ18 and the flowering time-related regulatory proteins.【Result】OsFLZ18 was ubiquitously expressed in various rice tissues, with the highest expression level in 14 day-old seedling, followed by leaf sheaths and leaf blades at the tillering stage, and stem and young panicles at reproductive stages. The OsFLZ18-CRISPR vector was constructed and transformed into Nipponbare. Two independent homozygous OE lines (OE-2, OE-3) with higher OsFLZ18 expression level and two homozygous mutants (CRISPR-21, CRISPR-25) were selected for further study. Phenotypic observation showed that the OE lines flowered later than the wild-type plants under both natural long-day and short-day conditions in Guangzhou, while the CRISPR lines had no obvious differences in heading date when compared to the wild-type plants. The expression levels of Ehd1, Hd3a and RFT1 were significantly decreased in OE-2 plants compared with those in the wild-type plants under artificial short-day conditions, but no significant difference in the expression level of Hd1 was observed between them. The results of Y2H experiment showed that OsFLZ18 interacted with OsMADS51, a positive regulator of rice flowering time. Furthermore, OsFLZ18 exhibits a diurnal rhythmic expression profile, showing lower expression levels in the daytime and higher expression levels at night with a peak at midnight. 【Conclusion】Overexpression of OsFLZ18 delays rice flowering time. Table and Figures | Reference | Related Articles | Metrics

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Identification of the Root-Specific Soybean GmPR1-9 Promoter and Application in Phytophthora Root-Rot Resistance YAN Qiang,XUE Dong,HU YaQun,ZHOU YanYan,WEI YaWen,YUAN XingXing,CHEN Xin Scientia Agricultura Sinica    2022, 55 (20): 3885-3896.   DOI: 10.3864/j.issn.0578-1752.2022.20.002 Abstract （356）   HTML （60）    PDF （3529KB）（198）       Knowledge map    Save 【Objective】The objective of this study is to identify the root-specific promotors and the core regulatory sequence of soybean. Then evaluate the potential application of the synthetic promoter in Phytophthora root-rot resistance. 【Method】The genes which specifically expressed in roots with high expression levels were screened based on the transcriptome date of soybean root, stem and leaf tissues in the seedling stage. Based on the distribution of the cis elements, the promoter truncation approach was used to map the minimal promoter controlling root specific expression in soybean hairy roots. The obtained minimal promoter fragment was concatenated with the Phytophthora inducible promoter elements p4XD to construct the synthetic promoter. The synthetic promoter driven over-expression of Phytophthora resistance related gene GmNDR1 in soybean hairy roots, then the resistance level of transgenic tissue to Phytophthora and the expression profiles of GmNDR1 during the interaction had been analyzed. Furthermore, the transgenic Nicotiana benthamiana plants were generated to evaluate the resistance at plant level. 【Result】Though screening, six soybean PR1 homologues with significant root specific expression manner were identified, and GmPR1-9 had the highest promoter activity. Numbers of root specific expression related cis elements were identified in promoter sequence using the online tool PLACE. Truncation analysis of the promoter showed that serial 5’ end deletions L1, L2, L3, L4 and L5 had different GUS activities. The L5 (-166 to -1) fragment had 80% activity of the full-length promoter, and was able to drive GUS expression in roots of transgenic N. benthamiana. GUS enzyme activity was almost undetectable in three 3’ end deletions R1, R2 and R3, and the double terminal deletion mutant M1. When the fusion promoter p4XD-L5 driven GmNDR1 expression in soybean hairy roots, the resistance to P. sojae was significantly enhanced. The disease severity and lesion length were significantly reduced in the over-expression hairy roots when compared with control, and the relative biomass of Phytophthora decreased by 66.5% at 48 h post inoculation. GmNDR1 maintained high expression level in over-expression tissues, with 39.2 times of that in control tissues. The expressions were further up-regulated after inoculation, and reached the highest level at 36 h. In p4XD-L5::NDR1 transgenic N. benthamiana plants, the expression of GmNDR1 was significantly higher in roots than that in stems and leaves. Fifteen days after P. capsica inoculation, the plant height, root length and fresh weight of GmNDR1 over-expression plants were significantly higher, and meanwhile the leaf wilting rate and lesion length were significantly lower. 【Conclusion】This study obtained a soybean root specific promoter and identified the core regulation sequence. The strategy which driven the expression of GmNDR1 by the synthetic promoter p4XD-L5 combined the inducible and tissue-specific promoter core elements can significantly enhance the resistance of transgenic soybean hairy roots and Nicotiana benthamiana plants to Phytophthora pathogens. Table and Figures | Reference | Related Articles | Metrics

