CN109486829B - Rice semi-dwarf gene sd1 allele and identification method thereof - Google Patents

Abstract

The invention discloses a rice semi-dwarf gene sd1 allele and an identification method thereof, which is formed by C-G mutation of 146 th base on the 3 rd exon of the rice semi-dwarf gene sd1 and is a gene for regulating and controlling the plant height property of rice. The functional molecular marker of the invention is based on PCR technology, can distinguish and identify the dwarf type of rice seed resources, is not influenced by environment and human factors, can be used for rice dwarf breeding and improving the rice plant type, and has wide application prospect in the field of rice breeding.

Description

Rice semi-dwarf gene sd1 allele and identification method thereof

Technical Field

The invention belongs to the technical field of rice breeding, and relates to a rice semi-dwarf gene sd1 allele and an identification method thereof.

Background

Rice is a main energy crop of human beings, and with the continuous increase of the global population and the rapid decrease of the cultivated land area, the food safety is becoming a serious global problem. The plant height is one of important agronomic traits of rice, the application of the semi-dwarf gene sd1 greatly improves the yield per unit of rice, solves the lodging problem of high-fertile rice varieties of the long-stalked rice, and plays a key role in the 'green revolution'. Short stalk varieties bred by taking short young plants, short foot south Tech or low foot Wujian tip as short sources have made great contribution to world rice production. However, the dwarf gene of indica rice varieties is too single in source at present, and is mainly concentrated in dwarf southern mutation types. Therefore, the problem to be solved is how to use the existing variety as a parent in breeding, and the breeding of new varieties is not limited by a single dwarf source.

Disclosure of Invention

The invention aims to solve the problem of providing the allele of the rice semi-dwarf gene sd1 and an identification method thereof, wherein the allele is selected from indica rice variety 9311, and the allele is used for rice cultivation to reduce the plant height of rice, especially the plant height of indica rice, and can further modify the plant type of japonica rice.

The invention is realized by the following technical scheme:

a rice semi-dwarf gene sd1 allele is an allele formed by C-G mutation of 146 th base on the 3 rd exon of rice semi-dwarf gene sd1 and is a gene for regulating and controlling the plant height property of rice.

The nucleotide sequence of exon 3 of the allele is shown in SEQ ID No. 1.

The C-G mutation of the allele causes the GA20 oxidase coding to terminate early, and causes the protein function to be deleted, thereby regulating and controlling the rice plant height trait.

The molecular marker is used for detecting whether the 146 th base on the 3 rd exon of the rice semi-dwarf gene sd1 has C-G mutation or not, and comprises primer pairs shown in SEQ ID NO.2 and SEQ ID NO. 3;

SEQ ID NO.2:CGCGGTTTGTCCTTGTCG；

SEQ ID NO.3:AAATCGGCTTCTGTTCGTTCC。

the invention also provides an identification method of the rice semi-dwarf gene sd1 allele, which comprises the following operations:

performing PCR amplification by taking the extracted total DNA of the rice as a template and taking a primer pair shown in SEQ ID NO.2 and SEQ ID NO.3 as a primer, performing Ncil enzyme digestion on an amplification product, performing electrophoresis detection, and identifying alleles according to the electrophoresis detection result:

if the detection result only has a 486bp strip, the 146 th base is shown to generate C-G mutation;

if the detection result has two bands of 184bp and 302bp, the C-G mutation does not occur at the 146 th base.

The PCR reaction system during PCR amplification is as follows:

DNA template 1. mu.l, 2.5mM dNTPs 5. mu.l, 2 XKOD buffer 12.5. mu.l, 10. mu.M forward primer 1. mu.l, 10. mu.M reverse primer 1. mu.l, 1U/. mu.l KOD enzyme 0.5. mu.l, H₂O 4μl；

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 60sec, annealing at 65 ℃ for 60sec, and extension at 72 ℃ for 120sec for 34 cycles; finally, extension is carried out for 10min at 72 ℃.

The rice semi-dwarf gene sd1 allele is applied to cultivation of dwarf rice varieties or strains.

