CN110894542A - Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer - Google Patents

Abstract

The invention discloses a primer for identifying GS5 gene and GLW7 gene types and application thereof, belonging to the technical field of rice variety identification. The invention identifies the types of the GS5 gene and the GLW7 gene by converting simple repeated sequences of the GS5 gene and the GLW7 gene of rice into enzyme digestion molecular markers, wherein the nucleotide sequences are shown as SEQ ID No.: 1 to 6. By utilizing the molecular marker disclosed by the invention, the favorable variation types of GS5 and GLW7 genes can be accurately identified, and the breeding and screening efficiency and progress are accelerated; the enzyme digestion molecular marker designed by the invention has good amplification effect, and the selected enzyme is a common enzyme with lower price, so that the identification cost can be greatly reduced, and the enzyme digestion molecular marker is suitable for analyzing genotypes of a large number of varieties.

Description

Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer

Technical Field

The invention belongs to the technical field of rice variety identification, and particularly relates to a primer for identifying types of GS5 genes and GLW7 genes of rice and application thereof.

Background

Rice is an important grain crop, and higher requirements are put forward on rice varieties along with the economic development of China and the improvement of the living standard of people. However, the time period of the conventional variety improvement is long, a large number of groups need to be planted, and the target characters need to be screened by means of visual observation, and with the improvement of rice genome information and the continuous breakthrough of functional gene research, the method for directionally improving varieties by using molecular markers for assistance becomes an effective means. SSR markers are relatively common molecular markers characterized by a simple repeat of a nucleic acid base sequence. The marker can be distinguished by adopting a PCR amplification mode, if the number of the repeated sequences of two varieties is very different, the fragment difference of corresponding PCR amplification products is also larger, the difference is generally larger than 10 bases, the amplified fragments are not more than 10 times of the number of the corresponding different bases, the fragments can be directly distinguished by adopting an agarose gel electrophoresis method, when the number of the SSR bases is less than 10 bases, although PCR can be amplified, the fragments are difficult to distinguish on the agarose gel, the fragments are necessarily distinguished by means of acrylamide gel, and the latter has the defects of complicated gel preparation process, long electrophoresis time and high gel component toxicity. Some key character variations are found in rice at present and are caused by SSR repeated sequence differences, and if the SSRs are effectively identified, related characters can be directly tracked without worrying about the problem that target characters are lost due to chromosome recombination.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a primer for identifying types of rice GS5 genes and GLW7 genes by converting simple rice repeated sequences into enzyme digestion molecular markers and application thereof.

The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:

a primer for identifying GS5 gene and GLW7 gene types comprises the following primers:

(1) primers for identifying the type of the GS5 gene: the nucleotide sequence is shown as SEQ ID No.: 1-2;

(2) primers for identifying the type of GLW7 gene: the nucleotide sequence is shown as SEQ ID No.: 3-4 or SEQ ID No.: 5 to 6.

An agent for identifying the GS5 gene or GLW7 gene type, comprising SEQ ID No.: 1 to 6.

A method for identifying the gene type of GS5 gene comprises the following steps:

(1) extracting the genome DNA of the rice to be detected;

(2) taking the DNA obtained in the step (1) as a template and SEQ ID No.: 1-2 as primers to carry out PCR amplification respectively;

(3) SEQ ID No.: 1-2, performing enzyme digestion treatment on a product obtained by amplification of the primers shown in the specification by using an AluI endonuclease, and performing agarose gel electrophoresis on the enzyme digestion product;

(4) if SEQ ID No.: 1-2, performing enzyme digestion on a PCR product of the primer shown in the specification by using an AluI endonuclease to obtain a fragment with the size of 229bpbp, wherein the genotype of GS5 in the rice is AAAC; if the size of the fragment obtained after the digestion by the AluI endonuclease is 262bp, the GS5 genotype in the rice is AAACAAC.