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Maize Transcription Factor ZmEREB93 Negatively Regulates Kernel Development PANG HaoWan,FU QianKun,YANG QingQing,ZHANG YuanYuan,FU FengLing,YU HaoQiang Scientia Agricultura Sinica    2022, 55 (19): 3685-3696.   DOI: 10.3864/j.issn.0578-1752.2022.19.001 Abstract （454）   HTML （70）    PDF （4112KB）（195）       Knowledge map    Save 【Objective】 Maize, a kind of crucial crop, is widely used in food supply, livestock feed, and industry. AP2/EREBP (APETALA2/ethylene response element-binding protein) transcription factor (TF) plays an important role in plant growth, development, and stress response. Previous study showed that ZmEREB93 might regulate seed size as a target gene of ZmBES1/BZR1-5 TF. ZmEREB93 was cloned and used to analyze its expression pattern and function, which lays foundation to clarify the function and mechanism of ZmEREB93 regulating seed size. 【Method】 The full length of ZmEREB93 was cloned from maize inbred line B73 by PCR. The characters of nucleotide and amino acid sequences were analyzed by informatic methods. Subsequently, the tissue expression specificity of ZmEREB93 was analyzed via quantitative real time PCR (qRT-PCR). The expression vector in plant and yeast was constructed and used for subcellular localization and transcription activation assay, respectively. ZmEREB93 was transformed into Arabidopsis mediated by agrobacterium transformation. The seed phenotype of transgenic lines was analyzed. Finally, the potential target genes of ZmEREB93 were screened by chromatin immunoprecipitation sequencing (Chip-seq) and co-expression analysis, and further confirmed by yeast one hybrid (Y1H). 【Result】 The ZmEREB93 gene was cloned by PCR. Sequence analysis showed that ZmEREB93 had no intron and a 618 bp ORF, encoding 205 amino acids with a highly conserved AP2 domain and belongs to the ERF subclade of AP2 family. The results of qRT-PCR showed that the ZmEREB93 gene highly expressed in kernels of 15 and 25 days after pollination (DAP), and slightly expressed in stem and root, but did not express in tassel, silk and bract. The expression level of ZmEREB93 was the highest in 25 DAP kernels reached 11 times of that in 15 DAP kernels. The results of transcriptional activation and subcellular localization assay exhibited that ZmEREB93 protein had no transcriptional activation activity in yeast cells and was localized in the nucleus, respectively. Compared to wild type, the seeds of transgenic lines were significantly smaller and showed lower thousand-seed-weight. Chip-seq and co-expression analysis suggested that the Zm00001d013611, Zm00001d006016, Zm00001d027448 and Zm00001d03991 genes were candidate target genes regulated by ZmEREB93 TF. The result of Y1H showed that ZmEREB93 directly bind to Zm00001d013611 promoter. 【Conclusion】 Maize ZmEREB93 TF specifically expressed in seeds and negatively regulated seed size. Table and Figures | Reference | Related Articles | Metrics

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Cloning and Functional Analyses of MsCIPK2 in Medicago sativa SU Qian,DU WenXuan,MA Lin,XIA YaYing,LI Xue,QI Zhi,PANG YongZhen Scientia Agricultura Sinica    2022, 55 (19): 3697-3709.   DOI: 10.3864/j.issn.0578-1752.2022.19.002 Abstract （358）   HTML （72）    PDF （5750KB）（141）       Knowledge map    Save 【Objective】 CIPKs are a group of important protein kinase involved in signaling pathway of plant in response to stress. They can form CBL-CIPK complex with CBL, to activate the expression of related responsive genes to cope with various abiotic stresses in cells. Exploration and study on the molecular mechanism of MsCIPK genes in alfalfa in response to abiotic stress will help to reveal the biological basis of stress resistance in alfalfa, and to provide new gene resources for alfalfa breeding with enhanced stress resistance. 【Method】 The MsCIPK2 gene was cloned by using PCR, the sequence was analyzed by bioinformatics tools, and the expression level of MsCIPK2, MsCBL2, MsCBL6, MsCBL7 and MsCBL10 genes in various tissues were analyzed by using qRT-PCR. The pCAMBIA1302-GFP-MsCIPK2 vector was transiently expressed in tobacco leaf epidermal cells, and the subcellular localization was observed under laser confocal microscope. Yeast two-hybrid assay was used to analyze interaction between MsCIPK2 and four MsCBLs proteins. Agrobacterium rhizogenes was used to generate alfalfa hairy roots over-expressing MsCIPK2. qRT-PCRs were used to analyze the expression levels of related genes in transgenic hairy root lines. 