FRE7 molecular marker of rice fragrance gene OsBadh2 and application

Abstract

An FRE7 molecular marker of a rice fragrance gene OsBadh2 and application thereof belong to the technical field of crop molecular breeding, the FRE7 molecular marker is designed in a 7th exon mutation region of the OsBadh2 gene, and primer sequences of the FRE7 molecular marker are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses an application of the FRE7 molecular marker. The FRE7 molecular marker of the rice fragrance gene OsBadh2 provided by the invention can be applied to fragrant rice breeding, the genotype of an E7 type fragrant rice variety can be accurately identified by utilizing the FRE7 molecular marker, and different genotypes of an E7 type fragrant rice / non-fragrant rice hybrid F2 population can also be accurately identified, so that fragrant rice offspring materials are efficiently screened through the coincidence of the genotype and phenotype, and the breeding efficiency of the fragrant rice is improved. The fragrant rice identification and breeding efficiency can be improved, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of crop molecular breeding, and in particular relates to a FRE7 molecular marker of a rice aroma gene OsBadh2 and an application thereof. Background Art

[0002] Using molecular marker-assisted breeding methods to cultivate aromatic japonica rice varieties can shorten the breeding period and reduce the workload, and is an effective means to improve breeding efficiency.

[0003] 2-Acetyl-1-pyrroline (2-AP) is the main aromatic compound that produces fragrance in rice. The functional loss of the gene encoding betaine aldehyde dehydrogenase 2 (OsBadh2) on rice chromosome 8 promotes the accumulation of 2-AP. The OsBadh2 gene is a recessive nuclear gene. The homozygous mutant genotype emits fragrance, while the heterozygous genotype does not show fragrance. There are many types of mutations in this gene, including 8bp deletion and 3 base mutations in exon 7 (E7 type), 7bp deletion in exon 2 (E2 type), 8bp insertion in the promoter region, 3bp deletion in the 5'UTR region, etc. Among them, E7 type and E2 type are the main mutation types of aromatic rice.

[0004] High-quality aromatic japonica rice varieties such as Wuyoudao No. 4, Jijing 816 and Jijing 830 all belong to the E7 type with 8bp deletion and 3 base mutations in exon 7. The identification method of aromatic traits in the process of aromatic rice cultivation has problems such as strong subjectivity and heavy workload. The development and application of aromatic gene molecular markers can improve the efficiency of hybrid offspring selection, and the operation is simple and has strong practicality.

[0005] Wang Jun et al. (2008) developed the E7-type molecular marker InDel-E7 (fragrant type 196bp / non-fragrant type 204bp) of the OsBadh2 gene, and used this marker to detect the genotypes of 24 test materials, accurately detecting the homozygous, heterozygous and non-fragrant genotypes of the fragrance type. Yan Ying et al. (2015) used the amplification refractory mutation system (ARMS) technology to perform allele-specific amplification based on the sequence characteristics of the E7 type with 8bp deletion and 3bp mutation, and developed new functional markers Y5-Y8. Li Xiaoqiong et al. (2023) developed the E7-type molecular marker FG-Badh2 (fragrant type 272bp / non-fragrant type 280bp), and used this molecular marker combined with conventional breeding methods to cultivate the high-quality indica fragrant rice variety "Naguxiang".

[0006] Since the genome sequence of E7 aromatic rice and non-aromatic rice differs by only 8bp, when using the developed molecular markers for genotype detection, there is not only difficulty in judging the size difference of PCR amplification fragments, but also the generation of non-specific amplification fragments, resulting in low accuracy, time-consuming, labor-intensive and impractical genotype identification of offspring. Summary of the invention

[0007] The present invention discloses a molecular marker for a rice aroma gene OsBadh2. The FRE7 molecular marker is designed in the mutation region of the 7th exon of the OsBadh2 gene. Three PCR bands of different sizes are amplified by PCR technology, which can distinguish the wild type, heterozygous type and homozygous mutant type of the mutation type (E7 type). The primer sequences of the FRE7 molecular marker are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

[0008] The invention discloses an application of a FRE7 molecular marker of a rice aroma gene OsBadh2 in aromatic rice breeding, specifically, an application of the FRE7 molecular marker in the identification of the genotype of E7 aromatic rice and the genotype identification of the offspring of the hybrid F2 population of E7 aromatic rice / non-aromatic rice. The FRE7 molecular marker can be used to accurately identify the genotype of the E7 aromatic rice variety, and can also accurately identify the different genotypes in the offspring of the F2 population, and then the aromatic rice offspring materials can be efficiently screened through the consistency of the genotype and the phenotype, which can improve the efficiency of aromatic rice identification and breeding, and has broad application prospects.

