A PARMS primer set for detecting specific mutations of rice aroma gene Fgr and its application

Abstract

The invention discloses a PARMS primer set and application for detecting a specific mutation of a rice aroma gene Fgr, the primer set includes a specific primer and a universal primer, the specific primer includes a nucleotide sequence such as Primer Seq Fgr-M8Fa shown in SEQ ID NO.1 and a nucleotide sequence such as Primer Seq Fgr-M8Ft shown in SEQ ID NO.2, and the universal primer is a nucleotide sequence such as PrimerFgr-M8R shown in SEQ ID NO.3; the specific mutation site is located at the 20380297th base of the Fgr gene of chromosome No. 8 of rice Nipponbare msu7.0, and there is an insertion of a base T; the primer set can be used to identify the rice Fgr genotype. The special primer provided by the present invention has strong specificity, high detection efficiency, simple procedures during the detection process, and clear results. These markers can be used to quickly, easily and accurately identify and analyze the genotype of the aroma gene Fgr-E2, and the difference in fluorescent signals between the aroma type and the non-aroma type can be more clearly identified, and can be better applied to the breeding of aroma rice varieties.

Description

Technical Field

[0001] The invention relates to the field of rice breeding, in particular to a PARMS primer set for detecting a specific mutation of a rice aroma gene Fgr and an application thereof. Background Art

[0002] Rice is an important food crop in China. With the continuous improvement of people's living standards, people's demand for rice is not only limited to quantity, but also to quality. Fragrance is one of the criteria for measuring rice quality. Fragrant rice has a unique fragrance and high nutritional value. It is rich in vitamins and amino acids, and is favored by consumers. It has high production and market value. Therefore, fragrance traits have also become a hot topic for breeders and an important research content for rice quality improvement.

[0003] Traditional manual tasting and identification of rice aroma germplasm resources has the disadvantages of being time-consuming and subjective, and the identification results are prone to errors, which is time-consuming and labor-intensive. Molecular marker-assisted selection breeding is simple and effective, which can save labor costs, shorten the breeding cycle, and can also carry out purposeful multi-gene aggregation, improve breeding efficiency, and bring huge social and economic benefits. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a PARMS primer set for detecting a specific mutation of a rice aroma gene Fgr and its application.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the purposes of the present invention is to provide a PARMS primer set for detecting a specific mutation of a rice aroma gene Fgr, the primer set comprising a specific primer and a universal primer, wherein the specific primer comprises a Primer Seq Fgr-M8Fa having a nucleotide sequence as shown in SEQ ID NO.1 and a PrimerSeqFgr-M8Ft having a nucleotide sequence as shown in SEQ ID NO.2, and the universal primer is a Primer Fgr-M8R having a nucleotide sequence as shown in SEQ ID NO.3; the specific mutation site is located at the 20380297th base of the Fgr gene of chromosome 8 of rice Nipponbare msu7.0, and there is an insertion of a base T.

[0007] Furthermore, the 5' end of the Primer SeqFgr-M8Fa is connected to a FAM fluorescent sequence, and the 5' end of the Primer Seq Fgr-M8Ft is connected to a HEX fluorescent sequence; or the 5' end of the Primer Seq Fgr-M8Fa is connected to a HEX fluorescent sequence, and the 5' end of the Primer Seq Fgr-M8Ft is connected to a FAM fluorescent sequence.

[0008] The second object of the present invention is to provide an application of a primer set in identifying the Fgr genotype of rice, comprising the following steps:

[0009] S1. Extract genomic DNA from rice leaves;

[0010] S2. Using the genomic DNA extracted in step S1 as a template, using the primer set for detecting the PARMS molecular marker of the rice aroma gene Fgr, and using the PARMS technology to detect the Fgr-M8Ft molecular marker; if the sequence of Fgr-M8Ft is T insertion, the tested rice sample is determined to be an Fgr functional type, a homozygous aroma Fgr genotype; if the sequence of Fgr-M8Ft is T deletion, the tested rice sample is determined to be a homozygous non-aroma Fgr genotype; if T / - is detected at the polymorphic site of Fgr-M8Ft at the same time, the tested rice is determined to be a heterozygous Fgr genotype.

