CN113151575B - An InDel molecular marker GW6a-InDel, a major QTL for grain shape in rice, and its detection primers and applications - Google Patents

Abstract

The invention relates to an InDel molecular marker GW6a-InDel of the main QTL of rice grain shape, a detection primer and application thereof, and belongs to the technical field of rice breeding. The present invention provides an InDel molecular marker GW6a-InDel of the main QTL of rice grain shape, and the nucleotide sequence of the GW6a-InDel is shown in SEQ ID NO: 1; in the SEQ ID NO: 1, there are consecutive 2 or 3 repeat sequence; the repeat sequence is CGTCTT. The molecular marker of the invention is closely linked with the grain shape QTL, and can be used to identify and breed rice varieties with novel allelic variation gw6a-2, so as to realize the selection of target rice varieties with slender grain phenotype.

Description

An InDel molecular marker GW6a-InDel for rice grain shape major QTL and its detection Primer and application

technical field

The invention relates to the technical field of rice breeding, in particular to an InDel molecular marker GW6a-InDel of the main QTL of rice grain shape, and a detection primer and application thereof.

Background technique

Rice is one of the most important cereal crops in the world and the main source of food for about half of the world's population. The steady increase in rice production is of great significance to the food production and stability of my country and the world. Rice yield is mainly determined by the number of panicles per plant, the number of grains per panicle and grain weight. Among them, grain shape is one of the most important factors affecting the grain weight of rice, and it also affects the quality of rice. The three main characters related to grain shape are grain length, grain width and thousand-grain weight. Rice grain shape not only directly determines the appearance quality of rice varieties, but also plays an important role in the high yield of rice varieties. Therefore, exploring and utilizing new allelic variation of grain shape genes is also one of the research goals of rice molecular breeding.

According to the data published on the Gramene website (http://www.gramene.org/qtl/), there are currently 18 major QTLs related to grain shape in rice, which are distributed on each chromosome of rice. However, in these studies, most of the QTLs were only found and located in specific genetic backgrounds, and allelic variants in other breeds were not found and studied, leading to the study of the gene distribution and function of these QTLs Not good enough. At present, molecular sequencing methods are mainly used to detect and analyze these major QTLs in some specific germplasms. Through the strategy of gene sequence analysis, the novel allelic variants of the cloned major QTLs were mined, in order to improve the distribution and functions of these major QTLs in different materials.

Grain shape is an important yield trait in rice. Therefore, discovering the allelic variation of new main grain shape QTL loci in rice can provide theory and practice for further elucidating the genetic mechanism and regulatory network of rice grain shape and high-yield and broad-adaptive rice breeding. support.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of InDel molecular marker GW6a-InDel of the main QTL of rice grain shape and its detection primer and application. The molecular marker of the invention is closely linked with the grain shape QTL, and can be used for identifying and breeding rice varieties with novel allelic variation gw6a-2, so as to realize the selection of target rice varieties with slender grain phenotype.

The present invention provides an InDel molecular marker GW6a-InDel of the main QTL of rice grain shape, and the nucleotide sequence of the GW6a-InDel is shown in SEQ ID NO: 1; 2 or 3 repeat sequences; the repeat sequence is CGTCTT.

The present invention also provides a reagent for detecting the InDel molecular marker GW6a-InDel described in the above technical solution, the reagent comprises a primer pair; the primer pair comprises a positive nucleotide sequence as described in SEQ ID NO:2 The primer and reverse primer whose nucleotide sequence is shown in SEQ ID NO:3 were used.

The present invention also provides a kit for detecting grain shape traits of rice, including the reagent described in the above technical solution.

Preferably, the kit further includes dNTPs, Taq DNA polymerase and 10×Taqbuffer containing magnesium ions.

The present invention also provides the application of the InDel molecular marker GW6a-InDel described in the above technical solution or the reagent described in the above technical solution or the kit described in the above technical solution in detecting rice grain shape.

The present invention also provides the application of the InDel molecular marker GW6a-InDel described in the above technical solution or the reagent described in the above technical solution or the kit described in the above technical solution in breeding rice varieties with slender grain shape.

