Detection primer for identifying polymorphism of rice mesocotyl length regulation gene PAO5 and application thereof

Abstract

The invention belongs to the field of genetics and plant breeding, and particularly relates to a detection primer for identifying polymorphism of a rice mesocotyl length regulation gene PAO5 and application thereof, and the detection primer in the technical scheme is shown as SEQ ID NO: 1-2. By adopting the detection primers as shown in SEQ ID NO: 1-2, the polymorphism and mesocotyl length type of the rice mesocotyl length regulation gene PAO5 can be rapidly and accurately identified, the polymorphism and mesocotyl length type of the mesocotyl length regulation gene PAO5 in parents, hybridized, backcrossed and selfing offspring can be rapidly identified, rapid and effective breeding of new long mesocotyl rice products is realized, and the breeding efficiency is improved. The important application value is realized on rice cultivation and screening.

Description

Technical Field

[0001] The present invention belongs to the field of genetics and plant breeding, and specifically relates to a detection primer for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5 and its application. Specifically, the present invention screens for molecular markers of rice gene OsPAO5 alleles with relatively long mesocotyl length, and primers for amplifying or detecting molecular markers and a detection method, which are used to assist conventional breeding in quickly, simply and accurately breeding rice with longer mesocotyl length. Background Art

[0002] Rice (Oryza sativa) is the staple food of most people in China, and rice production plays a crucial role in the country. With the development of the times, direct seeding of rice has gradually replaced manual sowing and become a widely adopted technology. Direct seeding of rice is an efficient and rapid sowing method that can effectively address labor shortages. However, direct seeding of rice often results in low seedling emergence rates, significantly reducing rice yields. Rice seedling emergence rates are affected by multiple factors, and rice mesocotyl elongation is the primary driving force for rapid rice seedling emergence. Increasing rice mesocotyl length can effectively address this low seedling emergence rate during direct seeding.

[0003] OsPAO5 expression in rice seedlings is enhanced in the presence of light and repressed in darkness. Nucleotide polymorphism analysis revealed that a single nucleotide polymorphism (SNP) located 578 bp upstream of the OsPAO5 start codon (PAO5 578G / A) alters its expression during rice mesocotyl domestication. The OsPAO5 578G gene, which is associated with long mesocotyls, is primarily found in wild rice, most Australian accessions, and some japonica rice accessions. Compared to the OsPAO5 578A rice genotype, the OsPAO5 578G rice genotype exhibits relatively longer mesocotyls, releases less hydrogen peroxide, and synthesizes more ethylene. The mutant exhibits higher and more uniform seedling emergence, suggesting its potential application in direct seeding.

[0004] Rice varieties with shorter mesocotyls have a low seedling emergence rate, necessitating hybridization with other rice varieties with longer mesocotyls. These varieties are then selected through backcrossing or self-pollination to obtain varieties with longer mesocotyls. In the past, rice breeders primarily determined whether rice varieties with longer mesocotyls were long by measuring the length of their mesocotyls individually. However, this method was time-consuming and labor-intensive. With the development of various molecular markers, molecular markers for many important agronomic and quality traits have been developed for use in rice breeding.

[0005] Therefore, it is very important to provide a molecular marker, corresponding primers and detection methods to assist in the breeding and screening of varieties with relatively longer mesocotyl lengths, thereby increasing rice yield. Summary of the Invention

[0006] The invention provides a detection primer for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5.

[0007] The present invention also provides an application of primers for detecting polymorphism of rice mesocotyl length regulating gene PAO5.

[0008] The technical solution of the present invention is a detection primer for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5, as shown below:

[0009] OsPAO5-578SNP-F:5'-ACCCCTCTCATACCCAAACC-3'(SEQ ID NO:1)

[0010] OsPAO5-578SNP-R:5'-GCAACACTCCAACACACAGG-3'(SEQ ID NO:2)

[0011] The detection primers for identifying the polymorphism of the rice mesocotyl length regulatory gene PAO5 provided by the present invention can be prepared into products such as reagents, kits, test strips, gene chips, devices or equipment, which are used to detect the type of the rice mesocotyl length regulatory gene PAO5 allele, identify the rice mesocotyl length, cultivate or screen rice with different mesocotyl lengths, prepare products for detecting the type of the rice mesocotyl length regulatory gene PAO5 allele, prepare products for identifying the rice mesocotyl length, and prepare products for cultivating or screening rice.