A method for identifying GLW7 gene types comprises the following steps:

(1) extracting the genome DNA of the rice to be detected;

(2) taking the DNA obtained in the step (1) as a template and SEQ ID No.: 3-4 as primers to respectively carry out PCR amplification;

(3) SEQ ID No.: 3-4, performing enzyme digestion treatment on a product obtained by amplification of the primers shown in the specification by using HinfI endonuclease, and performing agarose gel electrophoresis on the enzyme digestion product;

(4) if SEQ ID No.: 3-4, obtaining 99bp after enzyme digestion of a PCR product of the primers shown in the specification by HinfI endonuclease, wherein the GLW7 genotype in the rice is CTTCCACTTCCA, and obtaining a fragment with the size of 133bp after enzyme digestion of the HinfI endonuclease, wherein the GLW7 genotype in the rice is CTTCCACTTCCACTTCCA.

Alternatively, the first and second electrodes may be,

(1) extracting the genome DNA of the rice to be detected;

(2) taking the DNA obtained in the step (1) as a template and SEQ ID No.: 5-6 as primers for PCR amplification;

(3) if SEQ ID No.: 5-6, if the size of the obtained fragment obtained by PCR is 98bp, the GLW7 genotype in the rice is CTTCCACTTCCA, and if the size of the obtained fragment is 107bp, the GLW7 genotype in the rice is CTTCCACTTCCACTTCCA.

Wherein, in the step (2), the PCR amplification conditions are as follows: denaturation at 94 ℃ for 3 min, followed by 35 cycles of "denaturation at 94 ℃ for 20 sec-55 ℃ annealing for 30 sec-72 ℃ extension for 20 sec", and finally extension at 72 ℃ for 5 min.

Has the advantages that:

the invention discloses a primer for identifying types of GS5 gene and GLW7 gene of rice and application thereof, aiming at SSR sequence difference with smaller base number difference, the primer is effectively converted into a molecular marker based on enzyme digestion, and can be distinguished by agarose gel, so that the difficulty of molecular identification is reduced; by utilizing the molecular marker disclosed by the invention, the favorable variation types of GS5 and GLW7 genes can be accurately identified, and the breeding and screening efficiency and progress are accelerated; the enzyme digestion molecular marker designed by the invention has good amplification effect, and the selected enzyme is a common enzyme with lower price, so that the identification cost can be greatly reduced, and the enzyme digestion molecular marker is suitable for analyzing genotypes of a large number of varieties.

Drawings

FIG. 1 shows SSR sequence differences between GS5 and GLW7 genes.

FIG. 2GS5 gene SSR and its flanking sequence comparison in two varieties.

FIG. 3GLW7 gene SSR and its flanking sequence comparison in two varieties.

FIG. 4 is a schematic diagram of the amplified sequence of the enzyme-cleaved identity tag GS 5.

FIG. 5 shows the amplified sequences of the marker identified by the enzyme digestion and the non-enzyme digestion of GLW 7.

FIG. 6GS5 restriction identification tag gel electrophoresis image.

FIG. 7 shows the electrophoresis of GLW7 non-restriction enzyme identification marker gel.

FIG. 8 shows the gel electrophoresis of the marker identified by the cleavage with GLW 7.

Detailed Description

Example 1:

the grain shape is an important character for determining the yield and the quality of rice, the grain weight can be increased by increasing the grain shape, so that the yield is increased, the grain shape can be increased by increasing the grain shape, the grain filling degree can be improved, the chalkiness degree is reduced, and the appearance quality of rice is improved. GS5 and GLW7 are two key grain type genes which are cloned in rice, correspond to gene numbers Os05g0158500 and Os07g0505200 respectively, the core variation of the two key grain type genes is mainly reflected in the simple repeat sequence (SSR) difference of a promoter region or a 5UTR region, as shown in figure 1, the GS5 functional variation is the AAAC sequence repeat difference at 324 to 327bp at the upstream of ATG, the variation 1 is CAAA, the two key grain type genes correspond to broad grain and heavy grain phenotypes, the variation 2 is AAACAAC, and the two key grain type genes correspond to normal phenotypes. GLW7 functional variation is the repeated difference of TCCACT sequence at 133-150 bp upstream of ATG, variant 1 is TCCACTTCCACT corresponding to long-grain and heavy-grain phenotype, variant 2 is TCCACTTCCACTTCCACT corresponding to normal phenotype.

