Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (10): 2064-2072.doi: 10.3864/j.issn.0578-1752.2021.10.003


Creation of New Maize Variety with Fragrant Rice Like Flavor by Editing BADH2-1 and BADH2-2 Using CRISPR/Cas9

ZHANG Xiang(),SHI YaXing(),LU BaiShan(),WU Ying,LIU Ya,WANG YuanDong(),YANG JinXiao(),ZHAO JiuRan()   

  1. Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097
  • Received:2021-01-25 Accepted:2020-02-20 Online:2021-05-16 Published:2021-05-24
  • Contact: YuanDong WANG,JinXiao YANG,JiuRan ZHAO;;;;;


【Objective】Fragrance is an important trait for quality improvement of crops. The 2-acetyl-1-pyrroline (2-AP) is the major component of the aroma flavor. BADH2 controls fragrance in plants, and its null or weak alleles lead to 2-AP accumulation. In this study, the fragrance related genes were modified in JING724, a maize elite inbred line invented by Beijing Academy of Agriculture and Forestry Sciences, using CRISPR/Cas9 to improve its trait of fragrance. 【Method】To find BADH gene family of target species, OsBADH2 protein sequence was used to search against protein databases of Arabidopsis, rice and maize with the Ensembl online BLAST tool. All BADH family members were verified by protein domain information in UniProt database. Furthermore, phylogenetic analysis conducted in MEGA software was used to search for maize BADH2 homologs as gene-editing targets. Based on principles of CRISPR/Cas9, we designed highly specific sgRNA to target the candidate genes. The CRISPR/Cas9 vector containing this sgRNA was introduced into the maize variety JING724 by Agrobacterium-mediated transformation. We obtained transgenic maize plants with PMI resistance. Sanger sequencing was used to confirm the CRISPR/Cas9-mediated mutations. Finally, we used gas chromatography-mass spectrometry (GC-MS) to measure the 2-AP content in T1 seeds of the gene-editing lines. 【Result】Phylogenetic analysis showed that there were two BADH2 homologs in the maize gnome, subsequently they were named ZmBADH2-1 and ZmBADH2-2. ZmBADH2-1 is located in chromosome 4, whereas ZmBADH2-2 was in chromosome 1. Both genes have 15 exons and 14 introns. The 4th exon of ZmBADH2-1 shares high nucleotide identity with that of ZmBADH2-2. A specific target sequence, which is located in the 4th exons of both genes, was designed and introduced into a CRISPR/Cas9 vector. Using this vector, 10 gene-editing lines were acquired after maize transformation. PCR amplification and sanger sequencing revealed that, in each of the 10 gene-editing lines, different type of insertions or deletions were introduced into the target sites of both ZmBADH2 genes successfully. Genotypes of mutations included biallelic and multi-allelic mutations. Mess spectra analysis showed that Zmbadh2-1/Zmbadh2-2 double mutants had 2-AP, which is the same substance of flavor with that in fragrant rice. Using GC-MS, we found that 2-AP contents in grains gathered from four randomly selected T0 gene-editing lines were 438.29, 404.63, 348.65 and 161.82 μg·kg-1, respectively. On the contrary, no 2-AP was detected in JING724 wild type. 【Conclusion】With site specific mutations introduced into ZmBADH2-1 and ZmBADH2-2 simultaneously using CRISPR/Cas9, new maize variety with fragrant rice like flavor was created successfully.

Key words: maize elite inbred line JING724, CRISPR/Cas9, genome editing technology, BADH2, fragrance, 2-acetyl-1-pyrroline

Supplementary Fig.1

Diagram of the map of CRISPR/Cas9"

Table 1

Primers used in this study"

Primer name
Primer sequence (5’-3’)
Construction of cassette2
Construction of cassette3
Mutation detection of ZmBADH2-1 target site
Mutation detection of ZmBADH2-2 target site
T0 Transgenic plants detection
Mutation detection of ZmBADH2-2 target site

Fig. 1

The phylogenetic tree of Oryza sativa, Zea mays and Arabidopsis thaliana BADH gene family Red words represented maize ZmBADH2-1 (Zm00001d050339) and ZmBADH2-2 (Zm00001d032257)"

Fig. 2

Diagram of the sgRNA design (A) and mutation analysis (B) on ZmBADH2-1 and ZmBADH2-2 The nucleotides with underline represented target sequence, the nucleotides with red font represented PAM sequence, the nucleotides with green font indicated bases of insertion, -: Base deletions, blue words indicated base substitutions"

Supplementary Fig.2

Gene-editing induced mutations caused abnormalities of ZmBADH2-1and ZmBADH2-2 protein sequences"

Supplementary Fig.3

T1 seeds of Zmbadh1/Zmbadh2 double mutants"

