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Knockout of miR396 genes increases seed size and yield in soybean

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Journal of Integrative Plant Biology 2024年第6期66卷 1148-1157页

作者： Hongtao Xie Fei Su Qingfeng Niu Leping Geng Xuesong Cao Minglei Song Jinsong Dong Zai Zheng Rui Guo Yang Zhang Yuanwei Deng Zhanbo Ji Kang Pang Jian-Kang Zhu Jianhua Zhu School of Life Sciences Anhui Agricultural University Research Center for Biological breeding Technology Research Institute of Frontier Science Anhui Agricultural University Bellagen Biotechnology Co.Ltd Institute of Crop Sciences/National Nanfan Research Institute Chinese Academy of Agricultural Sciences Key Laboratory of Gene Editing Technologies (Hainan) Ministry of Agriculture and Rural Affairs Southern University of Science and Technology Hainan Yazhou Bay Seed laboratory

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. mi R396 genes have been shown to negatively regulate grain size in rice,but whether mi R396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean mi R396genes through genome editing with clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas)12SF01 in the elite soybean cultivar Zhonghuang 302(ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants(mir396-abcd and mir396acfi), and two quintuple mutants(mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfiplants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems.Taken together, our study demonstrated that loss-of-function of mi R396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

关键词： Cas12SF01 CRISPR/Cas miR396 seed size soybean yield

J-family genes redundantly regulate flowering time and increase yield in soybean

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The Crop Journal 2024年第3期12卷 944-949页

作者： Haiyang Li Zheng Chen Fan Wang Hongli Xiang Shuangrong Liu Chuanjie Gou Chao Fang Liyu Chen Tiantian Bu Fanjiang Kong Xiaohui Zhao Baohui Liu Xiaoya Lin Guangdong key laboratory of Plant Adaptation and Molecular Design/Guangzhou key laboratory of Crop gene editing/Innovative Center of Molecular genetics and Evolution School of Life SciencesGuangzhou UniversityGuangzhou 510006GuangdongChina

Soybean(Glycine max)is a short-day crop whose flowering time is regulated by photoperiod.The longjuvenile trait extends its vegetative phase and increases yield under short-day conditions.Natural variation in J,the major locus controlling this trait,modulates flowering time.We report that the three J-family genes influence soybean flowering time,with the triple mutant Guangzhou Mammoth-2 flowering late under short days by inhibiting transcription of E1-family genes.J-family genes offer promising allelic combinations for breeding.

关键词： Soybean Flowering time Yield J-family genes

Molecular Mechanism of Rice Necrotic Lesion for Optimized Yield and Disease Resistance

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Rice science 2024年第3期31卷 285-299页

作者： HOU Xinyue WANG Yuping QIAN Qian REN Deyong State key laboratory of Rice Biology and breeding China National Rice Research InstituteHangzhou 310006China State key laboratory of Crop gene Exploration and Utilization in Southwest China Rice Research InstituteSichuan Agricultural UniversityChengdu 611130China

How to balance rice resistance and yield is an important issue in rice breeding.Plants with mutated necrotic lesion genes often have persistent broad-spectrum resistance,but this broad-spectrum resistance usually comes at the expense of yield.Currently,many necrotic lesion mutants in rice have been identified,and these genes are involved in disease resistance pathways.This review provides a detailed introduction to the characteristics,classification,and molecular mechanisms of necrotic lesion formation.Additionally,we review the molecular regulatory pathways of genes involved in rice disease resistance.Concurrently,we summarize the relationship between resistance and yield in rice using newly developed gene editing methods.We discuss a rational and precise breeding strategy to better utilize molecular design technology for breeding disease-resistant and high-yield rice varieties.