The SSR sequence differences of the two genes are respectively 4bp and 6bp, a target band can be amplified by adopting a conventional PCR marker, but cannot be effectively distinguished by adopting agarose gel, and Sanger sequencing can accurately identify the variation, but has higher identification cost and poor effect on distinguishing heterozygous variation, so that the application of the two genes in molecular marker-assisted selective breeding is limited.

Based on this current situation, we explored a molecular identification method that converts sequence differences of corresponding small fragments into significantly distinguishable differences. Because the sequence variation belongs to a simple repetitive sequence, the variation type cannot generate enzyme cutting site difference, and therefore cannot be converted into the Caps label based on enzyme cutting, and a new solution is required to be found.

In order to solve the problems, the variant types of two genes are firstly subjected to Sanger sequencing, and the sequenced varieties are rice reference genome varieties Nipponbare (NIP) and indica rice variety YYP 1. As shown in fig. 2 and 3, the YYP1 variety GS5 gene is AAAC variant, and the NIP variety is aaacaac variant; the YYP1 variety GLW7 gene is a CTTCCACTTCCA variant, while the NIP variety is a CTTCCACTTCCACTTCCA variant. In addition, we also found that there is a CTC simple repeat sequence upstream of the GLW7 SSR variant, compared with YYP1, one CTC is added to NIP, and combined with the above 6bp deletion, YYP1 has 9bp of overall deletion compared with NIP.

We compared the SSR flanking sequence characteristics of two parental GS5 genes (fig. 2), the right side of the AAAC sequence of YYP1 is TGCC, and the right side of the NIP AAAC sequence is AAAC, so the bases immediately adjacent to the right side of the first AAAC sequence of two varieties are T and a, respectively, we can introduce base mutation into the PCR amplification forward primer, which combines with T or a to generate enzyme cutting site difference, the recognition sequence of the AluI endonuclease is AGCT, based on the sequence recognition characteristics, we select AAAC and its upstream 26bp sequence as primers, and replace the third a in AAAC with G, the corresponding primer sequence is ACTCCCATGGAATTACTAGAGAAGCCAAGC, the recognition ability of the primers to the two parental DNAs is completely the same, but after extension, they form an AluI enzyme cutting site with the T base of YYP1, and the enzyme cutting site is not generated in the extension product of NIP, so two variation types can be distinguished by enzyme cutting PCR product.

Similarly, we compared the SSR flanking sequence characteristics of GLW7, where the immediate base on the right of TCCACTTCCACT of YYP1 is C, the immediate base on the right of NIP is T, and the recognition sequence of HinfI endonuclease is gatnc (N represents any base), so we replaced the C at the fifth position of the second CTTCCA with G, and designed forward primer CCTCCTCCTCCTCCGCCTTCCACTTCGACT, which contains a 3bp deletion but also has a CCT sequence upstream of the primer, thus ensuring perfect primer matching and identical recognition ability to the NIP sequence, when YYP1 is used as template, the primer is extended to bind C to generate HinfI site, when NIP is used as template, the primer extension binds T to generate no corresponding site, and the two parental amplification products are digested to generate fragment size difference.

Then, reverse primers are respectively designed aiming at two forward primers by copying the forward primers and a downstream 300bp sequence thereof to Premier Primer 5.0 software, wherein the range of the forward primers is still within the original 30bp interval, the reverse primers are arranged outside the range of the 30bp interval, the highest scoring Primer after operation is used as the reverse Primer, and finally, a GS 5-marked reverse Primer sequence AGGAGAAGAAAAGGTGAAAAGT and a GLW 7-marked reverse Primer sequence TGCTACTGTGTGCTGTGTGCT are obtained. As shown in FIG. 4 and FIG. 5, the amplification sizes of the GS5 marker pair YYP1 and NIP are 258 bp and 262bp respectively, the size of the YYP1 fragment after the AluI enzyme digestion is about 229bp, and the size of the NIP is still 262 bp; the amplification sizes of the GLW7 marker pair YYP1 and NIP are 127bp and 133bp respectively, the size of YYP1 fragment after HinfI enzyme digestion is about 99bp, and the size of NIP fragment is still 133 bp; YYP1 is less than NIP by 9bp on the whole at a GLW7 sequence, we also design an amplification Primer containing two deletion sequences to generate a molecular marker for directly identifying deletion variants (figure 5), the Primer design method is to directly copy a deletion sequence containing region and 100bp at the upstream and downstream thereof to Premier Primer 5.0 software, search the optimal forward Primer and reverse Primer at the upstream and downstream respectively, finally obtain a forward Primer sequence TCCCTTTCAACCTTTTCCA and a reverse Primer sequence CTCGAGCTCGAGCTCATG, the size of the pair of Primer amplification YYP1 fragments is 98bp, and the size of the amplified NIP fragment is 107 bp; to this end, we succeeded in designing three pairs of markers for identifying SSR sequence variations of GS5 and GLW 7.