[0011] Compared with the prior art, the PARMS primer set for detecting specific mutations of the rice aroma gene Fgr of the present invention is used to detect the Fgr gene of rice materials by applying the PARMS technical reaction, so that the rice aroma gene Fgr can be quickly and accurately detected in different germplasm resources such as indica rice and japonica rice. The present invention uses the PARMS technology to type the Fgr gene. Compared with the most commonly used SSR technology based on PCR+electrophoresis, the PARMS detection technology eliminates the time-consuming and labor-intensive electrophoresis technology, and directly reads the genotype signal of the allele by fluorescence scanning. With the data analysis software, the genotype analysis work can be completed quickly, and the work efficiency is improved by an order of magnitude; the PARMS technology is very suitable for detection automation, and with the GeneMatrix high-throughput genotyping system, it can achieve ultra-high-throughput Fgr gene identification and aroma resource screening of 100,000 data points per day. PARMS is perfectly compatible with the LGC KASP SNP detection hardware system and primers, and can achieve seamless conversion. The present invention does not require cumbersome procedures such as enzyme cutting, electrophoresis and sequencing during the detection process, reduces the pollution of PCR product aerosol and the use of toxic substances such as EB, and can also perform prospect selection in the early stage of molecular marker-assisted breeding, reduce the scale of breeding populations, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The positions of specific mutations in the embodiments of the present invention.

[0013] Figure 2 PARMS detection technology flow chart.

[0014] Figure 3 This is a typing diagram of the molecular marker Fgr-M8Ft of different varieties in the examples of the present invention. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0016] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0017] Example 1: Design of PARMS primer set for detecting specific mutations in rice aroma gene Fgr

[0018] The present embodiment discloses a PARMS primer set for detecting a specific mutation of the rice aroma gene Fgr, the primer set comprising a specific primer and a universal primer, wherein the specific primer comprises a Primer SeqFgr-M8Fa having a nucleotide sequence as shown in SEQ ID NO.1 and a Primer Seq Fgr-M8Ft having a nucleotide sequence as shown in SEQ ID NO.2, and the universal primer is a PrimerFgr-M8R having a nucleotide sequence as shown in SEQ ID NO.3; the specific mutation site is located at the 20380297th base of the Fgr gene on rice chromosome 8, and there is an insertion of a base T, and the position on the rice chromosome is as shown in Figure 1 The specific design process is as follows. Figure 2 As shown, the physical location of the cloned target gene Fgr is determined, the polymorphic site and flanking sequence are extracted, the primer sequence of the marker is designed and synthesized, and the marker is screened and tested, as follows:

[0019] 1) Primer design

[0020] According to the positioning marker information in the literature, the physical location of Fgr (GenBank: AB096083.1) was determined to be the 20379794-20386061 interval of chromosome 8 of Nipponbare (MSU7.0), and the polymorphic sites and flanking sequences in this interval were extracted, and primers were designed using the online primer design website http: / / www.snpway.com / . The primer design of Fgr-M8Fa is shown in Table 1. Each group of markers has three primers, and the 5' end of Primer SeqFgr-M8Fa is connected to the FAM fluorescent sequence, and the 5' end of Primer SeqFgr-M8Ft is connected to the HEX fluorescent sequence; or the 5' end of Primer Seq Fgr-M8Fa is connected to the HEX fluorescent sequence, and the 5' end of Primer Seq Fgr-M8Ft is connected to the FAM fluorescent sequence. The primers were commissioned to Wuhan Jingpeptide Biotechnology Co., Ltd. for synthesis.

[0021] Table 1

[0022]

[0023] Example 2: Identification of rice Fgr genotype using the primer set of Example 1

[0024] DNA extraction: Genomic DNA was extracted from rice leaves using the simplified CTAB method.

[0025] PARMS reaction test: 10ul PCR reaction system. Add 20ng DNA sample to the PCR reaction plate, dry it and add PARMS MasterMix reaction mixture (produced by Wuhan Jingpeptide Biotechnology Co., Ltd.). The reaction system is shown in Table 2.

[0026] Table 2

[0027] volume Final concentration 10 μM Primer Fgr-M8Fa 0.15μL 150nM 10 μM Primer Fgr-M8Ft 0.15μL 150nM 10μM PrimerFgr-M8R 0.4μL 400nM 2xPARMSMasterMix 5μL 1x Ultrapure water Make up to 10 μL

[0028] PCR amplification was completed in the ABI 7500 fluorescence quantitative PCR system. The Touchdown PCR reaction conditions were as follows: 94°C pre-denaturation for 15 minutes; the first amplification reaction, 94°C denaturation for 20 seconds, 65°C-57°C annealing and extension for 60 seconds, 10 cycles, and the annealing and extension temperature was reduced by 0.8°C in each cycle; the second amplification reaction, 94°C denaturation for 20 seconds, 57°C annealing and extension for 60 seconds, 32 cycles. After the PCR reaction was completed, the fluorescence data was read. If the sequence of Fgr-M8Ft was T inserted, the tested rice sample was determined to be Fgr functional type, homozygous fragrance Fgr genotype; if the sequence of Fgr-M8Ft was T deleted, the tested rice sample was determined to be homozygous non-fragrant Fgr genotype; if T / - was detected at the polymorphic site of Fgr-M8Ft at the same time, the tested rice was determined to be heterozygous Fgr genotype.

