应用CRISPR/Cas9技术与分子标记辅助选择创制广东丝苗米新种质

doi:10.16819/j.1001-7216.2023.220705

摘要/Abstract

摘要： 目的创制优质香型丝苗米水稻新种质，探索水稻育种改良新途径，助力广东丝苗米品牌建设。方法以广东省农业主导品种粤农丝苗（YNSM）与优质稻粤王丝苗（YWSM）为材料，利用CRISPR/Cas9基因编辑技术定点编辑上述品种的香味基因Badh2，创制香稻新品系，随后通过分子标记辅助选择（MAS）技术定向导入GW7/GL7位点，系谱选育优质香型丝苗米水稻新种质。结果利用CRISPR/Cas9基因编辑技术成功创制两个香稻新品系yn-ko^badh2与yw-ko^badh2，其2-AP含量均极显著提高，达到239.39~440.79 μg/kg，而有效穗数、每穗实粒数、糙米外观品质、千粒重与产量等主要农艺性状均未受到显著影响；MAS技术结合系谱选育的方法成功选育两个丰产性好、籽粒长宽比超过4.3的优质香型丝苗米水稻新品系NW^badh2GW7-1与NW^badh2GW7-2，达到广东丝苗米品种关于香味与外观粒型的认定标准。结论利用CRISPR/Cas9基因编辑与分子辅助选择技术相结合可精准、高效地创制新的优异水稻种质，为广东丝苗米品种选育提供重要的种质储备与遗传改良资源。

关键词: 广东丝苗米, 香味, 粒形, CRISPR/Cas9, 分子标记辅助选择

Abstract:

【Objective】 To generate new germplasms of high-quality and fragrant Simiao rice, explore new ways of rice breeding and improvement, and build the Guangdong Simiao Rice brand. 【Method】 Using the leading variety Yuenongsimiao (YNSM) and high-quality rice variety Yuewangsimiao (YWSM) as materials, the fragrance gene Badh2 of these varieties was knocked out by CRISPR/Cas9 gene editing technology to generate new fragrant rice lines. Subsequently, the excellent GW7/GL7 was introduced into these new fragrant rice lines by marker-assisted selection (MAS). 【Result】 Two new fragrant rice lines, yn-ko^badh2 and yw-ko^badh2, were successfully generated by CRISPR/Cas9 gene editing technology. 2-AP contents of these lines were significantly increased, ranging from 239.39 to 440.79 μg/kg, other main agronomic traits, including effective panicle number, grain number per panicle, appearance quality of brown rice, 1000-grain weight, and grain yield, were not significantly affected. Two novel fragrant Simiao rice lines, NW^badh2GW7-1 and NW^badh2GW7-2, with high grain yield and slender grain size of length to width ratio more than 4.3, were selected by MAS combined with pedigree breeding, which met the identification standards of Guangdong Simiao rice varieties on aroma and appearance grain size. 【Conclusion】 Combination of CRISPR/Cas9 gene editing and MAS technology contribute to accurate and efficient breeding of new excellent rice germplasms. The new germplasms developed can provide important germplasms and genetic resources for breeding of new high-quality fragrant Simiao Rice varieties in Guangdong Province.

Key words: Guangdong Simiao rice, aroma, grain shape, CRISPR/Cas9, marker-assisted selection

王石光, 陆展华, 刘维, 卢东柏, 王晓飞, 方志强, 巫浩翔, 何秀英. 应用CRISPR/Cas9技术与分子标记辅助选择创制广东丝苗米新种质[J]. 中国水稻科学, 2023, 37(1): 29-36.

WANG Shiguang, LU Zhanhua, LIU Wei, LU Dongbai, WANG Xiaofei, FANG Zhiqiang, WU Haoxiang, HE Xiuying. Generating Guangdong Simiao Rice Germplasms by Applying CRISPR/Cas9 Gene Editing and Marker-assisted Selection Technology[J]. Chinese Journal OF Rice Science, 2023, 37(1): 29-36.