【Result】 The coding sequence of MsCIPK2 gene was obtained by using PCR, and it is 1 230 bp in length, encoding 409 amino acids. The deduced MsCIPK2 protein contained typical ATP binding site, activation loop, NAF motif and PPI motif as for the CIPK family genes. The expression level of MsCIPK2 gene was the highest in roots, and the lowest in the flowers of alfalfa. Subcellular localization results showed that MsCIPK2 protein was localized in the endoplasmic reticulum. Yeast two-hybrid assays showed that MsCIPK2 protein interacted with MsCBL2, MsCBL6, MsCBL7 and MsCBL10 proteins, showing stronger interaction with MsCBL10 than with other MsCBLs. The expression levels of MsCBL2, MsCBL6, and MsCBL10 were the highest in roots of alfalfa, and the expression level of MsCBL7 was the highest in pods. qRT-PCR results showed that the expression levels of abiotic stress-associated genes ATPase, P5CS, CYP705A5, COR47, HAK5 and RD2 were significantly up-regulated in hairy roots over-expressing MsCIPK2. Under the treatment of 200 mmol·L-1 NaCl and 20% PEG, when compared with the control hairy root line, hairy roots over-expressing MsCIPK2 had lower MDA content, and higher POD activity, proline content and soluble sugar content. 【Conclusion】 MsCIPK2 can interact with CBL protein, and responded to salt and drought stress in roots of alfalfa. Over-expression of MsCIPK2 can improve salt and drought stress resistance in alfalfa, and MsCIPK2 can be used as candidate gene for alfalfa breeding with improved abiotic stress resistance. Table and Figures | Reference | Related Articles | Metrics

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QTL Mapping of Thousand-Grain-Weight and Its Related Traits in Zhou 8425B × Xiaoyan 81 Population and Haplotype Analysis LinHan ZOU,XinYing ZHOU,ZeYuan ZHANG,Rui YU,Meng YUAN,XiaoPeng SONG,JunTao JIAN,ChuanLiang ZHANG,DeJun HAN,QuanHao SONG Scientia Agricultura Sinica    2022, 55 (18): 3473-3483.   DOI: 10.3864/j.issn.0578-1752.2022.18.001 Abstract （441）   HTML （57）    PDF （3981KB）（222）       Knowledge map    Save 【Objective】Zhou 8425B is one of the most important founder parents in China and Xiaoyan 81 is an elite cultivar with high yield and good quality. Thousand-grain weight (TGW) is an important factor that affects wheat yield. Identification of QTL associated with grain related traits from Zhou 8425B and Xiaoyan 81, and haplotype analysis of these QTL in wheat cultivars from different ecological regions would be beneficial for yield improvement by molecular marker-assisted selection.【Method】In this study, a RIL population (F8) derived from Zhou 8425B × Xiaoyan 81 was planted in Yangling during 2015 and 2016 cropping seasons to evaluate grain related traits. Using a high-density genetic map constructed by 90K SNP markers, QTL mapping of thousand-grain weight, grain length, grain width and thickness was performed under three environments. Simultaneously, the KASP markers linked to the identified QTL were developed and molecular detection was carried out among 479 wheat accessions worldwide. Moreover, haplotype analysis of target QTL was performed in 106 current wheat commercial cultivars from Yellow and Huai River Valley Winter Wheat Region selected from 479 wheat accessions.【Result】A total of 22 QTL on 8 chromosomes were detected, and the phenotypic variation explanation (PVE) ranged from 4.77% to 19.95%. Among them, 12 QTL are major QTL (PVE>10%) and Qkgw.nwafu-6B is a new QTL. QTL on chromosomes 4A, 6A, 6B, and 7D were detected in multiple environments, of which, the QTL on chromosomes 4A and 7D are same as previously reported ones. Compared to TaGW2-6A using molecular detection, both Zhou 8425B and Xiaoyan 81 carried the same allele of TaGW2. Based on haplotype result of Qkgw.nwafu-6A, Zhou 8425B and Xiaoyan 81 were placed in different groups. Therefore, Qkgw.nwafu-6A tends to be a new one. Haplotype analysis showed that there were five haplotypes for Qkgw.nwafu-6A and there were eight haplotypes for Qkgw.nwafu-6B. 6A_h1 and 6B_h6 accounted for over 20% in different ecological regions. In addition, a co-segregated KASP marker was developed for Qkgw.nwafu-6B and was significantly associated with the grain weight in the 479 wheat accessions.【Conclusion】Qkgw.nwafu-6A and Qkgw.nwafu-6B are possible new QTL associated with thousand-grain-weight, and 6A_h1 and 6B_h6 are likely favorable haplotypes. A molecular marker KASP_IWA349 co-segregated with Qkgw.nwafu-6B was developed and will be useful for marker assisted selection. Table and Figures | Reference | Related Articles | Metrics