The present invention also provides a method for detecting grain shape of rice, comprising the following steps:

1) Extract the DNA of the rice to be tested;

2) using the DNA extracted in step 1) as a template, performing PCR amplification with the primer pair in the reagent described in the above technical solution to obtain an amplification product;

3) Analyze the amplified product in step 2), and judge the grain shape of the rice to be tested according to the length of the amplified product: when the length of the amplified product is 130bp, the genotype of the rice to be tested is GW6a, and the grain shape is GW6a. is short grain; if the length of the amplified product is 136bp, the genotype of the rice to be tested is gw6a-2, and the grain type is slender; if the amplified product is heterozygous, the rice to be tested is The genotype is GW6a/gw6a-2, and the grain shape is between the two.

Preferably, the reaction system for PCR amplification in step 2) is as follows: 1 μL DNA, 0.5 μL 10 mmol forward primer, 0.5 μL 10 mmol reverse primer, 1 μL 10×Taq buffer containing 20 mM Mg ²⁺ , 0.2 μL 10 mM dNTPs, 0.2 μL 2U/μL Taq DNA polymerase and 6.6 μL _ddH2O .

Preferably, the reaction procedure of PCR amplification in step 2) is pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 32 cycles; and a final extension at 72°C for 10 minutes.

Preferably, the varieties of rice to be tested include Nipponbare, 9311, Zhonghui 9308, Zhonghui 8015, Long Grain Japonica, BG1, IR56, Huazhan, Zhonghua 11, 02428, Nanjing 11, Yuzhenxiang, IR24, IR26 and IR64.

The invention provides an InDel molecular marker GW6a-InDel of the main QTL of rice grain shape. The present invention discovers for the first time a new allelic variation of the main gene GW6a that controls grain shape located on the long arm of rice chromosome 6, and obtains the InDel molecular marker GW6a-InDel that can be used to distinguish the two. It is only necessary to detect the characteristics of the amplified bands of the above markers to determine whether the GW6a locus in the target material is a new allelic variation gw6a-2, which can be used to guide the breeding work for improving the grain shape of rice. Screening of phenotypic varieties. The molecular marker of the invention can not only distinguish the genotypes of rice varieties at the seedling stage, but also can conveniently, quickly and directly realize the identification of target genes in rice germplasm resources and breeding offspring, which greatly reduces labor costs, Save time and not be affected by environmental and human factors.

Description of drawings

Figure 1 is the identification results of grain shape of different rice varieties using the molecular marker GW6a-InDel provided by the present invention; wherein 1 to 15 represent: 1, Nipponbare; 2, 9311; 3, Zhonghui 9308; 4, Zhonghui Hui8015; 5, Long-grain Japonica; 6, BG1; 7, IR56; 8, Huazhan; 9, Zhonghua 11; 10, 02428; 11, Nanjing 11; 12, Jade Needle Fragrance; 13, IR24; 14, IR26 ; 15, IR64.

Detailed ways

The present invention provides an InDel molecular marker GW6a-InDel of a major QTL for rice grain shape, and the nucleotide sequence of the GW6a-InDel is shown in SEQ ID NO: 1: GCTCGACGCAGAGAGACATGCCCGCCTTCTTCTTCTT(CGTCTT) _{2 or 3} GGTCTTCTTCTTCTCCTTGGCGGCGGCGCCGTCGCCGTCGTCGGCGTGTCGTGAACCGCCGCCATGTCGCCGGACAGGCC; There are 2 or 3 consecutive repeating sequences in the SEQ ID NO: 1; the repeating sequence is CGTCTT. Based on the precise positioning of GW6a, the present invention performs sequencing identification analysis on multiple varieties (specifically, second-generation sequencing is performed on the GW6a locus in large-grain indica varieties BG1 and XLJ, and the genome sequence of the reference variety Nipponbare is compared with the genome sequence of the reference variety Nipponbare. Right), a new allelic variation was found, that is, a repeat sequence of CGTCTT was inserted at the +202 position of the first exon, and the major particle shape QTL was named gw6a-2. The GW6a gene sequences of large grain indica varieties BG1 (SEQ ID NO. 4, gw6a-2), XLJ (SEQ ID NO. 5, gw6a-2) and Nipponbare (SEQ ID NO. 6, GW6a) are provided in the sequence listing. Compared with the coding region sequence of GW6a (corresponding to 2 repeat sequences), the new allelic variation (corresponding to 3 repeat sequences) is at a CGTCTT repeat sequence (+202 site) in the first exon, with multiple insertions A section of CGTCTT, the original two repeats, will generate three repeats after insertion. The corresponding coding sequence transcript is an amino acid sequence rich in lysine (K) and threonine (T) at the original corresponding site, and the transcript of the new allelic variation has an additional pair of lysines here. acid (K) and threonine (T) to increase the TK enrichment. When the GW6a gene contains 3 repeats, the grain shape of rice is elongated, and when the GW6a gene contains 2 repeats, the grain shape of rice is short. The invention amplifies the InDel molecular marker GW6a-InDel of the main QTL of rice grain shape by PCR, and judges the grain shape character of rice according to the length of the amplified product. The invention aims at the above variation and obtains molecular markers, which can greatly improve the identification and breeding efficiency of the rice grain shape gene GW6a and accelerate the breeding progress.