Further, the rice semi-dwarf gene sd1 of the rice variety or strain needing to be improved is identified by the molecular markers shown in SEQ ID NO.2 and SEQ ID NO.3, and if the 146 th base on the 3 rd exon does not generate C-G mutation, the rice semi-dwarf gene sd1 of the rice 9311 strain is introduced into the breeding strain by a hybrid backcross method, so that the plant height character of the new variety or strain is improved.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention provides a novel rice semi-dwarf gene sd1, and BC is screened out by comparing characters₁F₄Then to BC₁F₄The plant height character regulation gene is analyzed, and finally the candidate gene is positioned in a range of 189kb by combining with the phenotype verification of offspring populations, genome annotation information shows that the semi-dwarf gene sd1 is positioned in the interval, and the sd1 gene of Zhe japonica 22 and 9311 is sequenced, and sequencing results show that the C-G mutation of 146 th base positioned on the 3 rd exon leads the encoding of GA20 oxidase in 9311 to be terminated in advance, so that protein function is lost, which is consistent with the plant dwarfing effect of 9311 locus, and therefore, the sd1 is determined to be the candidate gene for regulating and controlling the plant height character of 9311. And its BC₁F₄The sd1 gene in the long-stalk strain is Zhe japonica 22 fragment, so the long-stalk strain shows the character of long stalk.

According to the molecular marker and the identification method of the rice semi-dwarf gene sd1, a new rice dwarf source can be identified by detecting the molecular marker linked with the sd1 allele, and the molecular marker and the identification method are used for rice dwarf breeding and rice plant type improvement. The sd1 locus of 9311 can reduce the plant height of rice, especially the plant height of indica rice, and can further transform japonica rice plant type to adapt to the cultivation conditions of specific regions, thereby improving the fertilizer resistance and lodging resistance of rice and finally increasing the yield.

Drawings

FIG. 1 shows BC₁F₄A comparison of the high culm lines in the population with 9311;

FIG. 2 shows the results of genetic background screening;

FIG. 3 shows the high contribution rate of 9311 site, Zhe Jing 22 site and heterozygous site to the plant;

FIG. 4 is a map of the location of a new allele of rice semi-dwarf gene sd 1;

FIG. 5 is a sequence alignment of the half dwarf alleles;

FIG. 6 shows the result of DNA detection of 48 indica rice varieties by specific molecular marker Sd1SNP, and arrow indicates 9311 dwarf type.

Detailed Description

The invention will be further described with reference to the following drawings and examples, which are intended to be illustrative and not limiting.

Example 1: novel allele of semi-dwarf gene sd1 in indica rice variety 9311

The invention uses round-grained nonglutinous rice Zhe-grained nonglutinous rice 22 as female parent and indica-grained nonglutinous rice 9311 as male parent for hybridization, and F obtained by hybridization₁Backcrossing the generations with 9311, and then continuously selfing for 4 generations to obtain BC₁F₄. At BC₁F₄Strains with the strain height remarkably higher than 9311 are screened in the population, and the phenotype observation shows BC₁F₄The plant height of the high-stalk line in the population is obviously higher than 9311 (shown as BC in figure 1)₁F₄The plant height of the high-stalk lines in the generation obviously exceeds 9311), further statistical analysis finds that the increase of the plant height of the high-stalk lines is caused by that each section is longer than 9311, but the ear length is not obviously changed, which indicates that Zhe japonica 22 and 9311 contain independent plant height genes.

Thus, for its BC₁F₄Genetic background screening is carried out on 1 extreme high-stalk plant in the high-stalk line by using molecular markers distributed throughout the rice genome (the genetic background screening method is shown in the specification: Mapping of qGL7-2, a grain length QTL onchromosome 7 of rice, J.Genet.genomics 37(2010)523-531), and the result shows that the BC is subjected to the genetic background screening₁F₄Most of the genome of the strain is replaced by the 9311 fragment, only 7 genome regions (marked by black fragments in figure 2) contain homozygous Zhe japonica 22 fragments, and corresponding progeny plants are high and stable and are all long-stalked plants, so that the replacement region of the Zhe japonica 22 fragment is presumed to contain plant height control genes. Further, mapping populations were identified using the end-arm markers A1 and A2 for chromosome 1Performing linkage analysis, and finding that the region is linked with the high stalk phenotype (figure 2); statistical analysis shows that the contribution rate of the long-arm end linked fragment (figure 2) of the No.1 chromosome to the phenotype reaches 44.5 percent (figure 3), and proves that the long-arm end linked fragment contains a plant high major effective site and influences the high stalk phenotype. Through comparing the locus sequence of Nipponbare and 9311, designing a new linkage marker identification positioning population, finally finding 4 key crossover individuals L30-11-5, L30-14-5, L30-16-1 and L31-12-2, positioning the candidate gene to the range of 189kb according to the genotype of the recombination position of the 4 key crossover individuals in combination with the phenotype verification of the progeny population (as shown in figure 4, L30-11-5 exchanges between A147.5-1 and A149.1-1 markers, L30-14-5, L30-16-1 and L31-12-2 exchanges between A149.1-1 and A150.7-1 markers, wherein the phenotype of L30-11-5 and L31-12-2 is high stalk, the black region is a high stalk regulatory region, i.e., between a147.5-1 and a 150.7-1), the genome annotation information shows that the semidwarf gene sd1 is located in the interval (fig. 4, the left side represents the names of four crossover individuals, the upper part is the name of a molecular marker, and the right side number is the number of progeny of the crossover individual).