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Deciphering of the Genetic Diversity After Field Late Blight Resistance Evaluation of Potato Breeds XiaoChuan LI,ChaoHai WANG,Ping ZHOU,Wei MA,Rui WU,ZhiHao SONG,Yan MEI Scientia Agricultura Sinica    2022, 55 (18): 3484-3500.   DOI: 10.3864/j.issn.0578-1752.2022.18.002 Abstract （335）   HTML （50）    PDF （3315KB）（193）       Knowledge map    Save 【Objective】To evaluate the late blight resistance in field. To use SNP markers to analyze the genetic diversity of late blight resistance of potato germplasm and discern the genetic segments that may affect the phenotype of potato late blight resistance, and to provide a theoretical basis for the innovation and utilization of potato late blight resistant germplasm.【Method】Field resistance to late blight was evaluated in potato germplasm at multiple locations and over the course of several years. SNP markers were detected using a dd-RAD simplified genome sequencing strategy. The population genetic structure was analyzed using Admixture, principal component analysis was conducted using GCTA, a phylogenetic tree was constructed using fastTree, population genetic diversity parameters were calculated using the populations command in the Stacks package, selective sweep parameters were calculated using vcftools, protein sequences were aligned using Clustal Omega, and a proteins phylogenetic tree was drawn using MEGA6. Genome-wide association analysis was constructed using GEMMA 0.98.1 and QQ and manhattan plots were drawn using CMplot.【Result】Through years of evaluation of late blight field resistance in multiple locations, potato germplasm of 101 late blight resistant varieties (lines) and 21 susceptible varieties were obtained. A total of 8 697 602 relatively evenly distributed SNPs were obtained using dd-RAD simplified genome sequencing on these germplasms. This germplasm can be further divided into 6 populations through structural analysis, principal component analysis and phylogenetic analysis. The average nucleotide diversity (π) within the 6 populations ranged from 0.2055 to 0.2572 and the fixation index (Fst) among the six populations ranged from 0.156909 to 0.187336, revealing a relatively large genetic diversity for these germplasms. The expected heterozygosity (He) within the 6 populations ranged from 0.187 to 0.2297 and the observed heterozygosity (Ho) ranged from 0.0829 to 0.1186. The values of Ho were less than those of He in all six populations. Meanwhile, the inbreeding coefficient (Fis) for the six populations ranged from 0.2412 to 0.3554, indicating inbreeding events during the breeding process. To identify the genetic segments that may affect the phenotype of potato late blight resistance, π ratios and Fst among different late blight resistance germplasms in the whole potato genome were calculated using 20 kb as the window length and 5 kb as the step length. The 745 genetic segments which had a π ratio value in the lowest 5% and a Fst value in highest 10% were further analyzed by performing a selective sweep analysis. These selected segments contain a total of 507 genes, including 4 NBS-LRR genes. A genome-wide association analysis was also conducted, yielding 9 SNP highly associated with late blight resistance. Of the 69 genes located in the genome within 50 kb around the 9 SNPs, 15 genes were predicted to be involved in stress response, and 12 genes were predicted to be involved in removing peroxide radicals.【Conclusion】Large amount of SNPs, which are relatively evenly distributed in the potato genome, can be genotyped by dd-RAD simplified genome sequencing. Potato late blight field resistance germplasm has a large genetic diversity, but had inbreeding events in the process of breeding. Population structure analysis can reveal the genetic relationship between potato germplasms, which can further provide a theoretical basis for parental selection in breeding. Selective sweep and genome-wide association analyses help to isolate genetic segments that may affect late blight resistance traits. Table and Figures | Reference | Related Articles | Metrics