The present invention also provides a reagent for detecting the InDel molecular marker GW6a-InDel described in the above technical solution, the reagent comprises a primer pair; the primer pair comprises a positive nucleotide sequence as described in SEQ ID NO:2 A forward primer (GGCCTGTCCGGCGAC) and a reverse primer (GCTCGACGCAGAGAGACA) whose nucleotide sequence is shown in SEQ ID NO:3. The source of the primer pair is not particularly limited in the present invention, and the source of the primer pair known in the art can be used. In the embodiment of the present invention, the preferred pair of primers is entrusted to Hangzhou Youkang Biotechnology Co., Ltd. to synthesize.

The present invention also provides a kit for detecting grain shape traits of rice, including the reagent described in the above technical solution. In the present invention, the kit preferably further includes dNTPs, Taq DNA polymerase and 10×Taqbuffer containing magnesium ions. The present invention does not specifically limit the sources of the dNTPs, Taq DNA polymerase and 10×Taq buffer containing magnesium ions, and reagents such as dNTPs, Taq DNA polymerase and 10×Taq buffer containing magnesium ions that are well known in the art are used source can be. In the examples of the present invention, the dNTPs, Taq DNA polymerase and 10×Taq buffer containing magnesium ions were purchased from Novozymes, respectively.

The present invention also provides the application of the InDel molecular marker GW6a-InDel described in the above technical solution or the reagent described in the above technical solution or the kit described in the above technical solution in detecting rice grain shape.

The present invention also provides the application of the InDel molecular marker GW6a-InDel described in the above technical solution or the reagent described in the above technical solution or the kit described in the above technical solution in breeding slender grain-shaped rice varieties.

The present invention also provides a method for detecting grain shape of rice, comprising the following steps:

1) Extract the DNA of the rice to be tested;

2) using the DNA extracted in step 1) as a template, performing PCR amplification with the primer pair in the reagent described in the above technical solution to obtain an amplification product;

3) Analyze the amplified product in step 2), and judge the grain shape of the rice to be tested according to the length of the amplified product: when the length of the amplified product is 130bp, the genotype of the rice to be tested is GW6a, and the grain shape is GW6a. is short-grain; if the length of the amplified product is 136bp, the genotype of the rice to be tested is gw6a-2, and the grain type is slender; if the amplified product is heterozygous, the rice to be tested is The genotype is GW6a/gw6a-2, and the grain shape is between the two.

The invention extracts the DNA of the rice to be tested. In the present invention, the DNA of rice is preferably the genomic DNA of rice. The present invention has no particular limitation on the method for extracting the DNA of the rice to be tested, and a method known in the art for extracting plant DNA may be used, such as the kit method and the CTAB method. The method of the present invention does not specifically limit the variety of rice to be tested, and the variety of rice well-known in the art can be used. In the present invention, the variety of rice to be tested preferably includes Nipponbare, 9311, Zhonghui 9308, Zhonghui 8015, Long Grain Japonica, BG1, IR56, Huazhan, Zhonghua 11, 02428, Nanjing 11, Jade Needle Fragrance, IR24, IR26 and IR64.

After extracting the rice DNA to be tested, the present invention uses the extracted DNA as a template and uses the primers for PCR amplification to obtain an amplification product. In the present invention, the PCR amplification reaction system is preferably as follows: 1 μL DNA, 0.5 μL 10 mmol forward primer, 0.5 μL 10 mmol reverse primer, 1 μL 10×Taq buffer containing 20 mM Mg ²⁺ , 0.2 μL 10 mM dNTPs, 0.2 μL 2U/μL Taq DNA polymerase and 6.6 μL ddH ₂ O. In the present invention, the reaction procedure of the PCR amplification is preferably pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 32 cycles; and a final extension at 72°C for 10 minutes. The present invention has no particular limitation on the PCR amplification instrument, and a PCR instrument well-known in the art may be used.