Fig. 3

Chromatography of 2-AP in Daohuaxiang seeds (A) and mass spectra of 2-AP in Daohuaxiang (B) and T1 seeds of BH02 line (C)"

Fig. 4

The chromatogram (A) and contents of 2-AP (B) in the seeds of gene-editing lines (n=3, mean±SD) Lower case letters represented significant difference (P<0.01) in 2-AP contents of different gene editing lines"

Supplementary Fig.4

Mutations and 2-AP content of Zmbadh2-2 mutants A: Diagram of the sgRNA design of ZmBADH2-2. Primers used to detect mutation were shown in supplementary table 1. B: Mutations of two gene editing line on ZmBADH2-2. C: 2-AP content of Nipponbare, Daohuaxiang, B104 maize inbred line, Zmbadh2-1/Zmnadh2-2 double mutant line BH02 and two gene editing line of Zmbadh2-2 mutant in B104 background. Lower case letters represented significant difference (P<0.01) in 2-AP contents of different gene editing lines"

[1] BRADBURY L M T, FITZGERALD T L, HENRY R J, JIN Q, WATERS D L. The gene for fragrance in rice. Plant Biotechnology Journal, 2005,3(3):363-370.
doi: 10.1111/pbi.2005.3.issue-3
[2] JEZUSSEK M, JULIANO B O, SCHIEBERLE P. Comparison of key aroma compounds in cooked brown rice varieties based on aroma extract dilution analyses. Journal of Agricultural and Food Chemistry, 2002,50(5):1101-1105.
doi: 10.1021/jf0108720
[3] HINGE V R, PATIL H B, NADAF A B. Aroma volatile analyses and 2AP characterization at various developmental stages in Basmati and Non-Basmati scented rice (Oryza sativa L.) cultivars. Rice, 2016,9(1):38.
doi: 10.1186/s12284-016-0113-6
[4] JUWATTANASOMRAN R, SOMTA P, CHANKAEW S, SHIMIZU T, WONGPORNCHAI S, KAGA A, SRINIVES P. A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety "Kaori" and SNAP marker development for the fragrance. Theoretical and Applied Genetics, 2011,122(3):533-541.
doi: 10.1007/s00122-010-1467-6
[5] JUWATTANASOMRAN R, SOMTA P, KAGA A, CHANKAEW S, SHIMIZU T, SORAJJAPINUN W, SRINIVES P. Identification of a new fragrance allele in soybean and development of its functional marker. Molecular Breeding, 2012,29(1):13-21.
doi: 10.1007/s11032-010-9523-0
[6] YUNDAENG C, SOMTA P, TANGPHATSORNRUANG S, CHANKAEW S, SRINIVES P. A single base substitution in BADH/AMADH is responsible for fragrance in cucumber (Cucumis sativus L.), and development of SNAP markers for the fragrance. Theoretical and Applied Genetics, 2015e128(9):1881-1892.
[7] YUNDAENG C, SOMTA P, TANGPHATSORNRUANG S, WONGPORNCHAI S, SRINIVES P. Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench). Theoretical and Applied Genetics, 2013,126(11):2897-2906.
doi: 10.1007/s00122-013-2180-z
[8] KHANDAGALE K S, CHAVHAN R, NADAF A B. RNAi-mediated down regulation of BADH2 gene for expression of 2-acetyl-1- pyrroline in non-scented indica rice IR-64 (Oryza sativa L.). 3 Biotechnology, 2020,10(4):145.
[9] CHEN M, WEI X, SHAO G, TANG S, LUO J, HU P. Fragrance of the rice grain achieved via artificial microRNA-induced down- regulation of OsBADH2. Plant Breeding, 2012,131(5):584-590.
doi: 10.1111/pbr.2012.131.issue-5
[10] NIU X, TANG W, HUANG W, REN G, WANG Q, LUO D, XIAO Y, YANG S, WANG F, LU B R, GAO F, LU T, LIU Y. RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.). BMC Plant Biology, 2008,8:100.
doi: 10.1186/1471-2229-8-100
[11] DONG L, QI X, ZHU J, LIU C, ZHANG X, CHENG B, MAO L, XIE C. Supersweet and waxy: meeting the diverse demands for specialty maize by genome editing. Plant Biotechnology Journal, 2019,17(10):1853-1855.
doi: 10.1111/pbi.v17.10
[12] SHI J, GAO H, WANG H, LAFITTE H R, ARCHIBALD R L, YANG M, HAKIMI S M, MO H, HABBEN J E. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal, 2017,15(2):207-216.