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Effects of Seedlings Enriched with Zinc on Cadmium Accumulations and Related Transporter Genes Expressions in Different Rice Cultivars JIANG XiaoTing,HUANG GaoXiang,XIONG XiaoYing,HUANG YunPei,DING ChangFeng,DING MingJun,WANG Peng Scientia Agricultura Sinica    2022, 55 (17): 3267-3277.   DOI: 10.3864/j.issn.0578-1752.2022.17.001 Abstract （328）   HTML （68）    PDF （533KB）（174）       Knowledge map    Save 【Objective】 Rice seedlings enriched with zinc (Zn) is a new method to antagonize cadmium (Cd) within rice, studying the various effects and underlying mechanisms among different rice cultivars is useful to provide a basis for the application of this method. 【Method】 Hydroponic and pot experiments were conducted to test the effects of the Zn enrichment in four cultivars including the Wuyunjing21, Zhejingyou1578, Huanghuazhan, and Huiliangyousimiao. Dynamics of Zn accumulation in different cultivars, and variations of Cd uptake and transport in rice tissues were investigated. Furthermore, the expressions of the Cd-related transporter genes of the root were analyzed to explore the underlying mechanisms responsible for the various effects in different cultivars. 【Result】 Results showed that the Zn cultivation didn’t affect the growth of all rice seedlings. Zn accumulations varied obviously among different cultivars, the Zhejingyou1578 had the highest Zn level (230 μg Zn per plant), followed by the Wuyunjing21 (124 μg Zn per plant), the Huanghuazhan and Huiliangyousimiao had the low levels (85.1-95.0 μg Zn per plant). Zn enrichment greatly influenced the expression of Cd-related transporters in the Zhejingyou1578, the OsZIP7 was down-regulated by 48.7% and OsZIP1 was up-regulated by 81.3%. The expressions of the OsIRT1 and OsZIP7 in the Wuyunjing21 were also down-regulated by 35.9% and 35.0%, respectively, and the OsZIP1 was up-regulated by 31.1%. However, the Cd-related transporters in the Huanghuazhan and Huiliangyousimiao were insensitive to Zn enrichment. As a result, Zn enrichment significantly reduced Cd concentrations in the root and shoot by 26.7% and 36.7% of the Wuyunjing21, and 32.0% and 40.0% of the Zhejingyou1578 with a 12.0% inhibition on the Cd transport, respectively. Furthermore, brown rice Cd were reduced by 37.5%, 36.7% and 25.3% in the Wuyunjing21, Zhejingyou1578, and Huanghuazhan, respectively, while no difference occurred in the Huiliangyousimiao, revealing various effects on reducing Cd among different cultivars induced by the Zn enrichment. Correlation analysis showed that the brown rice Cd was negatively correlated with the shoot Cd which was negatively correlated with the root and shoot Zn, revealing a significant antagonism between Zn and Cd within rice. However, the root and shoot Zn were negatively correlated with the OsZIP7 and OsIRT1 and positively correlated with the OsZIP1 expression, indicating that the Zn enrichment within seedlings influenced the brown rice Cd mainly through regulating the expressions of the OsZIP1, OsZIP7 and OsIRT1. 【Conclusion】 The regulations on Cd-related transporters through Zn enrichment were varied among different cultivars, and the Zhejingyou1578 and Wuyunjing21 were more sensitive, so that the reductions of brown rice Cd were higher than other cultivars. Table and Figures | Reference | Related Articles | Metrics

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Leaf Stomatal Close and Opening Orchestrate Rhythmically with Cell Wall Pectin Biosynthesis and Degradation ZHANG XiaoPing,SA ShiJuan,WU HanYu,QIAO LiYuan,ZHENG Rui,YAO XinLing Scientia Agricultura Sinica    2022, 55 (17): 3278-3288.   DOI: 10.3864/j.issn.0578-1752.2022.17.002 Abstract （270）   HTML （30）    PDF （1672KB）（100）       Knowledge map    Save 【Objective】 Comparing on differential expression proteins between stomatal closing and opening at different leaf stomata-densities, it is to be revealed how pectin metabolism regulates stomata closing and opening. The result will play an essential role in understanding how stomata functions to environment adaptation.【Method】 Vectors, either over- or inhibiting-expression of StEPF-2 (Solanum tuberosum EPIDERMAL PATTERNING FACTOR 2) in vivo were constructed. The fusing genes were transformed into Solanum tuberosum cultivar Kexin 1. Transgenic potato lines, either rise or lower at leaf stomatal density were generated. Gene and protein expression profiles of leaves at various stomatal densities were assayed via RNA-seq and iTRAQ. Comparing differentiation expression proteins, pectin metabolic enzymes driving stomatal movement under light and darkness were identified and confirmed by the Pulldown and LC-MS/MS. A pectin metabolism pathway regulating stomatal movement was to be proposed.【Result】 At least 14 protein families, driving stomata closing and opening involved in pectin metabolism of the guard cell wall during stomatal mature. Five protein families were detected and confirmed only in the stomatal-closed leaves under darkness, including polygalacturonase inhibitor proteins (PGIP) and rhamnose synthase (RHM) for RG side-chain biosynthesis. Four protein families, polygalacturonase (PG), pectate lyase-like (PLL), pectinmethylesterase (PME) and α-galactosidase (AGAL) were identified only in leaves at various stomatal densities under light. Additionally, five protein families were concurrently identified in both leaves of stomata closing and opening, including pectinacetylesterase (PAE) and subtilase (SBT). 