Supplementary data

Materials and methods

Rice cultivation

All cultivars and their homozygous derivatives were grown in 2016. Wild-type varieties of

different accumulated temperature zones were grown in parallel as controls. Seeds were sown

in seed beds in a greenhouse in early April and transplanted to the paddy field in the middle of

May. Plants were grown in Harbin (45° N) with natural long-day condition. Rice cultivation

followed the normal agricultural practices in the paddy field. Heading date was recorded from

sowing to the appearance of the first panicle (Li et al., 2015). For Nanjing9108 and its

derivatives, seeds were sown in a paddy field of the Jiangsu Academy of Agricultural

Sciences at Nanjing (31° N).

Construction of CRISPR/Cas9 vector for heading date genes

Under the guidance of CRISPR Primer Designer (http://www.plantsignal.cn), target

sequences were selected and target sequence-containing chimeric primers were designed (Yan

et al., 2015). We designed two sgRNAs for Hd2 (LOC_Os07g49460), one for Hd5

(LOC_Os08g07740), and two for Hd4 (LOC_Os07g15770) (Fig S1). The corresponding

primers are listed in Table S1. For the construction of CRISPR/Cas9 vector, multiple sgRNA

expression cassettes and CRSPR/Cas9 binary vector pYLCRISPR/Cas9Pubi-H were used

following the protocol previously described (Ma et al., 2015b). Simply, target sequences

flanked by diverse joint sequences were biosynthesized and inserted in the Bsa I sites of

different sgRNA expression cassettes. The integrated sgRNA expression cassettes were

amplified by nested PCR, and digested with Bsa I. Then, five sgRNA expression cassettes

were sequentially ligated into the binary vector pYLCRISPR/Cas9Pubi-H using the golden gate

cloning technique. The vectors were confirmed by enzyme digestion and sequencing.

Rice transformation and mutation detection

The CRISPR/Cas9 construct was introduced into Agrobacterium tumefaciens strain EHA105

by electroporation. Rice transformation was performed as described previously (Hiei et al.,

1994; Tian et al., 2015).

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At T0 generation, genomic DNA was extracted from leaves of transgenic rice plants. PCR

amplifications were carried out using sequencing primer pairs flanking the designed target

sites (Fig. S1 and Table S1). The PCR products were sequenced directly. Mutation types were

determined by analyzing the sequencing results using online Degenerate Sequence Decoding

(DSD) method (http://dsdecode.scgene.com) that will help decode automatically sequencing

chromatograms with biallelic, heterozygous, and homozygous mutations into allelic

sequences (Ma et al., 2015a). At T1 generation, based on the mutation type identified in the T0

generation, high-resolution melting analysis (HRM) were employed to analyze the statuses of

the edited heading date genes, and single plants in which the edited heading date genes are

homozygous were chosen for further analysis. Homozygous T2 lines were grown in a row to

investigate the heading date and other agronomic traits.

References

Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., 1994. Efficient transformation of rice (Oryza

sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

Plant J. 6, 271-282.

Li, X., Liu, H., Wang, M., Tian, X., Zhou, W., Lu, T., Wang, Z., Chu, C., Fang, J., Bu, Q.,

2015. Combinations of Hd2 and Hd4 genes determine rice adaptability to Heilongjiang

Province, northern limit of China. J. Integr. Plant Biol. 57, 698-707.

Ma, X., Chen, L., Zhu, Q., Chen, Y., Liu, Y.G., 2015a. Rapid decoding of sequence-specific

nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products.

Mol. Plant 8, 1285-1287.

Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y.,

Xie, Y., Shen, R., Chen, S., Wang, Z., Guo, J., Chen, L., Zhao, X., Dong, Z., Liu, Y.G., 2015b.

A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in

monocot and dicot plants. Mol. Plant 8, 1274-1284.

Tian, X., Wang, Z., Li, X., Lv, T., Liu, H., Wang, L., Niu, H., Bu, Q., 2015. Characterization

and functional analysis of pyrabactin resistance-like abscisic acid receptor family in rice. Rice

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Yan, M., Zhou, S.R., Xue, H.W., 2015. CRISPR Primer Designer: Design primers for

knockout and chromosome imaging CRISPR-Cas system. J. Integr. Plant Biol. 57, 613-617.

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Fig S1. Target sequences and partial genomic DNA sequences of Hd2 (A), Hd4 (B) and Hd5 (C). The PAM (NGG) sites are highlighted in red. The target sequences are highlighted in yellow. The sequencing primers for identifying gene edition are highlighted in green. Bold fonts indicate translation initiation site (ATG) of the corresponding genes.

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Fig. S2. CRISPR/Cas9-mediated mutation type in T0 generation

i, d, and s indicate insertion, deletion, and substitution, respectively. The number following

the i, d, and s is the base number of the insertion, deletion, and substitution respectively. None

indicate that there was no mutation. * indicate that those lines were chosen for further

examination in T2 generation

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Fig.S3. DNA sequences and mutation types of the three target genes in the T2 seedlings. Target sequences are highlighted in blue. The PAM site is underlined.

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Heinuomi Heinuomi-d1 Bijing45 Bijing45-d1Banpohe Banpohe-d1

Songjing19 Songjing19-d1

Dongnong429

Longdao16 Longdao16-d2

Dongnong429-d1

Nanjing9108 NJ9108-d1

Longdao18 Longdao18-d1Longqingdao2 LQD2-d1

Fig. S4. Representative pictures of T2 lines and their parents.

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Table S1. Primers used in this study.

Primer Sequence (5′‒3′)U3-Hd2-1-LP ggcATTGATAGCGATGACTCCACCU3-Hd2-1-RP aaacGGTGGAGTCATCGCTATCAAU6c-Hd2-2-LP tcaGCATACATGCAGTGACGAAGCU6c-Hd2-2-RP aaacGCTTCGTCACTGCATGTATGU6a-Hd4-1-LP gccGGAGAAGGATGTGGCCTGTGU6a-Hd4-1-RP aaacCACAGGCCACATCCTTCTCU3-Hd4-2-LP ggcAACTGGAACTCGTGCACCGGU3-Hd4-2-RP aaacCCGGTGCACGAGTTCCAGTU6b-Hd5-LP gttGTCGCCGGACTCGTTGTCCAAU6b-Hd5-RP aaacTTGGACAACGAGTCCGGCGASequencing-Hd2-F AACCGCTCATCACAACSequencing-Hd2-R GGTAGAAGGAGGAAAGASequencing-Hd4-F AAGTGACCTCACCTGCTASequencing-Hd4-R CGGTGTTCCTCCTGAASequencing-Hd5-F TCCTCACCTCCTTTCCTSequencing-Hd5-R GATGGTCTTCCGCTTCT

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