图/表 5

图1 亲本糙米粒型比较分析 YNSM―粤农丝苗；YWSM―粤王丝苗；XYXZ―象牙香占。数据均为平均值±标准差，n=10；图中大、小写字母分别表示差异达0.01、0.05显著水平（t检验）。

Fig. 1. Comparative analysis of grain size of parental brown rice. YNSM, Yuenongsimiao; YWSM, Yuewangsimiao; XYXZ, Xiangyaxiangzhan. Data are shown as means±SD, n=10; The uppercase and lowercase letters above the bars indicate statistical significance at the P=0.01 and P=0.05 level, respectively (t-test).

图2 Badh2基因敲除植株突变位点分析 A?Badh2基因结构及敲除靶点所在位置；B?Badh2基因靶点测序分析；C?粤农丝苗背景Badh2基因两种突变方式的测序峰；D?转基因成分检测，上下图片分别为Cas9与gRNA特异引物检测结果；YNSM―粤农丝苗；YWSM―粤王丝苗；M―DL2000 marker。

Fig. 2. Mutation types of Badh2 in gene knockout plants. A, Badh2 gene structure and location of the gene editing target. B, Sequence analysis of Badh2 gene target; C, Target region sequencing of Badh2 gene in the editing lines of Yuenongsimiao background. D, Detection of transgenic vector framework by specific Cas9 and gRNA primers. YNSM, Yuenongsimiao; YWSM, Yuewangsimiao; M, DL2000 DNA marker.

表1 野生型与创制株系的农艺性状

Table 1. Agronomic traits of the wild type and generated lines.

株系 Line	株高 Plant height / cm	有效穗数Effective panicle number	穗长 Panicle length / cm		每穗实粒数 Grain number per panicle		谷粒长度 Grain length / mm		谷粒长宽比 Grain length to width ratio
粤农丝苗YNSM	94.2±3.6 CDde	7.00±1.00 Ab	22.81±0.72 Bc		120.7±13.0 ABab		9.34±0.10 De		3.81±0.04 Cd
yn-ko^badh2-1	90.2±1.5 Df	6.67±0.58 Ab	24.65±0.36 ABab		120.8±24.0 ABab		9.79±0.11 BCcd		3.90±0.10 Cd
yn-ko^badh2-2	92.6±2.4 CDef	7.33±1.53 Aab	23.39±1.44 ABbc		111.1±24.9 ABabc		9.56±0.06 CDde		3.78±0.08 Cd
粤王丝苗YWSM	105.0±2.5 Aa	8.00±1.00 Aab	24.60±0.57 ABab		99.5±8.9 Bbc		10.12±0.03 ABab		4.35±0.04 ABbc
yw-ko^badh2-1	100.4±1.5 ABbc	8.33±0.58 Aab	25.62±0.34 Aa		91.1±8.8 Bc		10.28±0.19 Aab		4.50±0.04 ABab
yw-ko^badh2-2	103.4±3.2 Aab	8.33±0.58 Aab	24.73±0.63 ABab		94.1±10.0 Bc		10.38±0.11 Aa		4.55±0.09 Aa
NW^badh2GW7-1	103.0±4.5 Aab	9.00±1.73 Aa	24.74±1.53 ABab		100.6±13.9 ABbc		10.01±0.05 ABc		4.31±0.04 Bc
NW^badh2GW7-2	97.2±2.9 BCcd	8.33±0.58 Aab	25.46±1.11 Aa		135.7±5.0 Aa		10.35±0.37 Aa		4.36±0.21 ABbc
株系 Line	糙米长度 Grain length of brown rice / mm	糙米长宽比Grain length to width ratio of brown rice		千粒重 1000-grain weight / g		产量 Grain yield / (kg·hm^-2)		2-AP含量 2-AP content / (μg·kg^-1)
粤农丝苗YNSM	6.92±0.07 Bb	3.30±0.01 Bc		21.08±0.38 ABab		6256.9±247.5 ABab		0.00±0.00 De
yn-ko^badh2-1	7.01±0.13 Bb	3.32±0.13 Bc		21.43±0.45 Aa		6203.6±1356.8 ABb		440.79±23.69 Aa
yn-ko^badh2-2	6.88±0.20 Bb	3.37±0.11 Bc		20.82±0.67 ABCabc		5885.4±782.6 ABb		306.07±30.18 Bc
粤王丝苗YWSM	8.40±0.18 Aa	4.68±0.09 Aa		20.30±0.39 ABCDbcd		5723.5±149.6 ABb		0.00±0.00 De
yw-ko^badh2-1	8.28±0.12 Aa	4.62±0.10 Aa		19.94±0.83 BCDcde		5402.4±694.2 Bb		239.39±5.51 Cd
yw-ko^badh2-2	8.25±0.12 Aa	4.45±0.14 Aab		20.07±0.56 BCDcd		5612.9±740.1 Bb		276.67±13.68 BCc
NW^badh2GW7-1	8.18±0.30 Aa	4.36±0.31 Ab		19.70±0.46 CDde		6342.1±1480.3 ABab		439.99±21.78 Aa
NW^badh2GW7-2	8.36±0.21 Aa	4.58±0.06 Aab		19.16±0.15 De		7708.4±298.4 Aa		402.95±13.97 Ab