[0029] Table 3

[0030] serial number Variety name Test results Fgr genotype serial number Variety name Test results Fgr genotype 1 New Rice 11 FAM -- 25 Thousand Waves 2 FAM -- 2 New Rice 44 FAM -- 26 Liaojing1415 FAM -- 3 New Rice 45 FAM -- 27 Shenyang Agricultural University 265 FAM -- 4 Jingning 33 FAM -- 28 Iron 4 FAM -- 5 Ningjing 48 FAM -- 29 Jinjingyou 919 FAM -- 6 Youyou 41 FAM -- 30 Port Source 8 FAM -- 7 Fujitsu FAM -- 31 Hong Kong Yuk 129 FAM -- 8 Liaoxing 1 FAM -- 32 Tiejing 9 FAM -- 9 Rice Flower Fragrance FAM -- 33 Meigu No. 4 FAM -- 10 Jindao 108 FAM -- 34 Liaojing 169 FAM -- 11 Jijing812 FAM -- 35 Yanfeng 48 FAM -- 12 Liaojing 439 FAM -- 36 Liaojing1305 FAM -- 13 Hunjing 31 FAM -- 37 Liaojing 419 FAM -- 14 Panjing 16 FAM -- 38 Beijing1702 FAM -- 15 Liaoxing 21 FAM -- 39 Beijing1705 FAM -- 16 Shen Dao 7 FAM -- 40 Liaojing 212 FAM -- 17 Koshihikari FAM -- 41 Zhongkefa No.6 FAM -- 18 Salt Japonica 939 FAM -- 42 Liaojing 294 FAM -- 19 Liaoyan 241 FAM -- 43 Brazilian land rice FAM -- 20 Yanfeng 47 FAM -- 44 LXX1-1 HEX T 21 Liaojing 9 FAM -- 45 LXX1-1 HEX T 22 Liaojing 101 FAM -- 46 LXX1-1 HEX T 23 Yanfeng 456 FAM -- 47 Blank control Blank control Negative control 24 Liaojing 371 FAM -- 48 Blank control Blank comparison Negative control

[0031] As can be seen from Table 3, HEX signals were detected at the Fgr-M8Ft test site in varieties carrying the Fgr gene, such as LXX1-1; FAM signals were detected at the Fgr-M8Ft test site in varieties not carrying the Fgr gene, such as Liaoxing 1 and Yueguang; all blank controls were negative controls. The Fgr gene is the main gene that affects the aroma of rice. The aroma of rice can be improved through the Fgr gene in the future. Fgr-M8Ft can be used for efficient detection of rice Fgr gene typing; it can be better applied to the breeding of aromatic rice varieties in the future.

[0032] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A PARMS primer set for detecting a specific mutation of a rice aroma gene Fgr, characterized in that: The primer set includes specific primers and universal primers, wherein the specific primers include Primer Seq Fgr-M8Fa with a nucleotide sequence as shown in SEQ ID NO.1 and Primer Seq Fgr-M8Ft with a nucleotide sequence as shown in SEQ ID NO.2, and the universal primer is Primer Fgr-M8R with a nucleotide sequence as shown in SEQ ID NO.3; the specific mutation site is located at the 20380297th base of the Fgr gene of chromosome 8 of rice Nipponbare msu7.0, and there is an insertion of a base T.

2. The PARMS primer set for detecting a specific mutation of the rice aroma gene Fgr according to claim 1, characterized in that: The 5' end of the Primer Seq Fgr-M8Fa is connected to a FAM fluorescent sequence, and the 5' end of the Primer Seq Fgr-M8Ft is connected to a HEX fluorescent sequence; or the 5' end of the Primer Seq Fgr-M8Fa is connected to a HEX fluorescent sequence, and the 5' end of the Primer Seq Fgr-M8Ft is connected to a FAM fluorescent sequence.

3. Use of the primer set as claimed in claim 2 in identifying the Fgr genotype of rice.

4. Use of the primer set according to claim 3 in identifying the Fgr genotype of rice, characterized in that: The steps include: S1. Extract genomic DNA from rice leaves; S2. Using the genomic DNA extracted in step S1 as a template, using the primer set for detecting the PARMS molecular marker of the rice aroma gene Fgr, and using the PARMS technology to detect the Fgr-M8Ft molecular marker; if the sequence of Fgr-M8Ft is T insertion, the tested rice sample is determined to be an Fgr functional type, a homozygous aroma Fgr genotype; if the sequence of Fgr-M8Ft is T deletion, the tested rice sample is determined to be a homozygous non-aroma Fgr genotype; if T / - is detected at the polymorphic site of Fgr-M8Ft at the same time, the tested rice is determined to be a heterozygous Fgr genotype.

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