Example 2:

in order to verify the PCR amplification and enzyme digestion identification effects of three pairs of markers, eight varieties were selected and analyzed, which are YYP1, Nipponbare (NIP), Zhenshan 97(ZS97), Daohuaxiang (DHX), Kasalath (Kasa), WY3, Baoyao (BDL) and Katy, respectively.

Accelerating germination and sprouting are carried out on the variety, leaves are sampled after two weeks, the leaves are placed into a 2ml centrifugal tube with steel balls, DNA is extracted by adopting a TPS small-amount extraction method, and the specific steps are as follows:

1. the leaves are vibrated and crushed by a ball mill, 500ul TPS buffer solution is added, and the mixture is placed for 45 minutes at 65 ℃;

2. centrifuging at 12000 speed for 10 min, sucking 300ul of supernatant into a new centrifuge tube, adding isopropanol with the same volume, and standing at room temperature for 45 min;

3. centrifuging at 12000 speed for 10 min to obtain DNA precipitate, pouring out supernatant, and adding 500ul 75% ethanol;

4. 7500 centrifuging for 5 min, removing supernatant, draining, standing at room temperature for 30 min, and adding 100ul double distilled water to obtain DNA solution.

Then, the extracted DNA is taken as a template to respectively carry out PCR amplification on three pairs of primers, and the reaction system is 20ul and comprises 2ul of DNA template, 2ul of PCR buffer (Beijing Dingguo organism), 0.5ul of forward primer, 0.5ul of reverse primer, 0.3ul of Taq polymerase (Beijing Dingguo organism) and 14.7ul of double distilled water. The PCR reaction conditions are as follows: denaturation at 94 ℃ for 3 min, followed by 35 cycles of "denaturation at 94 ℃ for 20 sec-55 ℃ annealing for 30 sec-72 ℃ extension for 20 sec", and final extension at 72 ℃ for 5 min to complete the reaction. Then, the PCR product of the enzyme digestion identification mark is subjected to enzyme digestion, wherein the reaction system comprises 20ul of the PCR product, 0.5ul of endonuclease (the endonuclease is purchased from Thermo Scientific company), 2ul of corresponding buffer, 7.5ul of double distilled water and enzyme digestion for 1 hour at 37 ℃.

After PCR and enzyme digestion are finished, 10ul of PCR is absorbed for agarose gel electrophoresis with the concentration of 3 percent, two enzyme digestion identification marks are photographed after electrophoresis is carried out for 20 minutes, the electrophoresis is carried out for 30 minutes for GLW7 non-enzyme digestion identification marks to ensure that two types of bands are distinguished, then three pairs of marks are compared for PCR amplification of different varieties and band type differences after enzyme digestion, as shown in figures 6, 7 and 8, clear bands can be amplified in eight varieties by the three marks, wherein the GS5 marks have the amplification size of 262bp of six varieties of bands in 8 varieties, cannot be subjected to enzyme digestion and corresponds to wild type variation, only PCR products of YYP1 and Zhenshan 97 varieties can be subjected to enzyme digestion, the band size is about 229bp, and corresponds to mutant type variation and is consistent with expectation (figure 6); the result of the GLW7 non-enzyme digestion identification marker shows (figure 7) that three varieties of NIP, DHX and WY3 have upward bands corresponding to 107bp in size, and the rest five varieties have downward bands corresponding to 98bp in size, although the marker can distinguish 9bp insertion deletion variation, the band difference is too small, and the identification is relatively difficult; the enzyme cutting marker identification result of GLW7 is completely consistent with a non-enzyme cutting marker, the sizes of three variety bands of NIP, DHX and WY3 are 133bp, the sizes of the rest five variety bands are 99bp, but the identification result is obvious and stronger in practicability compared with the non-enzyme cutting marker, and the identification result is proved that the SSR markers with smaller differences can be effectively converted into the markers based on enzyme cutting, so that the identification effect is improved.