[0009] The FRE7 molecular marker of the rice aroma gene OsBadh2 provided by the present invention can not only accurately distinguish between aroma and non-aroma genotypes, but also facilitate PCR amplification, and the amplified band size is accurate, clear and clean, and no non-specific bands are generated. It can be applied to molecular marker-assisted selection of aromatic rice breeding and E7 genotype identification and screening of aromatic rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a schematic diagram of the location of the FRE7 molecular marker in the genome;

[0011] Among them: light gray thick arrows represent the 4 primers of FRE7 molecular marker; thin dotted double arrows represent PCR forward and reverse primer pairs and amplified band sizes; dotted lines represent 8 deletion sequences of E7 variant types, and 3 gray shading characters represent variant sequences;

[0012] Figure 2 This is the electrophoresis diagram for genotyping of E7 aromatic rice varieties using FRE7 molecular marker;

[0013] Where: M represents 100bp DNA standard molecular weight; NC represents blank control; 1-2 represents E2 type aromatic rice varieties; 3-7 represents E7 type aromatic rice varieties;

[0014] Figure 3 Electropherogram for genotyping of F2 segregating population using FRE7 molecular marker;

[0015] Wherein: M represents 100bp DNA standard molecular weight; NC represents blank control; FC represents fragrance control variety; NFC represents non-fragrance control variety; WT represents wild-type genotype (non-fragrance); HZ represents heterozygous genotype (non-fragrance); HM represents homozygous genotype (fragrance). DETAILED DESCRIPTION

[0016] The present invention is described below with reference to the accompanying drawings.

[0017] 1 Materials and methods

[0018] 1.1 Experimental Materials

[0019] The test rice materials of the present invention are: E2 type fragrant rice varieties (including: Jijing 813 and Danxiangjing 1); E7 type fragrant rice varieties (including: Daohuaxiang, Jijing 816, Jijing 830, Jijing 530, Jijing 561, Yujingxiang 1, Longxiangjing 1 and Songjingxiang 2); 236 individual plants of the E7 type fragrant rice / non-fragrant rice hybrid F2 generation (Jijing 830 / Songjing 20 combination) segregation population; the non-fragrant control variety is Jijing 88.

[0020] 1.2 Design of FRE7 molecular marker for OsBadh2 gene

[0021] The OsBadh2 (Os08g0424500) gene sequence was obtained from the National Rice Data Center (https: / / www.ricedata.cn / ) and the online software NCBI-Blast ( https: / / blast.ncbi.nlm.nih.gov / Blast.cgi ), the sequences of exon 7 of the OsBadh2 gene in aromatic rice and non-aromatic rice were compared, and a molecular marker was designed in the mutation region using a combination of Primer 5 software and manual methods, and named FRE7 (e.g. Figure 1 The FRE7 molecular marker consists of primers FRE7AF, FRE7BF, FRE7CR and FRE7DR.

[0022] Using the FRE7 molecular marker to perform PCR amplification on different rice materials, three types of PCR bands of different sizes can be amplified. FRE7AF and FRE7DR can amplify a PCR band of 452bp, FRE7BF and FRE7DR can amplify a band of 298bp, and FRE7AF and FRE7CR can amplify a band of 195bp. When the amplified products are 452bp and 195bp (a total of 2 bands), the rice material is a homozygous mutant (HM); when the amplified products are 452bp, 298bp and 195bp (a total of 3 bands), the rice material is a heterozygous mutant (HZ); when the amplified products are 452bp and 298bp (a total of 2 bands), the rice material is a wild type (WT).

[0023] 1.3 PCR amplification of FRE7 molecular marker

[0024] Leaves were taken at the seedling stage of rice, DNA was extracted by CTAB method, and PCR amplification was performed using the four primers of FRE7A-D (as shown in Table 1). The PCR reaction system was 25 μL, Kangwei Century 2×Es Taq MasterMix (containing Es Taq DNA Polymerase, PCR buffer, 3mmol / L MgCl2, 400μmol / L dNTP mix), 0.4μL of 10μmol / L primers (4 primers were added separately), and 100-200ng genomic DNA. The reaction conditions were: 94℃ PCR pre-denaturation for 2min, 94℃ denaturation for 30s, 50℃ annealing for 30s, 35 cycles, and 72℃ extension for 30s. The amplified product was detected by 2% agarose gel electrophoresis to detect the type and size of the PCR band.