表1 野生型与创制株系的农艺性状

Table 1. Agronomic traits of the wild type and generated lines.

株系 Line	株高 Plant height / cm	有效穗数Effective panicle number	穗长 Panicle length / cm		每穗实粒数 Grain number per panicle		谷粒长度 Grain length / mm		谷粒长宽比 Grain length to width ratio
粤农丝苗YNSM	94.2±3.6 CDde	7.00±1.00 Ab	22.81±0.72 Bc		120.7±13.0 ABab		9.34±0.10 De		3.81±0.04 Cd
yn-ko^badh2-1	90.2±1.5 Df	6.67±0.58 Ab	24.65±0.36 ABab		120.8±24.0 ABab		9.79±0.11 BCcd		3.90±0.10 Cd
yn-ko^badh2-2	92.6±2.4 CDef	7.33±1.53 Aab	23.39±1.44 ABbc		111.1±24.9 ABabc		9.56±0.06 CDde		3.78±0.08 Cd
粤王丝苗YWSM	105.0±2.5 Aa	8.00±1.00 Aab	24.60±0.57 ABab		99.5±8.9 Bbc		10.12±0.03 ABab		4.35±0.04 ABbc
yw-ko^badh2-1	100.4±1.5 ABbc	8.33±0.58 Aab	25.62±0.34 Aa		91.1±8.8 Bc		10.28±0.19 Aab		4.50±0.04 ABab
yw-ko^badh2-2	103.4±3.2 Aab	8.33±0.58 Aab	24.73±0.63 ABab		94.1±10.0 Bc		10.38±0.11 Aa		4.55±0.09 Aa
NW^badh2GW7-1	103.0±4.5 Aab	9.00±1.73 Aa	24.74±1.53 ABab		100.6±13.9 ABbc		10.01±0.05 ABc		4.31±0.04 Bc
NW^badh2GW7-2	97.2±2.9 BCcd	8.33±0.58 Aab	25.46±1.11 Aa		135.7±5.0 Aa		10.35±0.37 Aa		4.36±0.21 ABbc
株系 Line	糙米长度 Grain length of brown rice / mm	糙米长宽比Grain length to width ratio of brown rice		千粒重 1000-grain weight / g		产量 Grain yield / (kg·hm^-2)		2-AP含量 2-AP content / (μg·kg^-1)
粤农丝苗YNSM	6.92±0.07 Bb	3.30±0.01 Bc		21.08±0.38 ABab		6256.9±247.5 ABab		0.00±0.00 De
yn-ko^badh2-1	7.01±0.13 Bb	3.32±0.13 Bc		21.43±0.45 Aa		6203.6±1356.8 ABb		440.79±23.69 Aa
yn-ko^badh2-2	6.88±0.20 Bb	3.37±0.11 Bc		20.82±0.67 ABCabc		5885.4±782.6 ABb		306.07±30.18 Bc
粤王丝苗YWSM	8.40±0.18 Aa	4.68±0.09 Aa		20.30±0.39 ABCDbcd		5723.5±149.6 ABb		0.00±0.00 De
yw-ko^badh2-1	8.28±0.12 Aa	4.62±0.10 Aa		19.94±0.83 BCDcde		5402.4±694.2 Bb		239.39±5.51 Cd
yw-ko^badh2-2	8.25±0.12 Aa	4.45±0.14 Aab		20.07±0.56 BCDcd		5612.9±740.1 Bb		276.67±13.68 BCc
NW^badh2GW7-1	8.18±0.30 Aa	4.36±0.31 Ab		19.70±0.46 CDde		6342.1±1480.3 ABab		439.99±21.78 Aa
NW^badh2GW7-2	8.36±0.21 Aa	4.58±0.06 Aab		19.16±0.15 De		7708.4±298.4 Aa		402.95±13.97 Ab