[0012] The present invention provides the following product, which contains the detection primers for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5 provided above:

[0013] (1) Products for detecting the allele type of rice mesocotyl length regulating gene PAO5;

[0014] (2) Products for identifying the length of rice mesocotyl;

[0015] (3) Cultivate or screen products for rice.

[0016] The products include but are not limited to reagents, kits, test strips, gene chips, devices or equipment, etc.

[0017] The present invention provides a method for detecting the allele type of a rice mesocotyl length regulating gene PAO5, comprising the following steps:

[0018] (1) Using the obtained rice genomic DNA as a template, PCR amplification is performed using the detection primers for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5 provided by the present invention;

[0019] (2) The PCR amplification product was digested with restriction endonuclease BaeGI;

[0020] (3) The enzyme digestion products were subjected to electrophoresis and the following allele type identification was performed:

[0021] a. The molecular weight of the enzyme digestion product is 507 bp, and the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon is homozygous for A;

[0022] b. The molecular weights of the enzyme digestion products were 328 bp and 179 bp, indicating that the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon was homozygous for G;

[0023] c. The molecular weights of the enzyme digestion products were 507 bp, 328 bp, and 179 bp, and the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon was an A / G heterozygous.

[0024] In step (1), the PCR amplification program is as follows: pre-denaturation at 93-95°C for 4-7 min; denaturation at 93-95°C for 30-50 s, annealing at 51-55°C for 30-50 s, and extension at 70-73°C for 25-45 s, for a total of 30-36 cycles; and a final extension at 70-73°C for 5-12 min. More preferably, the PCR amplification program is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 45 s, for a total of 36 cycles; and a final extension of 5 min.

[0025] In step (2), the enzyme digestion reaction was carried out in a 37°C water bath for 3 hours.

[0026] In step (3), 2.5-3.5% agarose gel is used for electrophoresis, preferably 3% agarose gel.

[0027] The present invention provides a method for identifying rice mesocotyl length, comprising the steps of: detecting the allele type of the rice mesocotyl length regulating gene PAO5 at 578 bp upstream of the start codon, wherein rice containing the A homozygous allele corresponds to a relatively short mesocotyl length, and rice containing the G homozygous allele corresponds to a relatively long mesocotyl length.

[0028] The rice varieties containing the G homozygous allele include but are not limited to "kasalath", "Shanghai Normal University No. 18", "Nanjing 46", "Shanghai Normal University No. 19", "Jiahua No. 1", "Xiushui 123", "Nihonbare", "Yinxiang 38", "Jiahe 218", "Xiangruan Early Rice", "Xiushui 134", "Jia 58", "Puxiangjing No. 1", etc.

[0029] The rice varieties containing the A homozygous allele include but are not limited to "9311", "Zhongxiang No. 1", "Zhongzao 35", "Guangluai No. 4" and the like.

[0030] The present invention provides a method for cultivating or screening rice, comprising the following steps: using the detection primers for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5 provided by the present invention to detect the allele type of a rice sample, determining the rice sample suitable as a parent, and / or the allele type of the progeny in hybridization, backcrossing, and / or selfing, and then cultivating or screening the rice.

[0031] For example, when the detection primers for identifying the polymorphism of the rice mesocotyl length regulatory gene PAO5 provided above are used for detection, it is determined that the rice samples in which the allele types of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon are A homozygote and G homozygote respectively are used as parents, the allele type of the first generation of the hybrid offspring is A / G heterozygote. Then, the A / G heterozygote first generation offspring is used as the parent for self-pollination, and three different allele types of second generation offspring are obtained, namely A homozygote, G homozygote and A / G heterozygote.