关键词： rice necrotic lesion regulation mechanism high yield high resistance

Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

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The Crop Journal 2024年第2期12卷 321-329页

作者： Shaoshuai Liu Ke Wang Shuaifeng Geng Moammar Hossain Xingguo Ye Aili Li Long Mao Karl-Heinz Kogel State key laboratory of Crop gene Resources and breeding Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 100081China Institute of Phytopathology Centre for BioSystemsLand Use and NutritionJustus Liebig UniversityHeinrich Buff-Ring 26-32D-35392GiessenGermany

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

关键词： Agrobacterium tumefaciens Cereal species Genome editing genetic engineering Plant breeding

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Hi-TOM 2.0:an improved platform for high-throughput mutation detection

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Science China(Life Sciences) 2024年第7期67卷 1532-1534页

作者： Tingting Sun Qing Liu Xi Chen Fengyue Hu Kejian Wang State key laboratory of Rice Biology and breeding China National Rice Research InstituteChinese Academy of Agricultural SciencesHangzhou 310006China Hainan Seed Industry laboratory Sanya 572025China

Dear Editor,CRISPR/Cas systems,which can create novel mutations by manipulating the genome or transcriptome,have been developed as a powerful molecular tool for gene editing in various organisms(Cheng et al.,2022;Gao,2021).However,identifying mutations in large samples and decoding chimeric mutations are time-consuming,labor-intensive.

关键词： CRISPR/Cas consuming mutation

Simple method for transformation and gene editing in medicinal plants

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Journal of Integrative Plant Biology 2024年第1期66卷 17-19页

作者： Xuesong Cao Hongtao Xie Minglei Song Lianghui Zhao Hailiang Liu Guofu Li Jian‐Kang Zhu Institute of Advanced Biotechnology and School of Life Sciences Southern University of Science and TechnologyShenzhen 518055China Bellagen Biotechnology Co.Ltd. Jinan 250000China Institute for Regenerative Medicine Shanghai East HospitalTongji University School of MedicineShanghai 200123China Ministry of Agriculture and Rural Affairs key laboratory of gene editing Technologies Nanfan Research InstituteChinese Academy of Agricultural SciencesSanya 572024China

gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding.However,a critical challenge hindering the widespread adoption of these technologies is the efficient delivery of gene-editing tools.The predominant methods for introducing these tools into plants typically involve Agrobacterium tumefaciens-mediated transformation or particle bombardment(Mao et al.,2019).Unfortunately,these traditional gene delivery methods require delicate and timeconsuming tissue culture procedures and show limited success,especially in medicinal and other less studied plants.A recent breakthrough is the development of the Cut-Dip-Budding(CDB)gene delivery system.The CDB system is highly effective for plants with root-suckering capabilities.It allows the delivery of transgenes and gene-editing tools into plants through hairy root induction followed by shoot regeneration from transformed hairy roots,bypassing the need for tissue culture processes(Cao et al.,2023).

关键词： transformation culture breakthrough

GmAP1d regulates flowering time under long-day photoperiods in soybean

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The Crop Journal 2024年第3期12卷 845-855页

作者： Shiyu Guo Yanfei Li Hongmei Qiu Guoyu Hu Chaosen Zhao Ruizhen Wang Hao Zhang Yu Tian Xiaoyu Li Bin Liu Ying-hui Li Li-juan Qiu College of Agriculture Northeast Agricultural UniversityHarbin 150030HeilongjiangChina State key laboratory of Crop gene Resources and breeding/The National key Facility for Crop gene Resources and genetic Improvement(NFCRI)/key laboratory of Grain Crop genetic Resources Evaluation and Utilization(MARA) Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 100081China Jilin Academy of Agricultural Sciences/National Engineering Research Center for Soybean Changchun 130033JilinChina Crop Research Institute Anhui Academy of Agricultural SciencesHefei 230031AnhuiChina Crops Research Institute of Jiangxi Academy of Agricultural Sciences/Jiangxi Province key laboratory of Oilcrops Biology Nanchang 330200JiangxiChina key Lab of Chinese Medicine Resources Conservation State Administration of Traditional Chinese Medicine of the People’s Republic of ChinaInstitute of Medicinal Plant DevelopmentChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100193China