Example 3:

some varieties may have variation in primer binding sites, thereby affecting amplification effects, and we further define the identification effects of two gene recognition primers on a large number of varieties. 280 varieties distributed all over the world are collected and planted in the field, and leaves are taken at the seedling stage to extract DNA, and the extraction method is the same as the above. Then GS5 and GLW7 enzyme digestion identification markers are respectively used for PCR amplification and enzyme digestion identification, and finally the variation types of different varieties are obtained. The GS5 marker identification result shows (Table 1), 280 varieties all amplify bands, wherein 52 varieties comprise 'AAAC' deletion broad grain weight grain type variation, 224 varieties are wild types, 3 varieties are heterozygotes, and the marker is proved to be applicable to genotype identification of hybrid rice and F2 segregation populations; the GLW7 marker identification result shows (Table 2), bands are amplified from 279 varieties, wherein 204 varieties comprise 'TCCACT' deletion long grain weight grain type variation, 73 varieties are wild types, and 3 varieties are heterozygotes, so that the marker can be also used for genotype identification of hybrid rice and F2 segregation population; the results show that the two functional markers show good amplification effect in identifying a large number of varieties, almost no phenomenon that the amplification cannot be carried out occurs, and the method has wide applicability. The variety can be further divided into six subspecies, namely fragrant rice (AROMATIC), autumn rice (AUS), indica rice (IND), temperate japonica rice (TEJ), tropical japonica rice (TRJ) and mixed group (ADMIX, the genome of which comprises different subspecies blood margins), the wide-grain heavy-grain mutation of GS5 is found in the subspecies mainly concentrated in indica rice, but is distributed in other several subspecies rarely or not (table 1), and based on the mutation, the wide-grain heavy-grain rice variety can be effectively used for improving the japonica rice variety planted in a large area in China and increasing the yield; GLW7 long-grain heavy-grain variation is distributed in a large amount in several subspecies except for temperate japonica rice (Table 2), so the variation type is also mainly suitable for improving the main japonica rice variety in China and is used for simultaneously improving the rice yield and the appearance quality of rice. The results show that the two grain type mutation types are not widely used in breeding practice of temperate japonica rice cultivars and have great breeding application potential.

TABLE 1 distribution frequency of two types of variation of GS5 Gene in 280 varieties

TABLE 2 distribution frequency of two types of variation of GLW7 Gene in 280 varieties

Further, the analysis and comparison of the grain type distinguishing effect of the two markers are carried out, and the genotype-phenotype association analysis is carried out only in subspecies by considering the group structure influence of different subspecies. The performance of grain type and grain weight characters is easily influenced by the environment, so that character measurement is carried out in two completely different planting environments of Shanghai and Hainan, after the plants are completely mature, single plant harvesting and drying are carried out on different varieties, then ten thousand-deep SC-G automatic seed test analysis and thousand-grain weight instrument analysis are adopted, grain length, grain width, length-width ratio and thousand-grain weight data are automatically obtained, and then the character difference of two mutant types in subspecies is compared. The specific method comprises the steps of grouping the phenotype data according to two genotypes of each marker, calculating the average value of each group, and carrying out Student's T-test difference detection, wherein if P is less than 0.05, the marker is considered to be obviously related to the corresponding grain type character. Two mutation types of the GS5 gene show relatively balanced distribution in an IND subspecies (Table 1), the number of wild types and the number of mutant types are respectively 17 and 41, and statistical analysis shows that the deletion type mutation has obvious effects on the grain width and the length-width ratio under two planting conditions (Table 3), namely increasing the grain width but reducing the length-width ratio, increasing the thousand-grain weight but not reaching the significance level, and in addition, the deletion type mutation can reduce the grain length under the Shanghai planting condition; however, two mutation types of the GLW7 gene showed balanced distribution in the submix subspecies (table 2), the number of wild types and mutants were 19 and 30, respectively, statistical analysis showed that the deletion-type variation had a tendency to increase grain length and aspect ratio under both planting conditions, but did not reach significant levels (table 4), which indicates that the grain length promoting effect of GLW7 was influenced by the genetic background of the variety or population, and selection of this site could not be accurately screened by phenotypic observation and had to be performed using molecular marker assisted selection technology.