After the amplified product is obtained, the present invention analyzes the amplified product, and judges the grain shape of the rice to be tested according to the length of the amplified product: when the length of the amplified product is 130bp, the genotype of the rice to be tested is: GW6a, the grain type is short grain; if the length of the amplification product is 136bp, the genotype of the rice to be tested is gw6a-2, and the grain type is slender grain; if the amplification product is a heterozygous type, The genotype of the rice to be tested is GW6a/gw6a-2, and the grain shape is between the two. In the present invention, the method for analyzing the amplification product preferably includes PAGE gel electrophoresis or sequencing. The present invention has no particular limitation on the method of PAGE gel electrophoresis or sequencing, and PAGE gel electrophoresis or sequencing well-known in the art can be used.

The InDel molecular marker GW6a-InDel and its detection primers and applications of a rice grain shape major QTL described in the present invention will be further described in detail below with reference to specific examples. The technical solutions of the present invention include but are not limited to the following examples.

Example 1

1. Testing materials

The GW6a loci in the large-grain indica varieties BG1 and XLJ were sequenced by the second generation, and compared with the genome sequence of the reference variety Nipponbare. After the differences were found, the molecular markers of the present invention were designed. Nipponbare, 9311, Zhonghui 9308, Zhonghui Fifteen parents, including Hui 8015, Long Grain Japonica, BG1, IR56, Huazhan, Zhonghua 11, 02428, Nanjing 11, Yuzhenxiang, IR24, IR26 and IR64, were identified by markers.

2. Test method (DNA extraction and PCR amplification)

Young leaves were taken from each plant, and individual DNA was extracted by CTAB method.

PCR reaction: 1μL DNA (100ng), 1μL primers (10mmol, 0.5μL forward primer and 0.5μL reverse primer), 1μL 10×Taq buffer (20mM Mg ²⁺ ), 0.2μL dNTPs (10mM), 0.2μL Taq DNA Polymerase (2U/μL) and 6.6 μL _ddH2O .

The PCR reaction program was: pre-denaturation at 95°C for 4 min (denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 32 cycles), and a final extension at 72°C for 10 min. PCR amplification was performed on a PCR machine, and the amplified products were separated by electrophoresis on an 8% polyacrylamide gel.

(2) Sequencing results

Amplify and sequence the genome sequences of GW6a in Nipponbare, BG1 and XLJ by sequencing primers, and found that there is an insertion/deletion difference of a CGTCTT repeat sequence in the first exon, which can be assisted by designing an InDel molecular marker and using it for molecular marker assistance choose. The 15 conventional parents were identified by polypropylene gel electrophoresis experiment with molecular marker GW6a-InDel, and the identification results are shown in Figure 1.

Table 1 Molecular marker information

(4) Results and Analysis

The 15 conventional parents were screened with molecular marker GW6a, and sequenced and identified according to the difference results. It was found that the cultivar with a band of 130 bp did not have CGTCTT sequence insertion, which was a tandem repeat normal type; the cultivar with a band of 136 had CGTCTT sequence insertion, which was a tandem repeat. Repeat insert type.

Table 2 Rational examination of 15 parents and genotyping of GW6a locus

Note: TGW stands for thousand-grain weight, GL/GW stands for grain length/grain width, GL stands for grain length, and GW stands for grain width.

According to the grain type investigation data of the above 15 parents, it was found that the grain length-width ratio of the rice varieties genotyped as gw6a-2 was generally larger, which belonged to typical slender grains (GL/GW≥3.0) (Huang Haixiang, Qian Qian. (2017). Rice grain shape inheritance and breeding progress of long-grain high-quality japonica rice. China Rice Science (06), 665-672. doi: 10.16819/j.1001-7216.2017.7115). The allelic variation of gw6a-2 identified by the present invention results in increased grain length.

