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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (8): 2144-2159.doi: 10.3724/SP.J.1006.2023.22043

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice (Oryza sativa L.)

WEI Xin-Yu1,3(), ZENG Yue-Hui1,3(), YANG Wang-Xing2,3, XIAO Chang-Chun1,3, HOU Xin-Po2,3, HUANG Jian-Hong1,3, ZOU Wen-Guang2,3, XU Xu-Ming2,3,*()   

  1. 1 Biotechnology Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian, China
    2 Rice Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, Fujian, China
    3 Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area, Sanming 365500, Fujian, China
  • Received:2022-07-11 Accepted:2023-02-21 Online:2023-08-12 Published:2023-03-03
  • Contact: XU Xu-Ming E-mail:wxy1209@163.com;1_zengyuehui_1@163.com;fj63xxm@sina.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-01);Fujian Provincial Natural Science Foundation(2021J01535);Fujian Provincial Natural Science Foundation(2021J01536);Sanming Municipal Science and Technology Project(2019-N-4)

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Abstract:

Fragrance is one of the important traits for quality improvement in rice, which is mainly controlled by the Badh2 gene encoding a betaine aldehyde dehydrogenase on chromosome 8. The mutation of Badh2 gene can increase the content of 2-acetyl-1-pyrroline (2-AP) and promote the fragrance production in rice. In this study, the Badh2 gene in Mingtai B (MTB), an elite maintainer line of Indica hybrid rice showing high eating quality bred by Sanming Academy of Agricultural Sciences, was edited and knocked out by using CRISPR-Cas9 technology. Two T0 transgenic lines carrying homozygous mutation on the loci of Badh2 were generated, and 48 T1 individuals derived from these two plants were genotyped by PCR amplification and sequencing analysis. A mutant line named MTB-badh2, which had a single T nucleotide insertion in the second exon of Badh2 without the vector skeleton, was finally obtained. In our study, the expression of Badh2 and the content of 2-AP were determined by semi-quantitative RT-PCR, qRT-PCR, and gas chromatography-mass spectrometry (GC-MS), respectively. Simultaneously, the 48 pairs of SSR markers recommended by “Sector Standard of Agriculture (NY/T 1433-2014)” were used to further analyze the DNA fingerprint and genetic diversity of the MTB and the MTB-badh2. The results showed that the Badh2 RNA level was significantly decreased in the MTB-badh2 compared with the wild-type MTB. In addition, the 2-AP content was dramatically increased in MTB-badh2. DNA fingerprint analysis revealed that only the Rm571 primer pair was specific for identifying allelic variation between wild type and MTB-badh2, suggesting that the genetic diversity between wild type and MTB-badh2 was very low. Furthermore, agronomic phenotype, cooking and eating quality, appearance quality of wild type, and MTB-badh2 were investigated and analyzed. There was no significant difference between MTB and MTB-badh2. The corresponding stable fragrant CMS line Mingtai A-badh2 (MTA-badh2) carrying homozygous mutant on the locus of Badh2 was successfully generated by the conventional test-crossing and back-crossing techniques combined with Badh2 sequencing analysis. Derived from this CMS line, MTA-badh2/Minghui 1831, MTA-badh2/Minghui 703, and MTA-badh2/Minghui 3009 hybrid combinations had been bred. Among them, the grain yield of MTA-badh2/Minghui 703 and MTA-badh2/Minghui 3009 were significantly increased compared with that of Mingtai you 703 and Mingtai you 3009, which were registered and released by the National Crop variety Appraisal Committee. Moreover, MTA-badh2/Minghui 1831 dramatically increased 2-AP content in the grains compared with MTA/Minghui 1831. Therefore, the CRISPR-Cas9-mediated technology could be used to edit the Badh2 and improve the fragrance in rice, and it provides the theoretical guidance for development of fragrant indica CMS line, leading to an accelerated breeding process of fragrant hybrid rice.