图3 Badh2基因编辑香稻新种质的农艺性状比较分析 A?粤农丝苗与粤王丝苗背景编辑新品系的植株形态比较，标尺为20 cm；B?粤农丝苗与粤王丝苗背景编辑新品系的糙米粒型比较，标尺为1 cm；C~G分别为有效穗数、每穗实粒数、千粒重、产量与2-AP含量的统计分析。数据为平均数±标准差，n=3；图中大小写字母分别表示差异达0.01、0.05显著水平（t检验）；N.D表示未检测到。YNSM―粤农丝苗；YWSM―粤王丝苗。

Fig. 3. Agronomic traits of new fragrant rice germplasms through Badh2 gene editing. A, Plant morphology of new edited lines of Yuenongsimiao and Yuewangsimiao background, respectively. Scale bar: 20 cm. B, Brown rice grain size of new edited lines of Yuenongsimiao and Yuewangsimiao background, respectively. Scale bar, 1 cm. C-G, Statistical analysis of effective panicle number, grain number per panicle, 1000-grain weight, grain yield and 2-AP content, respectively. Data are shown as means ± SD, n=3. The uppercase and lowercase letters above the bars indicate significance at 0.01 and 0.05 level, respectively (t-test). N.D, Not detected. YNSM, Yuenongsimiao; YWSM, Yuewangsimiao.

图4 丝苗型香稻新株系的农艺性状比较分析 A?丝苗型香稻新株系的植株形态比较，标尺为20 cm；B?丝苗型香稻新株系的糙米粒型比较，标尺为1 cm；C?GW7位点电泳检测，从上到下依次为GL7_Dup、GL7_InDel1与GL7_InDel2引物检测结果。D~G分别为糙米粒长、粒宽、长宽比与产量的统计分析。YNSM?粤农丝苗；YWSM?粤王丝苗；XYXZ?象牙香占；R498?蜀恢498。数据为平均数±标准差，n=3；图中的大小写字母分别表示差异达0.01、0.05显著水平（t检验）。

Fig. 4. Agronomic traits of new fragrant Simiao rice lines. A, Plant morphology of new fragrant Simiao Rice lines. Scale bar, 20 cm. B, Brown rice grain size of new fragrant Simiao Rice lines. Scale bar, 1 cm. C, Genotype identification of the GW7 gene using the functional markers GL7_Dup, GL7_InDel1与GL7_InDel2, respectively. D-G, Statistical analysis of brown rice grain length, width, and length to width ratio, and grain yield, respectively. YNSM, Yuenongsimiao; YWSM, Yuewangsimiao; XYXZ, Xiangyaxiangzhan; R498, Shuhui 498. Data are shown as means ± SD, n=3. The uppercase and lowercase letters above the bars indicate statistical significance at the P=0.01 and P=0.05 level, respectively (t-test).

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