寒地粳稻种质资源香味基因<i>Badh2</i>变异分析及其KASP标记开发与应用

doi:10.7668/hbnxb.20196288

摘要/Abstract

摘要：

香稻种质资源的筛选和香味基因的快速鉴定与利用对香稻遗传育种具有重要意义。为了明确黑龙江省香稻材料的香味类型,提高香稻品种的育种效率,通过对黑龙江省审定推广的180个香稻品种的Badh2基因进行了测序,测序结果共分为2种突变类型,第一种类型为第7外显子8 bp缺失和3处碱基替换(Badh2-E7型),包含177个品种;第二种类型为第2外显子7 bp缺失(Badh2-E2型),仅包含3个品种。针对这2个突变类型设计了2个特异性KASP分子标记,通过180份香稻品种鉴定,分型结果与测序结果一致,同时分别利用五优稻4号(Badh2-E7型香稻)/唯农101(普通粳稻)、松科粳134(Badh2-E2型香稻)/东富114(普通粳稻)杂交构建的2个F₂群体进行了香味基因筛选鉴定,鉴定结果吻合率为100%,验证了KASP分子标记的准确性。进一步利用KASP标记对实验室自行创制、收集的620份粳稻亲本进行了鉴定,共筛选出248份Badh2-E7型香稻材料,同时对现有的1 220份以E7型香稻为亲本的F₇材料进行了分型检测,其中293份材料为香型、906份材料为非香型、21份材料为杂合型;随机抽取香和非香各5份材料进行测序验证,结果验证该KASP分子标记分型正确。为利用分子标记辅助选择方法培育寒地香稻新品种提供了新标记和新材料。

关键词: 水稻, 香味基因, Badh2, KASP, 分子标记辅助选择

Abstract:

The screening of aromatic rice germplasm resources and the rapid identification and utilization of aromatic genes have practical significance for the genetic breeding of aromatic rice.In order to clarify the aroma types of aromatic rice materials in Heilongjiang Province and improve the breeding efficiency of aromatic rice varieties,180 aromatic rice varieties approved and promoted in Heilongjiang Province were sequenced.Compared with the reference genome Nipponbare,the sequencing results were divided into two types of mutations.The first type was the 8 bp deletion of exon 7 and three base substitutions(Badh2-E7 type),including 177 varieties.The second type was the 7 bp deletion of exon 2(Badh2-E2 type),which contained only three varieties.According to these two mutation types,two specific KASP molecular markers were designed to accurately detect the aroma types of E7 and E2 in rice.Through the identification of 180 aromatic rice varieties,the typing results were consistent with the sequencing results.Two hybrid populations were constructed by using E7 aromatic rice variety Wuyoudao 4 and E2 aromatic rice variety Songkejing 134 with non-aromatic japonica rice varieties Weinong 101 and Dongfu 114,respectively.The F₂ generation of the hybrid population was screened and identified for aroma genes,and the identification results were consistent with 100%,which verified the accuracy of KASP molecular markers.The 620 japonica rice parents created and collected by our laboratory were further identified by KASP markers,and 248 aromatic rice materials were screened.It was found that these parents were all Badh2-E7 type genes.At the same time,the existing 1 220 F₇ generation materials with E7 aromatic rice as parents were genotyped,including 293 aromatic rice materials,906 non-aromatic rice materials and 21 heterozygous rice materials.Five aromatic and non-aromatic rice materials were randomly selected for sequencing verification.The sequencing results verified that the KASP molecular marker typing was correct.This study provided new markers and materials for breeding new fragrant rice varieties in cold regions by using molecular marker assisted selection.

Key words: Rice, Fragrance gene, Badh2, KASP, Molecular marker-assisted selection

中图分类号:

刘长林, 辛威, 吴梦, 王敬国, 刘化龙, 杨洛淼, 徐善斌, 邹德堂, 郑洪亮. 寒地粳稻种质资源香味基因Badh2变异分析及其KASP标记开发与应用[J]. 华北农学报, 2026, 41(1): 14-21. doi: 10.7668/hbnxb.20196288.