Key words: rice, CRISPR-Cas9, gene editing, fragrance, Badh2, 2-acetyl-1-pyrroline (2-AP)

Table 1

Primers used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途Usage
Target1-Fwd GGCATGGCCACGGCGATCCCGCAG 靶序列1构建
Construction of target site 1
Target1-Rev AAACCTGCGGGATCGCCGTGGCCA
Target 2-Fwd GGCAAGGCGAGATCCCGGCGGGCA 靶序列2构建
Construction of target site 2
Target 2-Rev AAACTGGCCACGGCGATCCCGCAG
HPT-F ATTTGTGTACGCCCGACAGT 鉴定HPT
PCR detection in HPT
HPT-R GTGCTTGACATTGGGGAGTT
Cas9 identify-F AGAACCTCTCCGATGCTATCC 鉴定Cas9
PCR detection in Cas9
Cas9 identify-R AGCAAGAGGACCAACGTAG
Badh2Seq-F CAAGGCAGCACAGAACAGA Badh2基因靶点测序检测
Sequencing for target in Badh2
Badh2Seq-R GTAGTCACCACCCTACCTTG
Badh2-Q-F TCCGGGCCAAGTACCTCC Badh2基因qPCR检测
qPCR detection in Badh2
Badh2-Q-R CGTCCATGTCCCATGCTG
Actin-F ACCTTCAACACCCCTGCTAT 内参基因Actin qPCR检测
qPCR detection in Actin (as internal standard)
Actin-R CACCATCACCAGAGTCCAAC

Fig. 1

CRISPR-Cas9-mediated Badh2 gene editing and its mutation analysis A: the vector map of Cas9/gRNA; B: PCR identification of the positive transgenic plant in the gene-editing plants of T0 generation with Cas9 identify-F/R; C: PCR identification of the positive transgenic plant in the gene-editing plants of T0 generation with HPT-F/R; D and E: mutation analysis of Target 1 (D) and Target 2 (E) in the gene-editing plants of T0 generation; F and H: PCR detection of Cas9 gene in the gene-editing plants of T1 generation with Cas9 identify-F/R; G and H: PCR detection of HPT gene in the gene-editing plants of T1 generation with HPT-F/R. LB, UBI, Cas9, gRNA, rU6, 35S, Hygro, and RB in Fig. A represent the vector left border, UBI promoter, Cas9 gene, guide RNA, rice U6 promoter, 35S promoter, hygromycin, and vector right border, respectively. “+” and “-” in Fig. B-C represent positive control and negative control, respectively. The nucleotides with blue and red font in Fig. D-E represent Target and PAM sequence, respectively,“-” and “+” in Fig. D-E represent base deletions and base insertion, respectively. M in Fig. B-C, F-H, and I all represent 2000 bp DNA marker."

Fig. 2

Badh2 RNA level and 2-acetyl-1-pyrroline (2-AP) content in Mingtai B (MTB) and Mingtai B-badh2 (MTB-badh2) A-B: the relative expression levels of Badh2 in MTB and MTB-badh2 analyzed by semi-quantitative RT-PCR (A) and qRT-PCR (B); C: 2-AP content in MTB and MTB-badh2; D-E: the total ion chromatograms (TIC) of 2-AP and TMP (as the internal standard) in MTB and MTB-badh2. OsActin in Fig. A was amplified as the control. Values in Fig. B-C are shown as means ± SDs of three biological replicates (Student’s t-test: *: P < 0.05, **: P < 0.01). MTB and MTB-badh2 in all figures represent Mingtai B and Mingtai B-badh2, respectively."

Fig. 3

Performance of phenotypic and agronomic traits in Mingtai B (MTB) and Mingtai B-badh2 (MTB-badh2) A-D: the phenotype comparison of plant, panicle, and grains between wild-type MTB and MTB-badh2 mutant at mature stage; E: plant height (cm); F: seed setting rate (%); G: the total grain number per panicle; H: 1000-grain weight (g); I: tiller number; J: panicle length (cm); K: flag leaf length (cm); L: flag leaf width (cm); M: grain length (cm); N: grain width (cm); O: length-width ratio. Bars: 10 cm in A, 3 cm in B, 0.5 cm in C and D. Values in Fig. E-O are shown as means ± SDs of five biological replicates (Student’s t-test: *: P < 0.05; **: P < 0.01). MTB: Mingtai B; MTB-badh2: Mingtai B-badh2."