Sequencing the sd1 genes of Zhe Jing 22 and 9311, wherein the two genes have SNP difference on 3 exons, and the 146 th base on the 3 rd exon has C-G mutation to cause the early termination of GA20 oxidase coding in 9311, thereby causing protein function loss, which is consistent with the plant dwarfing effect of the 9311 locus, thus determining sd1 as a candidate gene for regulating the 9311 plant height character. And its BC₁F₄The sd1 gene in the long-stalk strain is Zhe japonica 22 fragment, so the long-stalk strain shows the character of long stalk.

Sequencing results show that the nucleotide sequence of the 3 rd exon of the 9311 rice semi-dwarf gene sd1 gene is shown in SEQ ID No.1 (underline shows that C-G mutation occurs):

CGCTGTCGAACGGGAGGTATAAGAGCTGCCTGCACAGGGCGGTGGTGAACCAGCGGCGGGAGCGGCGGTCGCTGGCGTTCTTCCTGTGCCCGCGGGAGGACAGGGTGGTGCGGCCGCCGCCGAGCGCCGCCACGCCGCGGCACTAGCCGGACTTCACCTGGGCCGACCTCATGCGCTTCACGCAGCGCCACTACCGCGCCGACACCCGCACGCTCGACGCCTTCACGCGCTGGCTCGCGCCGCCGGCCGCCGACGCCGCCGCGACGGCGCAGGTCGAGGCGGCCAGCTGA。

example 2: functional molecular marker of sd1 allele and primer design and detection thereof

The mutation type of 146-bit base on the 3 rd exon can generate a dwarf phenotype, so that a molecular marker is designed according to the mutation characteristics of the mutation type and is used for detecting or identifying the allele.

Primer Sd1SNP amplification comprises 9311 early termination site, wherein the SNP of the primer Sd1SNP amplification causes deletion of Ncil enzyme cutting site, so that an amplification product cannot be cut by enzyme, and the primer Sd1SNP sequence is shown as SEQ ID NO.2 and SEQ ID NO. 3; the rice seed resource DNA of the dwarf southwestern and 9311 dwarf mutant types is amplified by the primer group to obtain an amplification product, and then enzyme digestion electrophoresis verification is carried out.

The specific operation comprises the following steps:

(1) taking fresh rice leaves about 5cm, putting the leaves into a centrifugal tube of 1.5ml, adding a proper amount of liquid nitrogen, and grinding the leaves into powder;

(2) add 500. mu.l of preheated CTAB extract to a 1.5ml centrifuge tube. Incubating at 65 deg.C for 3h (1h or more), cooling to room temperature, extracting with chloroform, turning upside down, standing at room temperature for 10min, and centrifuging at 13000rpm at room temperature for 10 min;

(3) sucking the supernatant into another sterilized 1.5ml centrifuge tube, and not sucking the lower chloroform;

(4) precipitating with isopropanol or anhydrous ethanol of the same volume, mixing, standing at room temperature for 10min, centrifuging at 13,000rpm at room temperature for 10min, collecting precipitate, and washing with 70% ethanol;

(5) centrifuging the washed precipitate instantly, removing 70% ethanol, and naturally drying

(6) After the precipitate was air-dried, 50. mu.l of sterilized TE buffer was added to the centrifuge tube;

(7) storing at-20 deg.C for use.

