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

doi:10.7668/hbnxb.20196288

Abstract

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

CLC Number:

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.

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Figures/Tables 7

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

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

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

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.

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

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.

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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