TABLE 3 grain type discrimination Effect of two types of variation of GS5 Gene in IND subspecies

TABLE 4 granulometric differentiation Effect of two types of variation of the GLW7 Gene in the ADMIX subspecies

By combining the experimental results, the invention provides a method for effectively converting SSR markers with smaller differences into molecular markers identified based on enzyme digestion, thereby realizing accurate identification of different variation types by agarose gel electrophoresis. Based on the method, the functional SSR mutation of GS5 and GLW7 is subjected to enzyme digestion identification marker conversion, a good identification effect is obtained, the price of endonucleases used by the two markers is extremely low, is about one tenth of the identification cost of the Sanger sequencing method, and the method has high popularization and utilization values.

Sequence listing

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Claims (4)

1. A primer for identifying GS5 gene and GLW7 gene types is characterized by comprising the following primers:

(1) primers for identifying the type of the GS5 gene: the nucleotide sequence is shown as SEQ ID No.: 1-2;

(2) primers for identifying the type of GLW7 gene: the nucleotide sequence is shown as SEQ ID No.: 3-4 or SEQ ID No.: 5 to 6.

2. An agent for identifying the GS5 gene or GLW7 gene type, comprising SEQ ID No.: 1 to 6.

3. A method for identifying the types of GS5 gene and GLW7 gene, which comprises the following steps:

(1) extracting the genome DNA of the rice to be detected;

(2) taking the DNA obtained in the step (1) as a template and SEQ ID No.: 1-2 or SEQ ID No.: 3-4 or SEQ ID No.: 5-6 as primers for PCR amplification;

(3) SEQ ID No.: 1-2, performing enzyme digestion treatment on a product obtained by amplification of a primer shown in SEQ ID No.: 3-4, performing enzyme digestion treatment on a product obtained by amplification of the primers shown in the specification by using HinfI endonuclease; and then, the enzyme digestion product is compared with SEQ ID No.: carrying out agarose gel electrophoresis on the PCR products of the primers shown in 5-6;

(4) if SEQ ID No.: 1-2, performing enzyme digestion on a PCR product of the primer shown in the specification by using an AluI endonuclease to obtain a fragment with the size of 229bpbp, wherein the genotype of GS5 in the rice is AAAC; if the size of the fragment obtained after the digestion by the AluI endonuclease is 262bp, the GS5 genotype in the rice is AAACAAC;

if SEQ ID No.: 3-4, performing enzyme digestion on a PCR product of the primers shown in the specification by using HinfI endonuclease to obtain 99bp, wherein the GLW7 genotype in the rice is CTTCCACTTCCA, and if the size of a fragment obtained after the enzyme digestion by using the HinfI endonuclease is 133bp, the GLW7 genotype in the rice is CTTCCACTTCCACTTCCA;

if SEQ ID No.: 5-6, if the size of the obtained fragment obtained by PCR is 98bp, the GLW7 genotype in the rice is CTTCCACTTCCA, and if the size of the obtained fragment is 107bp, the GLW7 genotype in the rice is CTTCCACTTCCACTTCCA.

4. The method for identifying the GS5 gene and the GLW7 gene types as claimed in claim 3, wherein in the step (2), the PCR amplification conditions are as follows: denaturation at 94 ℃ for 3 min, followed by 35 cycles of "denaturation at 94 ℃ for 20 sec-55 ℃ annealing for 30 sec-72 ℃ extension for 20 sec", and finally extension at 72 ℃ for 5 min.

Images