The PCR reaction system is as follows:

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 60sec, annealing at 65 ℃ for 60sec, and extension at 72 ℃ for 120sec for 34 cycles; finally, extension is carried out for 10min at 72 ℃.

Example 3: application of functional molecular marker of sd1 allele in identification of different rice dwarf types

The genomic DNA of 48 indica rice containing different dwarf types is adopted and used as a template, and the molecular marker Sd1SNP is used for PCR amplification, enzyme digestion and electrophoresis detection, and the electrophoresis result is shown in figure 6. FIG. 6 shows the result of DNA detection of 48 indica rice varieties by specific molecular marker Sd1SNP, which shows that only Sd1SNP amplification products of 6 varieties cannot be digested by Ncil endonuclease, and are 9311 dwarf types, and Sd1SNP amplification products of the other varieties can be digested by Ncil endonuclease, so that amplification bands become smaller, and thus, 48 indica rice varieties have fewer varieties containing 9311SNP mutation sites; it is stated that the 9311 locus has not been widely applied to breeding lines.

The result verifies the function of the SNP primer in variety identification, and rice resources containing the dwarf southbound mutation type and the 9311 mutation type can be identified and distinguished, so that the new allele of sd1 (the SNP type of the sd1 locus is identified) can be selected and identified by using the marker in an auxiliary way, and the plant height character is improved (firstly, the sd1 locus type of a variety or a strain needing to be improved is identified through a molecular marker, and if the sd1 locus of 9311 does not exist, the sd1 locus of 9311 can be introduced into a breeding strain through a traditional hybridization backcross method, so that the aim of improving the plant height is achieved).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> rice semi-dwarf gene sd1 allele and identification method thereof

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<213> indica rice variety 9311(Oryza sativa L. ssp. indica)

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cgagcgccgc cacgccgcgg cactagccgg acttcacctg ggccgacctc atgcgcttca 180

cgcagcgcca ctaccgcgcc gacacccgca cgctcgacgc cttcacgcgc tggctcgcgc 240

cgccggccgc cgacgccgcc gcgacggcgc aggtcgaggc ggccagctga 290

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Claims (6)

1. A rice half-dwarf gene sd1 allele is characterized in that the allele is formed by C-G mutation of 146 th base on the 3 rd exon of the rice half-dwarf gene sd1 and is a gene for regulating and controlling the plant height property of rice.

2. The rice semi-dwarf gene sd1 allele according to claim 1, wherein the nucleotide sequence of exon 3 of the allele is shown in SEQ ID No. 1.

3. The method for identifying the allele of the rice semi-dwarf gene sd1 as claimed in claim 1, which comprises the following operations:

performing PCR amplification by taking the extracted total DNA of the rice as a template and taking a primer pair shown in SEQ ID NO.2 and SEQ ID NO.3 as a primer, performing Ncil enzyme digestion on an amplification product, performing electrophoresis detection, and identifying alleles according to the electrophoresis detection result:

if the detection result only has a 486bp strip, the 146 th base is shown to generate C-G mutation;

if the detection result has two bands of 184bp and 302bp, the C-G mutation does not occur at the 146 th base.

4. The method for identifying the allele of the rice semi-dwarf gene sd1 according to claim 3, wherein a PCR reaction system during PCR amplification is as follows:

DNA template 1. mu.l, 2.5mM dNTPs 5. mu.l, 2 XKOD buffer 12.5. mu.l, 10. mu.M forward primer 1. mu.l, 10. mu.M reverse primer 1. mu.l, 1U/. mu.l KOD enzyme 0.5. mu.l, H₂O 4μl；

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 60sec, annealing at 65 ℃ for 60sec, and extension at 72 ℃ for 120sec for 34 cycles; finally, extension is carried out for 10min at 72 ℃.

5. The application of the rice semi-dwarf gene sd1 allele of claim 1 in breeding dwarf rice varieties or lines.

6. The use of claim 5, wherein the semi-dwarf gene sd1 of rice of the rice variety or line to be improved is identified, and if the 146 th base on the 3 rd exon does not generate C-G mutation, the semi-dwarf gene sd1 of rice 9311 line of rice is introduced into a breeding line by a cross backcross method to improve the plant height character of a new variety or line.

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