[0025] 1.4 Identification of E7 aromatic rice varieties using FRE7 molecular marker

[0026] Two types of aromatic rice varieties with mutations, E2 and E7, and a non-aromatic rice variety (CK) were selected, and the E7 mutation genotype was identified using the FRE7 molecular marker. The E2 aromatic rice varieties tested were Jijing 813 and Danxiangjing 1, and the E7 aromatic rice varieties were Daohuaxiang, Jijing 816, Jijing 830, Jijing 530, Jijing 561, Yujingxiang 1, Longxiangjing 1, and Songjingxiang 2. Jijing 88 was used as a non-aromatic rice variety control. Genomic DNA was extracted from rice seedling leaves, and PCR amplification, agarose gel electrophoresis, PCR amplification band reading, and band type analysis of the FRE7 molecular marker were performed.

[0027] 1.5 Identification of the genotype of the F2 hybrid of E7 mutant aromatic rice and non-aromatic rice using the FRE7 molecular marker

[0028] A total of 236 plants from the F2 generation of aromatic rice / non-aromatic rice hybrids (Jijing 830 / Songjing 20 combination) were selected, with Jijing 88 as the non-aromatic rice control variety and Daohuaxiang as the aromatic rice control variety. The E7 mutant genotype was identified using the FRE7 molecular marker. Genomic DNA from rice seedling leaves was extracted, and PCR amplification of the FRE7 molecular marker was performed, followed by agarose gel electrophoresis, PCR amplification band reading, and band pattern analysis.

[0029] According to the results of banding analysis, the WT:HZ:HM genotype segregation ratio was calculated, and the chi-square test was used to determine whether it conformed to Mendel's law of segregation.

[0030] 1.6 PCR product sequencing and genotype verification

[0031] From the PCR amplification results of 236 individual plants of the F2 generation of aromatic rice / non-aromatic rice hybrid, 15 HZ and HM genotypes were randomly selected, and the 452bp band was subjected to Sanger sequencing using the FRE7AF primer (Cooma Biotech, Changchun). The sequencing results were compared and analyzed using Snapgene to verify the accuracy of the PCR results.

[0032] 1.7 F2 generation seed aroma identification

[0033] After the F2 generation segregation population matured, according to the results of FRE7 molecular marker genotype identification and agronomic traits, 20 homozygous genotypes and heterozygous genotypes with excellent comprehensive traits were selected and harvested. The rice harvested from two different genotypes was identified by rice chewing method, with Daohuaxiang as the fragrant rice control variety and Jijing 88 as the non-fragrant rice control variety, and 10 grains were tasted from each plant. The specific operation steps of the rice chewing method are: organize a 5-person rice aroma tasting team, and each person tastes 2 grains from each plant. During the tasting, the mature rice seed husk is peeled off and put into the mouth. The fragrance is judged by chewing and tasting carefully, and the consistency between genotype and phenotype is investigated and analyzed.

[0034] 2 Results and analysis

[0035] 2.1 Design of FRE7 molecular marker for rice aroma gene OsBadh2

[0036] The present invention aims at the 8bp deletion and 3bp mutation in the 7th exon of OsBadh2 (Os08g0424500) gene, and designs the FRE7 molecular marker of this mutation type (such as Figure 1As shown). The molecular marker consists of 4 primer sequences (FRE7A-FRE7D). During PCR amplification, the external upstream and downstream sequences (FRE7AF and FRE7DR) can combine at the corresponding positions in the aromatic rice gene sequence and the non-aromatic rice gene sequence to amplify a 452bp PCR band, while the internal upstream sequence only combines with the corresponding position of the non-aromatic rice or non-E7 mutant type aromatic rice gene sequence, and amplifies a 298bp band with the external downstream sequence (FRE7BF and FRE7DR). The internal downstream sequence only combines with the corresponding position of the E7 mutant type aromatic rice gene sequence, and amplifies a 195bp band with the external upstream sequence (FRE7AF and FRE7CR) (as shown). Figure 1 The PCR amplification conditions of FRE7 molecular marker are simple and easy. The amplified product size is divided into three types (452bp, 298bp and 195bp) according to the identified rice material genotype. The band size difference is significant and can be clearly separated and easily distinguished by 2% agarose gel electrophoresis. These three different size bands are combined into three different genotypes, namely WT, HZ and HM, which can accurately determine different genotypes.

[0037] 2.2 Identification of E7 aromatic rice varieties using FRE7 molecular marker

[0038] In order to accurately identify E7-type aromatic rice varieties using FRE7 molecular markers, two mutant types, namely E2 and E7-type aromatic rice varieties, and a non-aromatic rice variety (CK) were selected, and leaf genomic DNA was extracted to perform PCR amplification of FRE7 molecular markers (such as Figure 2 As shown). The results showed that two bands were amplified in E7-type aromatic rice varieties (Daohuaxiang, Jijing 816 and Jijing 830, etc.), which were 452bp and 195bp, respectively, that is, HM genotype. Two bands were amplified in the control variety Jijing 88, E2-type aromatic rice varieties Jijing 813 and Danxiangjing No. 1, which were 452bp and 298bp, respectively, that is, WT genotype. The above results show that the FRE7 molecular marker can accurately identify E7-type aromatic rice varieties, non-aromatic rice varieties or non-E7-type aromatic rice varieties. No relevant bands can be amplified, indicating that the marker has E7 genotype specificity.