[0032] The beneficial effects of the present invention are:

[0033] The present invention provides an OsPAO5 (BaeGI) molecular marker, its detection primers, and a detection method that are simple to operate and easy to interpret. The present invention's method for detecting the OsPAO5 578A allele and the OsPAO5 578G allele allows for easy resolution using only a 3% agarose gel. This method not only allows for rapid and convenient identification of rice varieties with relatively long and short mesocotyl lengths, but also allows for rapid identification of the OsPAO5 578A and OsPAO5 578G genotypes in hybrid, backcross, and self-cross progenies. This method has significant application value in the cultivation and screening of rice varieties with relatively long mesocotyls. The present invention can directly detect the OsPAO5 allele, a gene regulating rice mesocotyl length, laying the foundation for the rapid and efficient selection of new rice varieties with long mesocotyls. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is an identification diagram of the OsPAO5 genotype of rice with a relatively long mesocotyl length, OsPAO5 578G, and OsPAO5 genotype of rice with a relatively short mesocotyl length, as well as the F1 plants of their hybridization and the F2 plants produced by self-pollination in Example 1. Wherein M is a 100 bp type standard molecular weight DNA; lane 1 corresponds to the enzyme digestion product after PCR amplification of the "9311" plant with relatively short mesocotyl length, containing a band with a molecular weight of approximately 507 bp; lane 2 corresponds to the enzyme digestion product after PCR amplification of the rice "kasalath" plant with relatively long mesocotyl length, containing two bands with molecular weights of approximately 328 bp and 179 bp; lane 3 is the enzyme digestion product after PCR amplification of the F1 plant hybridized with "9311" and "kasalath", containing three bands with molecular weights of approximately 507 bp, 328 bp and 179 bp, respectively; lanes 4-12 are the enzyme digestion products after PCR amplification of the F2 plant produced by self-pollination of the F1 plant, among which lanes 4-6 have a band with a molecular weight of approximately 507 bp, lanes 7-9 have two bands with molecular weights of approximately 328 bp and 179 bp, respectively, and lanes 10-12 have three bands with molecular weights of approximately 507 bp, 328 bp and 179 bp, respectively.

[0035] Figure 2This is a diagram illustrating the OsPAO5 gene identification in Example 2, showing the OsPAO5 578A rice genotype with relatively short mesocotyl length and the OsPAO5 578G rice genotype with relatively long mesocotyl length. M represents a 100bp standard molecular weight DNA. Lanes 1 to 3 represent three rice genotypes with relatively long mesocotyl lengths: "Shanghai Normal University 18," "Nanjing 46," and "Shanghai Normal University 19," containing two bands with molecular weights of approximately 328bp and 179bp, respectively. Lane 4 represents one rice genotype with relatively short mesocotyl length: "Zhongxiang 1," containing one band with a molecular weight of 507bp. Lanes 5 to 6 represent two rice genotypes with relatively long mesocotyl lengths: "Jiahua 1" and "Xiushui 123," containing two bands with molecular weights of approximately 328bp and 179bp, respectively. Lane 7 represents one rice genotype with relatively short mesocotyl length: "Zhongzao 35." , containing a band with a molecular weight of 507bp; lanes 8 to 12 represent five rice genotypes corresponding to relatively long mesocotyl lengths: "Nipponbare", "Yinxiang 38", "Jiahe 218", "Xiangruan Zaodao", and "Xiushui 134", containing two bands with molecular weights of approximately 328bp and 179bp; lane 13 represents one rice genotype corresponding to relatively short mesocotyl length: "Guanglu Ai 4", containing a band with a molecular weight of 507bp; lanes 14 to 15 represent two rice genotypes corresponding to relatively long mesocotyl lengths: "Jia 58" and "Puxiangjing 1", containing two bands with molecular weights of approximately 328bp and 179bp. DETAILED DESCRIPTION

[0036] The technical solutions of the present invention are described below with reference to specific embodiments, which are not intended to limit the scope of protection of the present invention.

[0037] Example 1 Establishment and Detection Method of Molecular Markers for Identifying Alleles of the OsPAO5 Gene Controlling Rice Mesocotyl Length

[0038] The rice variety "Kasalath" has a relatively long mesocotyl length, and the rice variety "9311" has a relatively short mesocotyl length.