Fig. 4

Performance of rice quality in Mingtai B (MTB) and Mingtai B-badh2 (MTB-badh2) A: gel consistency; B: alkali spreading value; C: amylose content (%); D: protein content (%); E: chalkiness degree; F: transparency degree; G: chalkiness rate (%); H: brown rice rate (%); I: white rice rate (%); J: head rice rate (%). Values are shown as means ± SDs of three biological replicates (Student’s t-test: *: P < 0.05, **: P < 0.01). MTB: Mingtai B; MTB-badh2: Mingtai B-badh2."

Fig. 5

DNA fingerprinting analysis of Mingtai B (MTB) and Mingtai B-badh2 (MTB-badh2) with 48 pairs of SSR primers Each pair of SSR primer had four samples, the first two were Mingtai B, and the last two were Mingtai B-badh2. M: 100 bp DNA ladder marker."

Fig. 6

Content of 2-AP in hybrid rice combination MTA-badh2/Minghui 1831 derived from MTA-badh2 A: plant morphology of MTA and MTA-badh2; B: plant morphology of MTA/MH1831 and MTA-badh2/MH1831; C: 2-AP content in MTA/MH1831, MTA-badh2/MH1831, and TGXXZ. Bars: 20 cm in A and B. Values in Fig. C are shown as means ± SDs of three biological replicates. Different letters indicate significantly different at the 0.01 probability level. MTA: Mingtai A, MTA-badh2: Mingtai A-badh2, MTA/MH1831: Mingtai A/Minghui 1831, MTA-badh2/MH1831: Mingtai A-badh2/Minghui 1831, TGXXZ: Taiguoxiaoxiangzhan."

Table 2

Agronomic characters and over-standard heterosis of hybrid rice combinations using Mingtai A-badh2 (MTA-badh2)"

杂交组合
Cross combination		 播始历期
Duration from seeding to heading (d)		 株高
Plant height
(cm)		 穗长
Length of main panicle
(cm)		 有效穗
Panicle number per plant		 穗粒数
Grain number per panicle		 结实率
Seed-settingrate (%)		 千粒重
1000-grain weight
(g)		 产量
Grain yield
(kg hm-2)		 比CK增产
Compared with control (%)
明太A-badh2/明恢703
MTA-badh2/Minghui 703		 74.7±0.6 a 113.2±2.5 a 25.5±0.3 A 12.7±0.6 a 210.0±5.0 A 78.1±0.1 a 25.5±0.2 A 10877.0±196.0 A 6.6
明太优703
Mingtaiyou 703		 74.7±0.6 a 114.1±1.4 a 25.3±0.2 A 13.0±0.0 a 212.7±5.0 A 77.4±0.4 a 25.3±0.2 A 10843.8±224.2 A 6.3
天优华占(CK)
Tianyouhuazhan (CK)		 74.3±0.6 a 110.9±2.2 a 23.8±0.3 B 13.3±0.6 a 193.3±4.2 B 76.6±0.5 b 23.3±0.2 B 10205.7±85.0 B 0.0
明太A-badh2/明恢3009
MTA-badh2/Minghui 3009		 75.0±0.0 a 118.4±2.1 a 24.5±0.3 A 12.7±0.6 a 196.3±8.1 A 76.9±0.4 a 25.9±0.4 A 9567.2±139.4 A 6.0
明太优3009
Mingtaiyou 3009		 75.3±0.6 a 119.1±2.0 a 24.9±0.3 A 12.3±0.6 a 195.3±6.5 A 77.1±0.7 a 25.5±0.7 A 9549.5±156.7 A 5.8
天优华占(CK)
Tianyouhuazhan (CK)		 75.6±0.6 a 111.9±2.4 b 23.0±0.4 B 13.0±0.0 a 171.7±3.1 B 75.3±0.2 b 23.5±0.2 B 9025.5±116.0 B 0.0
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