LIU Changlin, XIN Wei, WU Meng, WANG Jingguo, LIU Hualong, YANG Luomiao, XU Shanbin, ZOU Detang, ZHENG Hongliang. Analysis of the Fragrance Gene Badh2 Variation in Cold-resistant Japonica Rice Germplasm Resources and the Development and Application of KASP Markers[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 14-21. doi: 10.7668/hbnxb.20196288.

http://www.hbnxb.net/CN/Y2026/V41/I1/14

图/表 7

表1 Badh2测序引物

Tab.1 Badh2 sequencing primers

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
Badh2-1F	TCTCCCACCACCACTCCACA
Badh2-1R	AGAGGATACGCACTTCCGAT
Badh2-2F	GAACTGTCAAACCATCAATT
Badh2-3R	TAAGAAAGTATTGCTAATCC
Badh2-4F	GTCAGTAACTTGATATTGGC
Badh2-4R	TGCTCAAAGTAGGAAGACCC
Badh2-5F	TCCGTAGGTTTATCTTGTCA
Badh2-5R	TGATGTGGAAGTGAAGAGCA
Badh2-6F	TCTATGCATCTCCAGTTCTT
Badh2-6R	TTTATTTCCAGGCTCAGCAC

表2 Badh2基因的KASP引物

Tab.2 KASP primers for the Badh2 gene

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	备注 Remarks
Badh2-1F1	GAAGGTGACCAAGTTCATGCTGCCGCGACTGGGCGCGCGCGCCGG	Badh2-E2野生型
Badh2-1F2	GAAGGTCGGAGTCAACGGATTGCCGCGACTGGGCGCGCGCGCCGT	Badh2-E2突变型
Badh2-1R	TACCTTGGCCGCGATTGCGCGGAGGTAC	通用引物
Badh2-2F1	GAAGGTGACCAAGTTCATGCTGGGAGTTATGAAACTGGTAAAAAG	Badh2-E7野生型
Badh2-2F2	GAAGGTCGGAGTCAACGGATTTGGGAGTTATGAAACTGGTATATAT	Badh2-E7突变型
Badh2-2R	GAAACAAACCTTAACCATAGGAGCAGCTG	通用引物

图1 180份香稻品种的Badh2测序的部分结果

Fig.1 Partial sequencing results of the Badh2 gene in 180 fragrant rice varieties

图2 180份香稻品种的等位基因分型结果 A—B.Badh2-E7型基因的分型结果;C—D.Badh2-E2型基因的分型结果。图中横坐标表示FAM信号强度;纵坐标表示HEX信号强度;蓝色井号表示阴性对照,黄色表示香稻材料,绿色表示非香材料,浅蓝色的表示杂合。图3-4同。

Fig.2 The allelic genotyping results of 180 fragrant rice varieties A—B.The genotyping results of the Badh2-E7 type gene;C—D.The genotyping results of the Badh2-E2 type gene.In the figure,the abscissa represents the FAM signal intensity;the ordinate represents the HEX signal intensity;the blue hash mark represents the negative control,yellow represents the aromatic rice material,green represents the non-aromatic material,and light blue represents the heterozygous.The same as Fig.3-4.

图3 620份亲本材料的Badh2等位基因分型结果筛选的亲本材料均为Badh2-E7型。

Fig.3 The allelic genotyping results of Badh2 gene in 620 parental materials The selected parental materials are all of the Badh2-E7 type.

图4 F₇材料Badh2等位基因分型结果 F₇材料是本实验室自行收集、创制的香稻材料为亲本而构建,亲本均为Badh2-E7型材料。

Fig.4 Allelic genotyping results of Badh2 in the F₇ generation materials The F₇ generation materials are constructed using self-collected and self-developed fragrant rice materials as the parental lines in our laboratory,the parents are the Badh2-E7 type material.

图5 10份材料Badh2-E7型基因目标片段测序结果图中每一行是1份材料的测序结果。

Fig.5 The sequencing results of the target fragment of the Badh2-E7 type gene in 10 samples Each row in the figure represents the sequencing result of a material.

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寒地粳稻种质资源香味基因Badh2变异分析及其KASP标记开发与应用

Analysis of the Fragrance Gene Badh2 Variation in Cold-resistant Japonica Rice Germplasm Resources and the Development and Application of KASP Markers

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寒地粳稻种质资源香味基因Badh2变异分析及其KASP标记开发与应用

Analysis of the Fragrance Gene Badh2 Variation in Cold-resistant Japonica Rice Germplasm Resources and the Development and Application of KASP Markers

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