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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 827-836.doi: 10.3724/SP.J.1006.2021.02044

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

Construction and identification of haploid induction gene OsMATL mutants in rice

WEN Qin(), JIA Si-Si, WANG Jia-Feng, HUANG Cui-Hong, WANG Hui, CHEN Zhi-Qiang, GUO Tao*()   

  1. National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou 510642, Guangdong, China
  • Received:2020-06-29 Accepted:2020-11-13 Online:2021-05-12 Published:2020-12-23
  • Contact: GUO Tao E-mail:wenqin111222@163.com;guo.tao@vip.163.com
  • Supported by:
    Research and Development Plant for the Key Areas in Guangdong Province(2018B020206002);China Agriculture Research System(CARS-01-17)

Abstract:

MTL is the key gene for control haploid induction traits in maize, which is highly function conserved in crops. OsMATL is a homologous gene of MTL in rice. It has been confirmed that OsMATL mutation can induce haploid in rice, but the effect of OsMATL mutation in different genetic backgrounds remains to be determined. In this study, Nipponbare (Oryza sativa japonica) and Huahang 48 (Oryza sativa indica) were used as research materials. CRISPR/Cas9 technology was used to edit different sites in the promoter region and coding region of OsMATL, and a series of OsMATL mutations with different genetic backgrounds were successfully obtained. The effect of different mutation sites on seed setting rate was analyzed, and it was found that the seed setting rate was 2%-15% in the coding region of OsMATL. There were different types of abortion seeds, while large fragment deletion in the promoter region had no significant differences on seed setting rate. In addition, the background of indica rice mutants of seed setting rate was higher than japonica rice mutants. OsMATL mutants of different types created in this study provided basic materials for further research on the mechanism of parthenogenetic haploid induction in rice.

Key words: rice, haploid induction gene, mutant

Fig. 1

Sequence and positions of targets in OsMATL The black bold letters are the protospacer adjacent motif (PAM) sequences; the red triangle points to the mutation site in the native MTL allele in maize."

Table 1

Primer sequences used in this study"

引物名称 Primer name 引物序列Primer sequence (5'-3')
OsMATL-P1-F GCATCCATGTGTTCCTGATCC
OsMATL-P1-R GGTCCCATTAAGTTTGGCGG
OsMATL-P2-F GGGAGAGTTGGGAACGTTGA
OsMATL-P2-R TGTCGAGGTAGAAGCGGTTG
Recom-F GTGCAGATGATCCGT GGCAACAAAGCACCAGTGGT
Recom-R CTATTTCTAGCTCTAAAAC AAAAAAAAAAGCACCGACTCGGTG
OsMATL-Cas9-1-F taGGTCTCCCTACCTCGACAAgttttagagctagaa
OsMATL-Cas9-1-R cgGGTCTCAGTAGAAGCGGTTtgcaccagccgggaa
OsMATL-Cas9-3-F taGGTCTCCAGGTCGAACTCGgttttagagctagaa
OsMATL-Cas9-3-R cgGGTCTCAACCTCATCGACGtgcaccagccgggaa
OsMATL-Cas9-4-F taGGTCTCCCAACGTCGAGACgttttagagctagaa
OsMATL-Cas9-4-R cgGGTCTCAGTTGACCCTCGAtgcaccagccgggaa
OsMATL-Cas9-P1-F taGGTCTCCTTGCTGGAAGGAgttttagagctagaa
OsMATL-Cas9-P1-R cgGGTCTCAGCAAGGAAACTAtgcaccagccgggaa
OsMATL-Cas9-P2-F taGGTCTCCTAATCCCATATTgttttagagctagaa
OsMATL-Cas9-P2-R cgGGTCTCAATTAAACCCAGCtgcaccagccgggaa
Cas9-J-F AAGCATTTCGTAGTGGGCCA
Cas9-J-R TTTGGTGGTCGCCGTTAGGA
OsMATL-1-F GCATCAGGGGCCTCATCC
OsMATL-1-R GCCCAGCATCTTCCTGATCT
OsMATL-3-F GCGTTGATTCCATCCGCATT
OsMATL-3-R GCTCCTCCTTGTCCTTCACC
OsMATL-4-F GCGCCCATCATCGACATCTT
OsMATL-4-R TGCGTGTACAGTTACTAACGCT
OsMATL-P-F GATCGATCGCCCATAAGCCA
OsMATL-P-R TGACCAACTTGGCGTCATGA
q-ACTIN-F GAATGCTAAGCCAAGAGGAG
q-ACTIN-R AATCACAAGTGAGAACCACAG
q-OsMATL-F CCGAGGTACAACGGCAAGTA
q-OsMATL-R AGAAGATGGTTGGCTGGAGC

Fig. 2

Schematic diagram of the CRISPR/Cas9 vector construction A: single-target vector; B: dual-target vector."

Fig. 3

Multiple alignment of MTL homologous genes amino sequences in cereals and A. thaliana The red box highlights the mutation site in the native MTL allele of maize."

Fig. 4

Promoter sequence and major cis-acting elements of OsMATL The blue letters are TATA-box; the gray shadow shows CAAT-box; other elements are underlined whose name remarks under the sequence."