[0039] Genomic DNA was extracted from two rice varieties, and the sequences of the OsPAO5 gene, which regulates mesocotyl length, were cloned and sequenced. The OsPAO5 gene sequence of the rice variety "Nipponbare" (LOC_Os04g57560), published in GenBank, was also examined. Compared to the OsPAO5 sequence of the rice variety "kasalath," the OsPAO5 of "9311" had a GA mutation 578 bp upstream of the start codon. In the rice variety "kasalath," which has a longer mesocotyl, the OsPAO5 sequence has a "GGGCAC" sequence 576-581 bp upstream of the start codon, which is recognized and cleaved by the restriction endonuclease BaeGI. In contrast, the OsPAO5 sequence of the rice variety "9311," which corresponds to a shorter mesocotyl, has a "GGACAC" sequence 576-581 bp upstream of the start codon, which is not recognized by the restriction endonuclease BaeGI. Thus, a molecular marker was established to identify different alleles of the OsPAO5 gene: OsPAO5(BaeGI).

[0040] A pair of upstream and downstream primers were designed at both ends of the 578bp sequence upstream of the start codon of the rice mesocotyl length gene OsPAO5, and the nucleotide sequences were shown as OsPAO5-578SNP-F and OsPAO5-578SNP-R, respectively.

[0041] OsPAO5-578SNP-F:5'-ACCCCTCTCATACCCAAACC-3'(SEQ ID NO:1)

[0042] OsPAO5-578SNP-R:5'-GCAACACTCCAACACACAGG-3'(SEQ ID NO:2)

[0043] The above primers were used as amplification test using rice genomic total DNA extracted from a short mesocotyl rice variety "9311," a long mesocotyl rice variety "kasalath," and F1 hybrids of a short mesocotyl rice variety "9311" and a long mesocotyl rice variety "kasalath" as materials. The method is as follows:

[0044] (1) Extract rice genomic DNA as a template (CTAB method can be used), and add the above two primers for PCR amplification;

[0045] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension for 45 s, for a total of 36 cycles; and final extension for 5 min.

[0046] (2) PCR products were digested with BaeGI;

[0047] The reaction system was: 2.5 μl of R3.1 Express Enzyme Reaction Buffer (purchased from NEB), 5 μl of PCR product, 1 μl of BaeGI Express Enzyme (purchased from NEB) (10 enzyme units), and 17 μl of sterile water. Reaction conditions were: 37°C water bath for 3 hours;

[0048] (3) The enzyme-digested product was detected by electrophoresis in a 3% agarose gel.

[0049] The F1 plants of the rice genotype "kasalath" with OsPAO5 578G corresponding to a relatively long mesocotyl length were self-pollinated with the rice genotype "9311" with OsPAO5 578A corresponding to a relatively short mesocotyl length to obtain self-pollinated F2 plants. Total DNA was extracted and PCR amplification, enzyme digestion and electrophoresis detection were performed using the same method.

[0050] Figure 1 The following are the rice genotypes (OsPAO5 578G, corresponding to a relatively long mesocotyl length) and the rice genotype (OsPAO5 578A, corresponding to a relatively short mesocotyl length) described in Example 1, as well as their F1 hybrids. M represents a 100-bp standard molecular weight DNA. Lane 1 represents the enzyme digestion product of the "9311" plant, which has a relatively short mesocotyl length, and contains a single band with a molecular weight of approximately 507 bp. Lane 2 represents the enzyme digestion product of the "kasalath" plant, which has a relatively long mesocotyl length, and contains two bands with molecular weights of approximately 328 bp and 179 bp. Lane 3 represents the enzyme digestion product of the F1 hybrid of "9311" and "kasalath," and contains three bands with molecular weights of approximately 507 bp, 328 bp, and 179 bp, respectively.

[0051] The F1 plants of the rice variety "Kasalath" with the OsPAO5 578G genotype, corresponding to relatively long mesocotyl length, were self-pollinated with the rice variety "9311" with the OsPAO5 578A genotype, corresponding to relatively short mesocotyl length. Test results showed that the selfed progeny of both combinations contained plants of three different genotypes. Among them, only two bands of DNA molecular weight, approximately 328 bp and 179 bp, corresponded to the PCR products of plants homozygous for the OsPAO5 578G genotype in the selfed progeny. Three bands of approximately 507 bp, 328 bp, and 179 bp corresponded to the PCR products of plants heterozygous for the OsPAO5 mesocotyl gene in the selfed progeny. A single band of 507 bp corresponded to the PCR product of plants homozygous for the OsPAO5 578A genotype with short mesocotyls in the selfed progeny (e.g., Figure 1 ).