【Conclusion】 Under light, PMEs catalyze pectin demethylesterification, afterwards, pectin was exo- and endo-hydrolyzed by PG, PLL and AGAL. Pectin losing structure was split under turgor, results in stomatal opening. Reversely, under darkness, PGI inhibited pectin hydrolysis. Pectin side-chain biosynthesis was promoted by RHM. Therefore, stomata kept closing due to structurally-complete pectin with voluntary expending function. Table and Figures | Reference | Related Articles | Metrics

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Mining of Genetic Locus of Maize Stay-Green Related Traits Under Multi-Environments CHANG LiGuo,HE KunHui,LIU JianChao Scientia Agricultura Sinica    2022, 55 (16): 3071-3081.   DOI: 10.3864/j.issn.0578-1752.2022.16.001 Abstract （490）   HTML （269）    PDF （2062KB）（562）       Knowledge map    Save 【Objective】 Functional stay-green is generally considered a desirable trait in major crop varieties including maize. Finding new loci and candidate genes related to stay-green, and providing new theoretical basis for the genetic research on stay-green. 【Method】Using 150 recombinant inbred lines (RIL) populations derived from the cross between Xu 178 and K12, QTL mapping of three stay-green related traits (visual stay green (VSG), green leaf number at silking stage (GLNS) and green leaf number at mature stage (GLNM) were performed by the composite interval mapping（CIM）method of Windows QTL Cartographer V2.5. Besides, an association population, which composed of 139 natural materials genotyped with 50790 high-quality SNP markers, was used to dissect genetic locus of three traits by genome-wide association study (GWAS) based on the mixed linear model MLM). 【Result】Based on CIM, three traits (GLNM, GLNS and VSG)were mapped using phenotypic values in a single environment and best linear unbiased prediction (BLUP) value. A total of 37 QTLs were detected on all chromosomes except Chromosome 10, and the LOD score ranged from 2.58-11.36, with a phenotypic variation contribution rate of 4.34%-22.40%. Among them, 14, 12 and 11 loci were detected for GLNM, GLNS and VSG traits, respectively. Four of the QTLs, qGLNS2-1, qVSG1-1, qVSG1-2 and qVSG7-1, were genetically stable and were detected simultaneously in three or more different single environments. GWAS was performed on three stay-green related traits using MLM, and a total of 44 significant SNPs above the threshold line were detected. According to the physical position of SNP markers in the B73 reference genome, a total of 15 SNP were found to fall into the QTL interval mapped by linkage analysis. 【Conclusion】Combined with the results of QTL mapping and genome-wide association study, a total of 4 genetically stable colocalization genetic regions were detected (the corresponding physical position intervals on the B73 reference genome version 4 are 6.2-8.2 Mb on chromosome 1, 209.1-221.4 Mb on chromosome 2, 96.8-102.1 Mb on chromosome 6, and 4.9-11.4 Mb on chromosome 7), and four important candidate genes (Zm00001d006119, Zm00001d018975, Zm00001d006535 and Zm00001d036763) related to photosynthesis and stress response were mined. Table and Figures | Reference | Related Articles | Metrics

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The Functional Analysis of High Mobility Group MsHMG-Y Involved in Flowering Regulation in Medicago sativa L. ZHANG YunXiu,JIANG Xu,WEI ChunXue,JIANG XueQian,LU DongYu,LONG RuiCai,YANG QingChuan,WANG Zhen,KANG JunMei Scientia Agricultura Sinica    2022, 55 (16): 3082-3092.   DOI: 10.3864/j.issn.0578-1752.2022.16.002 Abstract （408）   HTML （235）    PDF （2298KB）（238）       Knowledge map    Save 【Objective】Flowering is an important signal indicating the transformation from vegetative growth to reproductive growth and has a significant effect on plant biomass. Alfalfa is one of the upmost forage crops worldwide, its yield and quality are closely related to flowering time. The optimum harvest time for alfalfa is during the early flowering stage, which could give the highest yield and the best quality. In the current study, an alfalfa flowering related gene, Medicago sativa High Mobility Group Y (MsHMG-Y), was cloned. The gene structure and expression pattern of MsHMG-Y were studied. Function of MsHMG-Y in alfalfa flowering regulation was analyzed. This work could provide theoretical support for mechanism study underlying flowering regulation. 