Table 2

Homozygous mutation in coding region and large fragment deletion mutation in promoter region types of T0 generation"

植株编号
Number of plants
品种
Variety
序列
Sequence (5′-3′)
突变类型
Mutation type
Exon 1 WT AACCGCTTCTACCTCGACAACGGCCCACTCATC
1-1-1 Nipponbare AACCGCTTCTACCTCGAACAACGGCCCACTCATC +1 bp
1-1-2 Nipponbare AACCGCTTCTACCTCGATCAACGGCCCACTCATC +1 bp
1-1-3 Nipponbare AACCGCTTCTACCTCGAATTCG --------------------------- ATC -8 bp and substitute 3 bp
1-1-4 Nipponbare AACCGCTT --------------------------------- AACGGCCCACTCATC -10 bp
1-2-1 Huahang 48 AACCGCTTCTACCTCGATCAACGGCCCACTCATC +1 bp
Exon 3 WT CGTCGATGAGGTCGAACTCGCGGACCTTGCCGG
3-1-1 Nipponbare CGTCGATGAGGTCGAACTTCGCGGACCTTGCCGG +1 bp
3-1-2 Nipponbare CGTCGATGAGGTCGA-CTCGCGGACCTTGCCGG -1 bp
3-1-3 Nipponbare CGTCGATGAGGTCGAA-TCGCGGACCTTGCCGG -1 bp
3-1-4 Nipponbare CGTCGATGAGGTCGA ------ TCGCGGACCTTGCCGG -2 bp
3-1-5 Nipponbare CGTCGATGAGGTCG -------------------- CGGACCTTGCCGG -6 bp
3-1-6 Nipponbare CGTCGATGAGG ------------------------------ CGGACCTTGCCGG -9 bp
3-2-1 Huahang 48 CGTCGATGAGGTCGAAACTCGCGGACCTTGCCGG +1 bp
3-2-2 Huahang 48 CGTCGATGAGGTCGAA-TCGCGGACCTTGCCGG -1 bp
3-2-3 Huahang 48 CGTCGATGAGGTCGA ----- TCGCGGACCTTGCCGG -2 bp
3-2-4 Huahang 48 CGTCGATGAGGTC ------------ TCGCGGACCTTGCCGG -4 bp
3-2-5 Huahang 48 CGTCGATGAGGT --------------- TCGCGGACCTTGCCGG -5 bp
Exon 4 WT TCGAGGGTCAACGTCGAGACCGGCAGGTACGTC
4-1-1 Nipponbare TCGAGGGTCAACGTCGAAGACCGGCAGGTACGTC +1 bp
4-1-2 Nipponbare TCGAGGGTCAACGTCGATGACCGGCAGGTACGTC +1 bp
4-1-3 Nipponbare TCGAGGGTCAACGTC-AGACCGGCAGGTACGTC -1 bp
4-1-4 Nipponbare TCGAGGGTCAACGTC ------- GACCGGCAGGTACGTC -2 bp
4-2-1 Huahang 48 TCGAGGGTCAACGTCGAAGACCGGCAGGTACGTC +1 bp
4-2-2 Huahang 48 TCGAGGGTCAACGTCGATGACCGGCAGGTACGTC +1 bp
4-2-3 Huahang 48 TCGAGGGTCAACGT ------- AGACCGGCAGGTACGTC -2 bp
4-2-4 Huahang 48 TCGAGGGTCAACGTC ------ GAACCGGCAGGTACGTC -2 bp
4-2-5 Huahang 48 TCGAGGGT --------------------------------------------------------------------- ACGTC -20 bp
Promoter WT CCATCCTTCCAGCAAGGAAACTA(293 bp)CCGAATATGGGATTAAAC
CCAGC
P-1 Huahang 48 CCATCCT ------------------------------------- (-292 bp) -------------- ATGGGATTAAACCCAGC -292 bp
P-2 Huahang 48 CCATCC ---------------------------------------- (-293 bp) ------------- ATGGGATTAAACCCAGC -293 bp
P-3 Huahang 48 CCA -------------------------------------------------- (-304 bp) --------------------------------------- AAACCCAGC -304 bp
P-4 Huahang 48 TTAGTTA -------------------------------------- (-306 bp) ------------------- GGGATTAAACCCAGC -306 bp
P-5 Huahang 48 TTAGT --------------------------------------------- (-315 bp) ------------------------------------------ AACCCAGC -315 bp
P-6 Huahang 48 TTAGT --------------------------------------------- (-363 bp) -------------------- AGGAGTATATAATTA -363 bp
P-7 Huahang 48 CCAT ------------------------------------------------ (-722 bp) --------------------------------- TAGTTACGGAG -722 bp

Fig. 5

Tertiary structure prediction and relative expression levels of OsMATL gene in wild type and mutant A: tertiary structure prediction of OsMATL gene in wild type; B: tertiary structure prediction of OsMATL gene in the first exon mutant; C: tertiary structure prediction of OsMATL gene in the third exon mutant; D: tertiary structure prediction of OsMATL gene in the fourth exon mutant; E: relative expression levels of OsMATL gene in promoter region 293 bp deletion mutant. WT is wild type and MT is mutant."

Fig. 6

Seed setting rate and abortion seed rate of wild type and OsMATL mutant ** indicates significant differences at P < 0.01 between OsMATL mutant and wild type."

Fig. 7

Seed setting phenotype and different types of abortion seed phenotypes A, B: the phenotype of seed setting; C, D: the phenotype of abortion seed; the red triangles point to solid grains. Bar = 0.5 cm."

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