[0052] Example 2 Identification of rice mesocotyl length using the detection method for the molecular marker of the rice mesocotyl length gene OsPAO5 allele established in Example 1

[0053] The method for detecting the molecular marker allele type of the rice mesocotyl length gene OsPAO5 is the same as that in Example 1. The rice samples include: "Shangshida No. 18", "Nanjing 46", "Shangshida No. 19", "Jiahua No. 1", "Xiushui 123", "Nihonbare", "Yinxiang 38", "Jiahe 218", "Xiangruan Zaodao", "Xiushui 134", "Jia 58", "Puxiangjing No. 1", "Zhongxiang No. 1", "Zhongzao 35", "Guangluai No. 4", etc. The results are shown in FIG. Figure 2 The detection method of the rice mesocotyl length gene OsPAO5 allele type molecular marker of the present invention is used to identify the rice mesocotyl length, which can quickly and accurately confirm the mesocotyl length gene OsPAO5 allele type of each rice variety.

[0054] Note: The above examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A detection primer for identifying polymorphism of the rice mesocotyl length regulating gene PAO5, characterized in that: The primers are as follows: OsPAO5-578SNP-F:5'-ACCCCTCTCATACCCAAACC-3'(SEQ ID NO:1) OsPAO5-578SNP-R: 5'-GCAACACTCCAACACACAGG-3' (SEQ ID NO: 2).

2. The detection primer according to claim 1 has at least one of the following applications: (1) Detect the allele type of the rice mesocotyl length regulatory gene PAO5; (2) Identification of rice mesocotyl length; (3) Cultivating or screening rice; (4) Preparing a product for detecting the allele type of the rice mesocotyl length regulating gene PAO5; (5) preparing a product for identifying rice mesocotyl length; (6) Preparation of products for cultivating or screening rice.

3. The use according to claim 2, characterized in that The products include reagents, and / or kits, and / or test strips, and / or gene chips, and / or devices, and / or equipment.

4. At least one of the following products contains the detection primer according to claim 1: (1) Products for detecting the allele type of rice mesocotyl length regulating gene PAO5; (2) Products for identifying the length of rice mesocotyl; (3) Cultivate or screen products for rice.

5. The product according to claim 4, characterized in that The products include reagents, and / or kits, and / or test strips, and / or gene chips, and / or devices, and / or equipment.

6. A method for detecting the allele type of the rice mesocotyl length regulating gene PAO5, characterized in that the steps include: (1) Using the obtained rice genomic DNA as a template, PCR amplification is performed using the detection primers for identifying the polymorphism of the rice mesocotyl length regulating gene PAO5 according to claim 1; (2) The PCR amplification product was digested with restriction endonuclease BaeGI; (3) The enzyme digestion products were subjected to electrophoresis and the following allele type identification was performed: a. The molecular weight of the enzyme digestion product is 507 bp, and the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon is homozygous for A; b. The molecular weights of the enzyme digestion products were 328 bp and 179 bp, indicating that the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon was homozygous for G; c. The molecular weights of the enzyme digestion products were 507 bp, 328 bp, and 179 bp, and the allele of the mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon was an A / G heterozygous.

7. The detection method according to claim 6, characterized in that In step (1), the PCR amplification program is as follows: pre-denaturation at 93-95°C for 4-7 min; denaturation at 93-95°C for 30-50 s, annealing at 51-55°C for 30-50 s, and extension at 70-73°C for 25-45 s, for a total of 30-36 cycles; and finally extension at 70-73°C for 5-12 min.

8. The detection method according to claim 6, characterized in that In step (3), 2.5-3.5% agarose gel is used for electrophoresis.

9. A method for identifying the length of a rice mesocotyl, characterized in that the steps include: The allele type of the rice mesocotyl length regulatory gene PAO5 at 578 bp upstream of the start codon was detected. Rice containing the A homozygous allele had a relatively short mesocotyl length, and rice containing the G homozygous allele had a relatively long mesocotyl length.

10. A method for cultivating or screening rice, characterized in that the steps include: The allele type of a rice sample is detected using the detection primer according to claim 1, or the product according to claim 4 or 5, or the detection method according to any one of claims 6 to 8, to determine rice samples suitable as parents, and / or the allele type of progeny in hybridization, and / or backcrossing, and / or selfing, and to carry out rice breeding or screening.

Images