【Method】MsHMG-Y was cloned by homology cloning strategy and the amino acid sequence was analyzed by multiple sequence alignment. The phylogenetic tree was also constructed. qRT-PCR analysis was used to detect the expression level of MsHMG-Y in different tissues and different flowering stages. The expression pattern of MsHMG-Y under light, gibberellin (GA3), salicylic acid (SA) or methyl jasmonate (MeJA) treatment were analyzed. The phenotype of MsHMG-Y-overexpressing alfalfa was analyzed, and the expression levels of flowering activators and suppressors were also analyzed. 【Result】Phylogenetic analysis showed that MsHMG-Y has the closest relationship with MtHMG-Y in Medicago truncatula. Spatial expression pattern analysis showed that MsHMG-Y was expressed in flowers, stems and leaves, with the highest expression level in flowers and the lowest expression level in leaves in both paternal and maternal alfalfa. In paternal alfalfa with early flowering phenotype, the expression level of MsHMG-Y was the highest at early flowering stage. The highest expression level of MsHMG-Y was detected at flower bud differentiation stage in maternal alfalfa with late flower phenotype. Photoperiod analysis showed that MsHMG-Y was down-regulated after 16-hour light treatment. After 28 hours of light treatment, the expression level of MsHMG-Y was continuously lower than that in the control group, indicating that MsHMG-Y was down-regulated after light treatment. After 50 μmol·L-1 GA3, 100 μmol·L-1 SA or 100 μmol·L-1 MeJA treatment, the expression level of MsHMG-Y was up-regulated compared with the mock treatment. In detail, the expression level of MsHMG-Y was the highest at 1 h under GA3 treatment, which was 3.5 folds higher than control. Under SA treatment, the expression level of MsHMG-Y was the highest at 6 h, which was 24 folds higher than the mock treatment. The expression level of MsHMG-Y was the highest at 3 h under MeJA treatment, which was 11 folds higher than the control. These results indicated that the expression of MsHMG-Y was inducible by the above three hormones. MsHMG-Y-overexpressing alfalfa has late flowering phenotype. The expression levels of flowering activator genes were down-regulated in MsHMG-Y-overexpressing alfalfa, while the expression levels of flowering inhibitor genes were up-regulated. Among these genes, expression of flowering activator genes MsPHYA, MsGI and MsFTa1 was significantly down-regulated by 4.9 folds, 3.9 folds and 2.8 folds respectively, and the expression level of flowering inhibitor genes MsTEM and MsSVP was increased by 2.5 folds and 1.9 folds, respectively. 【Conclusion】The expression of MsHMG-Y is inducible by photoperiod and exogenous hormone treatment, including GA3, SA and MeJA. Overexpression of MsHMG-Y in alfalfa resulted in delayed flowering time. MsHMG-Y plays an important role in regulatory mechanism underlying late flowering in alfalfa. Table and Figures | Reference | Related Articles | Metrics

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Genome-Wide Association Study of Ear Related Traits in Maize Hybrids LI Ting,DONG Yuan,ZHANG Jun,FENG ZhiQian,WANG YaPeng,HAO YinChuan,ZHANG XingHua,XUE JiQuan,XU ShuTu Scientia Agricultura Sinica    2022, 55 (13): 2485-2499.   DOI: 10.3864/j.issn.0578-1752.2022.13.001 Abstract （587）   HTML （88）    PDF （4472KB）（326）       Knowledge map    Save 【Objective】Ear traits are important components of grain yield in maize. Dissecting their genetic basis and mining significant SNPs using genome-wide association study (GWAS) can provide references for cloning functional genes and breeding high-yield maize varieties. 【Method】A total of 115 superior inbred lines from Shaan A group and Shaan B group, as well as four domestic backbone lines were selected as parents. Based on NCⅡ genetic design, an association population consisting of 442 hybrids was constructed, which was planted in two different environments to collect phenotype data of ear traits. Meanwhile, all parental lines were sequenced by the tunable genotyping by sequencing (tGBS) protocols. According to the genotype of inbred lines, altogether 19 461 high-quality SNPs were inferred in the association population. Then, GWAS was performed using 19 461 SNPs and phenotype data by three models including additive, dominance and epistasis, respectively. Combining with the transcriptome data of maize ear related tissues in the public database and the annotation information of genes, candidate genes were predicted. 