doi: 10.1111/pbi.2017.15.issue-2
[13] ZHANG J, ZHANG X, CHEN R, YANG L, FAN K, LIU Y, WANG G, REN Z, LIU Y. Generation of transgene-free semidwarf maize plants by gene editing of Gibberellin-Oxidase20-3 using CRISPR/Cas9. Frontiers in Plant Science, 2020,11:1048.
doi: 10.3389/fpls.2020.01048
[14] SVITASHEV S, SCHWARTZ C, LENDERTS B, YOUNG J K, MARK CIGAN A. Genome editing in maize directed by CRISPR- Cas9 ribonucleoprotein complexes. Nature Communications, 2016,7:13274.
doi: 10.1038/ncomms13274
[15] SHAN Q, ZHANG Y, CHEN K, ZHANG K, GAO C. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnology Journal, 2015,13(6):791-800.
doi: 10.1111/pbi.2015.13.issue-6
[16] WANG X, SHI Z, ZHANG R, SUN X, WANG J, WANG S, ZHANG Y, ZHAO Y, SU A, LI C, WANG R, ZHANG Y, WANG S, WANG Y, SONG W, ZHAO J. Stalk architecture, cell wall composition, and QTL underlying high stalk flexibility for improved lodging resistance in maize. BMC Plant Biology, 2020,20(1):515.
doi: 10.1186/s12870-020-02728-2
[17] WU Y, XU W, WANG F, ZHAO S, FENG F, SONG J, ZHANG C, YANG J. Increasing cytosine base editing scope and efficiency with engineered Cas9-PmCDA1 fusions and the modified sgRNA in rice. Frontiers in Genetics, 2019,10:379.
doi: 10.3389/fgene.2019.00379
[18] XIE X, MA X, ZHU Q, ZENG D, LI G, LIU Y G. CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing. Molecular Plant, 2017,10(9):1246-1249.
doi: 10.1016/j.molp.2017.06.004
[19] ISHIDA Y, HIEI Y, KOMARI T. Agrobacterium-mediated transformation of maize. Nature Protocals, 2007,2(7):1614-1621.
[20] BRINKMAN E K, VAN STEENSEL B. Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER. New York: Springer New York Press, 2019: 29-44.
[21] HILL J T, DEMAREST B L, BISGROVE B W, SU Y C, SMITH M, YOST H J. Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products. Developmental Dynamics, 2014,243(12):1632-1636.
doi: 10.1002/dvdy.v243.12
[22] HOOPES G M, HAMILTON J P, WOOD J C, ESTEBAN E, PASHA A, VAILLANCOURT B, PROVART N J, BUELL C R. An updated gene atlas for maize reveals organ-specific and stress-induced genes. The Plant Journal, 2019,97(6):1154-1167.
doi: 10.1111/tpj.2019.97.issue-6
[23] STELPFLUG S C, SEKHON R S, VAILLANCOURT B, HIRSCH C N, BUELL C R, DE LEON N, KAEPPLER S M. An expanded maize gene expression atlas based on RNA sequencing and its use to explore root development. Plant Genome, 2016,9(1):1-16
[24] 应兴华, 徐霞, 陈铭学, 欧阳由男, 朱智伟, 闵捷. 气相色谱-质谱技术分析香稻特征化合物2-乙酰基吡咯啉. 色谱, 2010,28(8):59-62.
YING X H, XU X, CHEN M X, OUYANG Y N, ZHU Z W, MIN J. Analysis of fragrant compounds in fragrant rice using gas chromatography-mass spectrometry. Chromatography, 2010,28(8):59-62. (in Chinese)
[25] 郑家团, 杨德卫, 董炼飞, 游晴如, 郑轶, 涂诗航, 周鹏. 香型水稻的遗传和育种现状. 福建农业学报, 2012,27(10):1134-1138.
ZEHGN J T, YANG D W, DONG L F, YOU Q R, ZHENG Y, TU S H, ZHOU P. Genetics and breeding of fragrant rice. Fujian Agricultural Journal, 2012,27(10):1134-1138. (in Chinese)
[26] WANG F, WANG C, LIU P, LEI C, HAO W, GAO Y, LIU Y G, ZHAO K. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS ONE, 2016,11(4):e0154027.
doi: 10.1371/journal.pone.0154027
[27] LI M, LI X, ZHOU Z, WU P, FANG M, PAN X, LIN Q, LUO W, WU G, LI H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science, 2016,7:377.
[28] ZHANG Y, LIANG Z, ZONG Y, WANG Y, LIU J, CHEN K, QIU J L, GAO C. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communications, 2016,7:12617.
doi: 10.1038/ncomms12617