Example 2

Using GW6a-InDel to discriminate whether rice varieties contain a novel allelic variant gw6a-2 of GW6a

Specific steps are as follows:

(1) DNA extraction: take the young leaves of rice, and extract genomic DNA by CTAB method;

(2) Establishment of a standard PCR amplification system: PCR amplification was carried out with the GW6a-InDel marker primer and the whole genome DNA of a single rice plant to be tested as a template;

(3) Detection and analysis of amplified DNA fragments: PCR products are detected by 8% polyacrylamide gel electrophoresis. If the marker can amplify a 130bp fragment, it means that the target variety does not have CGTCTT insertion, and its genotype is GW6a. If the marker can amplify a 136bp fragment, it means that there is a CGTCTT insertion in the target variety, and its genotype is gw6a-2. If the heterozygous band type can be amplified, it means that the genotype of the individual plant is GW6a/gw6a-2.

Table 3 Detection of newly developed markers in 24 varieties and investigation of grain shape

Note: TGW stands for thousand-grain weight, GL stands for grain length, GW stands for grain width, GL/GW stands for grain length/grain width, and - means the same genotype as the header.

It can be seen from the above examples that the above molecular markers and methods are mainly used to identify whether there is a CGTCTT repeat insertion in the first exon of the rice grain shape major gene GW6a. Through detection and identification, it can be determined whether there is a new allelic variation gw6a-2 in the target variety that causes slender grains, which improves the diversity locus of the rice grain shape gene GW6a, improves the selection efficiency, and accelerates the breeding progress.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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cgatgatatt accagcatga cgtgttccgg ggcgtggcgg actctggcga gcgggtcgcc 1320

gatctgctcg acgcagagag acatgctcgc cttcttcttc ttcgtcttcg tcttggtctt 1380

cttcttctcc ttggcggcgg cgccgtcgcc gtcgtcggcg tggtcgtgaa ccgcggccat 1440

gtcgccggac aggccgacct ggcaccgccg ctccagctcc tccaccgccg gcaagtcctt 1500

ctccacgtcg aactccctca ccctcaccaa caccttcatc atcgtcgtcg tctccaccat 1560

Claims (5)

1. The InDel molecular marker GW6a-InDel of a rice grain shape major QTL, characterized in that the nucleotide sequence of the GW6a-InDel is as shown in SEQ ID NO:1; in the SEQ ID NO:1 There are 2 or 3 consecutive repeats; the repeat is CGTCTT. 2. a method for detecting grain shape of rice, is characterized in that, comprises the following steps: 1) Extract the DNA of the rice to be tested; 2) Take the DNA extracted in step 1) as a template, and carry out PCR amplification with primer pairs to obtain amplification products; the primer pairs include forward primers with nucleotide sequences such as those described in SEQ ID NO: 2 and nucleotides The reverse primer whose sequence is shown in SEQ ID NO:3; 3) Analyze the amplified product in step 2), and judge the grain shape of the rice to be tested according to the length of the amplified product: when the length of the amplified product is 130bp, the genotype of the rice to be tested is GW6a, and the grain shape is GW6a. is short grain; if the length of the amplified product is 136bp, the genotype of the rice to be tested is gw6a-2, and the grain type is slender; if the amplified product is heterozygous, the rice to be tested is The genotype is GW6a/gw6a-2, and the grain shape is between the two. 3. the method for detecting rice grain shape according to claim 2 is characterized in that, the reaction system of PCR amplification described in step 2) is as follows: the forward primer of 1 μL DNA, 0.5 μL 10mmol, the reverse direction of 0.5 μL 10mmol Primers, 1 μL 10×Taq buffer with 20 mM Mg ²⁺ , 0.2 μL 10 mM dNTPs, 0.2 μL 2U/μL Taq DNA polymerase, and 6.6 μL ddH ₂ O. 4. The method for detecting grain shape of rice according to claim 2, wherein the reaction procedure of PCR amplification in step 2) is 95°C pre-denaturation for 4min; 95°C denaturation for 30s, 55°C annealing for 30s, 72°C The extension was carried out for 30 s for a total of 32 cycles; the final extension was at 72 °C for 10 min. 5. The method for detecting grain shape of rice according to any one of claims 2 to 4, wherein the varieties of the rice to be tested include Nipponbare, 9311, Zhonghui 9308, Zhonghui 8015, long-grain japonica, BG1, IR56, Huazhan, Zhonghua 11, 02428, Nanjing 11, Yuzhenxiang, IR24, IR26 and IR64.

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