【Result】Phenotypic data analysis showed that eight ear traits followed a continuous distribution, and there were 3.78%-45.25% of phenotypic variation. Analysis of variance indicated that environment and genotype effects reached an extremely significant level (P<0.001), and the range of broad-sense heritability was from 54.15% to 68.89%. And there were significantly positive or negative correlations among ear traits of hybrids. In total, 16, 3, 79 significant SNPs/pairs were identified under additive, dominant, and epistatic models, respectively. The significant loci detected by the three models cumulatively explained 38.21%-60.69% of the phenotypic variation of each trait. The cumulative phenotypic variation of significant SNP detected by additive model and epistatic model was 0.00-41.26% and 15.18%-45.36%, respectively. Effect analysis of significant SNPs identified by additive and dominant models showed most SNPs with additive or partial dominance effects, and only two with over-dominance effects. Further, only seven single-SNPs and five interaction pairs explained more than 5% of the phenotypic variation, and 17 candidate genes were predicted based on the SNP locations and gene expression information. 【Conclusion】Ear traits of maize hybrids were mainly affected by additive and epistasis effects, but less by dominance effects. Multiple SNPs identified by additive and dominant models showed additive and partially dominance effects, and aggregating favorable alleles of these SNPs could improve the target traits. Table and Figures | Reference | Related Articles | Metrics

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QTL Mapping for Traits Related to Seed Number Per Pod in Peanut (Arachis hypogaea L.) HAO Jing,LI XiuKun,CUI ShunLi,DENG HongTao,HOU MingYu,LIU YingRu,YANG XinLei,MU GuoJun,LIU LiFeng Scientia Agricultura Sinica    2022, 55 (13): 2500-2508.   DOI: 10.3864/j.issn.0578-1752.2022.13.002 Abstract （379）   HTML （46）    PDF （1382KB）（166）       Knowledge map    Save 【Objective】Peanut (Arachis hypogaea L.) is one of the important vegetable oil and cash crop. High yield is always the predominant objective in peanut breeding and determined by seed number per unit area and seed weight. Seed number per unit area is produced by planting density per unit area×number of pods per plant×number of seeds per pod. Therefore, the genetic dissection of the number of seeds per pod is helpful to explore the gene/locus related to this trait, which provides an important theoretical basis for the molecular breeding of yield in peanut.【Method】A RIL population, derived from Silihong×Jinonghei 3, were planted at Qingyuan experimental station of Hebei Agricultural University in Baoding city, Hebei province in 2018(E1) and 2020(E2). Phenotypic values of traits associated with the number of one seed pods per plant, two seeds pods per plant and multiple pods per plant were investigated at harvest stage. By using the genetic linkage map constructed by laboratory of Peanut innovation team, Hebei Agricultural University and software of QTL Icimapping V4.1(the Inclusive composite interval Mapping (ICIM)), QTL mapping for the number of seeds per pod was carried out under two environments.【Result】The results showed that the rates of one seed pods per plant and two seeds pods per plant were normal distribution, while the rate of multiple pods per plant was skewed normal distribution. A total of 11 QTLs were detected for the three traits, which could explain the phenotypic variation of 4.66%-22.34% and the additive effects of -9.35-9.42. Among of them, 5 QTLs for the rate of multiple pods per plant with explained 3.19% to 22.34% of phenotypic variation were obtained. The additive effect of one QTL from Jinonghei 3 was negative (-4.77), while the additive effect of the other four QTLs from Silihong was positive (3.59-9.42). Two QTLs for the rate of one seed pods per plant were mapped with explained 4.97%-6.43% of phenotypic variation. The additive effects of the two QTLs from Jinonghei 3 were negative (-4.45 and -4.54). Four QTLs for the rate of two seed pods per plant were located with explained 4.97%-6.43% of phenotypic variation. The additive effects of the four QTLs from Jinonghei 3 were negative (-9.35--3.84). Among of these QTLs, 6 QTLs were major QTLs, of which qRMSPA05 was repeatedly detected, and the heritable phenotypic variation was 16.58%-17.34%, and the additive effect was 7.69-8.12.【Conclusion】Six major QTLs and one major stable QTL for multiple pods per plant were identified, which will be helpful for improving the yield traits in peanut. The results can be used as important candidate segments for genetic improvement, and molecular marker assisted selection and fine mapping. Table and Figures | Reference | Related Articles | Metrics

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