[29] CAI Y, CHEN L, LIU X, GUO C, SUN S, WU C, JIANG B, HAN T, HOU W. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean. Plant Biotechnology Journal, 2018,16(1):176-185.
doi: 10.1111/pbi.2018.16.issue-1
[30] LV J, YU K, WEI J, GUI H, LIU C, LIANG D, WANG Y, ZHOU H, CARLIN R, RICH R, LU T, QUE Q, WANG W C, ZHANG X, KELLIHER T. Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3. Nature Biotechnology, 2020,38(12):1397-1401.
doi: 10.1038/s41587-020-0728-4
[1] YANG Min,XU HuaWei,WANG CuiLing,YANG Hu,WEI YueRong. Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize [J]. Scientia Agricultura Sinica, 2021, 54(4): 696-707.
[2] LI SongMei,QIU YuGe,CHEN ShengNan,WANG XiaoMeng,WANG ChunSheng. CRISPR/Cas9 Mediated Exogenous Gene Knock-in at ROSA26 Locus in Sheep Umbilical Cord Mesenchymal Stem Cells [J]. Scientia Agricultura Sinica, 2021, 54(2): 400-411.
[3] WU ShiYang,YANG XiaoYi,ZHANG YanWen,HOU DianYun,XU HuaWei. Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology [J]. Scientia Agricultura Sinica, 2021, 54(18): 3805-3817.
[4] LI ZhaoWei,LING DongLan,SUN CongYing,ZENG HuiLing,LIU KaiJi,LAN YingShan,FAN Kai,LIN WenXiong. CRISPR/Cas9 Targeted Editing of OsIAA11 in Rice [J]. Scientia Agricultura Sinica, 2021, 54(13): 2699-2709.
[5] QI YongBin,ZHANG LiXia,WANG LinYou,SONG Jian,WANG JianJun. CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice [J]. Scientia Agricultura Sinica, 2020, 53(8): 1501-1509.
[6] ZHANG Cheng,HE MingLiang,WANG Wei,XU FangSen. Development of an Efficient Editing System in Arabidopsis by CRISPR-Cas9 [J]. Scientia Agricultura Sinica, 2020, 53(12): 2340-2348.
[7] YANG Qiang, XU Pan, JIANG Kai, QIAO ChuanMin, REN Jun, HUANG LuSheng, XING YuYun. Targeted Editing of BMPR-IB Gene in Porcine Fetal Fibroblasts via Lentivirus Mediated CRISPR/Cas9 Technology and Its Effects on Expression of Genes in the BMPs Signaling Pathway [J]. Scientia Agricultura Sinica, 2018, 51(7): 1378-1389.
[8] WEI YingHui, LIU ZhiGuo, XU Kui, Evanna HUYHN, Paul DYCE, LI JiLiang, ZHOU WeiLiang, DONG ShuRen, FENG BaoLiang, MU YuLian, JuLang LI, LI Kui. Generation and Propagation of Cluster of Differentiation 163 Biallelic Gene Editing Pigs [J]. Scientia Agricultura Sinica, 2018, 51(4): 770-777.
[9] PU Yan, LIU Chao, LI Ji-Yang, AERZU GULI·TaShi, HU Yan, LIU XiaoDong. Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato [J]. Scientia Agricultura Sinica, 2018, 51(2): 315-326.
[10] SHENG XiaBing, TAN YanNing, SUN ZhiZhong, YU Dong, WANG XueFeng, YUAN GuiLong, YUAN DingYang, DUAN MeiJuan. Using CRISPR/Cas9-Mediated Targeted Mutagenesis of qSH1 Reduces the Seed Shattering in Rice [J]. Scientia Agricultura Sinica, 2018, 51(14): 2631-2641.
[11] HU ChunHua, DENG GuiMing, SUN XiaoXuan, ZUO CunWu, LI ChunYu, KUANG RuiBin, YANG QiaoSong, YI GanJun. Establishment of an Efficient CRISPR/Cas9-Mediated Gene Editing System in Banana [J]. Scientia Agricultura Sinica, 2017, 50(7): 1294-1301.
[12] JI Xin, LI Fei, YAN Yun, SUN HongZheng, ZHANG Jing, LI JunZhou, PENG Ting, DU YanXiu, ZHAO QuanZhi. CRISPR/Cas9 System-Based Editing of Phytochrome-Interacting Factor OsPIL15 [J]. Scientia Agricultura Sinica, 2017, 50(15): 2861-2871.
[13] JING Run-chun, LU Hong . The Development of CRISPR/Cas9 System and Its Application in Crop Genome Editing [J]. Scientia Agricultura Sinica, 2016, 49(7): 1219-1229.
[14] LIANG Ye, LI He, MA Yue, CAO Fei, DOU Yu-Juan, ZHANG Zhi-Hong. Comparative Analysis of Biological Characteristics and Quality in a White-Flesh Strawberry Mutant ‘Sachinoka’ and Its Wild Type [J]. Scientia Agricultura Sinica, 2012, 45(15): 3115-3123.
Full text



No Suggested Reading articles found!