Flowering time is important for adaptation of soybean(Glycine max)to different environments.Here,we conducted a genome-wide association study of flowering time using a panel of 1490 cultivated soybean accessions.We identified three strong signals at the qFT02-2 locus(Chr02:12037319–12238569),which were associated with flowering time in three environments:Gongzhuling,Mengcheng,and Nanchang.By analyzing linkage disequilibrium,gene expression patterns,gene annotation,and the diversity of variants,we identified an AP1 homolog as the candidate gene for the qFT02-2 locus,which we named GmAP1d.Only one nonsynonymous polymorphism existed among 1490 soybean accessions at position Chr02:12087053.Accessions carrying the Chr02:12087053-T allele flowered significantly earlier than those carrying the Chr02:12087053-A allele.Thus,we developed a cleaved amplified polymorphic sequence(CAPS)marker for the SNP at Chr02:12087053,which is suitable for marker-assisted breeding of flowering time.Knockout of GmAP1d in the‘Williams 82’background by gene editing promoted flowering under long-day conditions,confirming that GmAP1d is the causal gene for qFT02-2.An analysis of the region surrounding GmAP1d revealed that GmAP1d was artificially selected during the genetic improvement of soybean.Through stepwise selection,the proportion of modern cultivars carrying the Chr02:12087053-T allele has increased,and this allele has become nearly fixed(95%)in northern China.These findings provide a theoretical basis for better understanding the molecular regulatory mechanism of flowering time in soybean and a target gene that can be used for breeding modern soybean cultivars adapted to different latitudes.

关键词： Soybean Flowering time GWAS GmAP1d Long-day conditions

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

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Science China(Life Sciences) 2024年第3期67卷 555-564页

作者： Jilong Ren Tang Hai Yangcan Chen Ke Sun Zhiqiang Han Jing Wang Chongyang Li Qingwei Wang Leyun Wang Huabing Zhu Dawei Yu Wei Li Shanjiang Zhao State key laboratory of Animal Nutrition Key Laboratory of Animal GeneticsBreeding and Reproduction of Ministry of Agriculture and Rural AffairsInstitute of Animal ScienceChinese Academy of Agricultural SciencesBeijing 100193China State key laboratoryf Stem Cell and Reproductive Biology Instituteof ZooogyChinese Academyof SciencesBeijing 100101China Beijing Institute for Stem Cell and Regenerative Medicine Beijing 100101China Beijing Farm Animal Research Center Institute of ZoologyChinese Academy of SciencesBeijing 100101China

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

关键词： Cas12i^(Max) gene editing livestock multiple trait improvement

Identification and effective regulation of scarb1 gene involved in pigmentation change in autotetraploid Carassius auratus

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Zoological Research 2024年第2期45卷 381-397页

作者： Xi-Dan Xu Yue Zhou Chong-Qing Wang Xu Huang Kun Zhang Xiao-Wei Xu Li-Wen He Xin-Yue Zhang Xin-Zhu Fu Ming Ma Qin-Bo Qin Shao-Jun Liu State key laboratory of Developmental Biology of Freshwater Fish Engineering Research Center of Polyploid Fish Reproduction and Breeding of the State Education MinistryCollege of Life SciencesHunan Normal UniversityChangshaHunan 410081China Nansha-South China Agricultural University Fishery Research Institute GuangzhouGuangdong 511458China Hunan Yuelu Mountain Science and Technology Co.Ltd.for Aquatic breeding ChangshaHunan 410081China College of Chemistry and Chemical Engineering Hunan Normal UniversityChangshaHunan 410081China

The autotetraploid Carassius auratus(4nRR,4n=200,RRRR)is derived from whole-genome duplication of Carassius auratus red var.(RCC,2n=100,RR).In the current study,we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin(red in RCC,brownish-yellow in4nRR).To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR,we performed transcriptome profiling and molecular functional verification in RCC and 4nRR.Results revealed that scarb1,associated with carotenoid metabolism,underwent significant down-regulation in 4nRR.Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration.Subsequently,we identified four divergent scarb1 homeologs in 4nRR:two original scarb1 homeologs from RCC and two duplicated ones.Notably,three of these homeologs possessed two highly conserved alleles,exhibiting biased and allelespecific expression in the skin.Remarkably,after precise editing of both the original and duplicated scarb1homeologs and/or alleles,4nRR individuals,whether singly or multiply mutated,displayed a transition from brownishyellow skin to a cyan-gray phenotype.Concurrently,the proportional areas of the cyan-gray regions displayed a gene-dose correlation.These findings illustrate the subfunctionalization of duplicated scarb1,with all scarb1genes synergistically and equally contributing to the pigmentation of 4nRR.This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploidfish,substantiallyenrichingour understanding of coloration formation and change within this group of organisms.

关键词： Autopolyploidization Coloration change scarb1 Functional differentiation genetic changes

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