[0039] 2.3 Identification of the genotype of the F2 hybrid progeny of the E7 mutant aromatic rice / non-aromatic rice using the FRE7 molecular marker

[0040] In order to accurately identify the genotype of the F2 generation of hybrid offspring of fragrant / non-fragrant rice using the FRE7 molecular marker, genomic DNA of individual leaves of the F2 generation population of fragrant / non-fragrant rice hybrids was extracted and PCR amplification of the FRE7 molecular marker was performed. Some of the results are shown in Figure 2. Figure 3The results showed that three genotypes were amplified in the F2 generation plants, namely WT, HZ and HM. The genotype segregation ratio of WT:HZ:HM in the 236 plants tested was 1:2:1, which was in line with Mendel's segregation law (p value = 0.7990) (as shown in Table 1). The above results show that the FRE7 molecular marker can accurately identify the different genotypes of the F2 generation of the hybrid offspring of the E7 mutant type aromatic rice / non-aromatic rice.

[0041] Table 1. Separation ratio of aroma and non-aroma genotypes in F2 population

[0042]

[0043] Comparative analysis of 2.4E7 genotype sequencing results and molecular marker identification results

[0044] To further verify the accuracy of PCR identification of FRE7 molecular marker, 15 plants of each genotype HM and HZ in the results of 2.3 were randomly selected for sequencing of PCR products of FRE7 molecular marker, and compared with the sequence of non-fragrant rice varieties. The results showed that the WT gene sequence was a single peak, and its genotype was completely consistent with the sequence of non-fragrant rice Jijing 88; the HZ gene sequence showed overlapping peaks, which were WT type and E7 mutant sequence peaks, respectively, indicating that the plant was a heterozygous genotype; the HM gene sequence was a single peak, which was completely consistent with the fragrant rice variety rice flower fragrance sequence, indicating that the plant was a homozygous mutant genotype. The results of PCR results and sequence comparison analysis showed that the PCR results of 30 F2 generation plants and two control varieties were completely consistent with the sequencing results (as shown in Table 2), indicating that the FRE7 molecular marker can accurately identify different genotypes.

[0045] Table 2. Comparison of PCR results and sequencing results of F2 segregating population

[0046]

[0047] Note: WT represents non-fragrant genotype; HZ represents heterozygous genotype; HM represents homozygous fragrant genotype; Y represents match;

[0048] N represents mismatch; gray shaded characters represent HM genotype individuals.

[0049] Comparative analysis of aroma identification results of 2.5F2 seeds and molecular marker identification results

[0050] To further verify the accuracy of the FRE7 molecular marker results, the aroma of the 30 individual F2 rice plants screened in 2.3 was identified. The results showed that the rice harvested from 15 homozygous genotype plants all had aroma traits, while the rice harvested from 15 heterozygous genotype plants was separated into aroma and non-aroma traits. The rice aroma identification results were consistent with the genotype identification results of the FRE7 molecular marker (as shown in Table 3), indicating that the FRE7 molecular marker can accurately identify the E7 genotype.

[0051] Table 3. Comparison of F2 rice aroma identification results and F2 plant genotype identification results

[0052]

[0053] Note: WT represents non-fragrant genotype; HZ represents heterozygous genotype; HM represents homozygous fragrant genotype; Y represents match;

[0054] N represents mismatch; gray shaded characters represent HM genotype individuals.

[0055] in conclusion

[0056] The FRE7 molecular marker of the rice aroma gene OsBadh2 of the present invention can not only accurately identify rice E7 type aromatic rice and non-aromatic rice varieties, but also accurately determine the genotype of the hybrid F2 generation of E7 mutant aromatic rice / non-aromatic rice, and can be applied to aromatic rice breeding and E7 genotype identification and screening of aromatic rice varieties. The present invention provides a FRE7 molecular marker and a practical application method thereof for aromatic rice breeding. The FRE7 molecular marker can improve the efficiency of E-type aromatic rice identification and aromatic rice breeding, and has a wide range of application prospects.

Claims

1. A molecular marker FRE7 of rice aroma gene OsBadh2, characterized in that A FRE7 molecular marker was designed in the mutation region of exon 7 of the OsBadh2 gene. The primer sequences of the FRE7 molecular marker are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

4.

2. Use of the FRE7 molecular marker of the rice aroma gene OsBadh2 according to claim 1 in aromatic rice breeding.

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