CN118147358A - Molecular marker closely linked with rice tillering number and single plant yield and application thereof - Google Patents
Molecular marker closely linked with rice tillering number and single plant yield and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker of a gene D27 capable of identifying the tiller number and yield of improved rice, a forward and reverse primer thereof and an auxiliary breeding method capable of applying the molecular marker to the tiller number and yield of improved rice. The marker has no genetic exchange through sequence difference design, and has high accuracy; the method is directly used for agarose gel electrophoresis detection, and is simpler and more convenient; can shorten the breeding period and improve the breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of plant propagation, in particular relates to the technical field of agricultural biology, and in particular relates to a molecular marker for regulating and controlling the tiller number and the single plant yield of rice and application thereof.
Background
Rice is a staple food of nearly half of the population worldwide, and is also the most important food crop in China. The final yield of the rice is determined by three factors of thousand grain weight, effective spike number and spike number, and the tiller number of the rice directly influences the spike number of the rice, so that the tiller number of the rice is one of important factors influencing the yield of a single plant of the rice. The effective spike number of unit area can be improved by properly increasing the tiller number of the rice, so that the single plant yield of the rice in unit area is improved. Therefore, cultivation of rice varieties with a large tiller number has important significance for ensuring the improvement of the yield of single rice plants and the grain safety.
In recent years, with the development of genetic and molecular biological techniques, a gene MOC1(Li et al.,Nature,2003,422(6932):618-621)、MOC2(Koumoto et al.,Plant Biotechnology,2013,30(1):47-56),MOC3(Lu et al.,Journal of genetics and genomics,2015,42(2):71-78) or the like that regulates the tiller number of rice has been cloned. MOC1 is the first cloned gene in rice that positively regulates tillering. moc1 mutant lost the ability to produce tillered shoots, eventually with only one main stem. (Li et al, nature,2003,422 (6932):618-621). MOC1 protein can bind to DELLA protein SLR1 to form a trimer that avoids being degraded, exogenous GA can cause degradation of GA signaling pathway protein SLR1, resulting in hindered complex formation, MOC1 degradation, and ultimately reduced tillering (Liao et al, nature Communications,2019, 10:2738). moc2 mutants exhibited single-tillering, dwarfing, pale green leaves, short and narrow leaves, small spikes and reduced spike number (Koumoto et al., plant Biotechnology,2013,30 (1): 47-56). MOC3 encodes a WOX protein family member, a transcriptional repressor, which is a gene mutated to disrupt the tillering buds of plants during development and reduced tillering numbers (Lu et al, journal of GENETICS AND genomics,2015,42 (2): 71-78). Another gene D27 which is empty with the tiller number of rice is positioned on chromosome 11, the whole length of cDNA is 1254bp, and the cDNA contains 7 exons, and encodes an ferritin which consists of 278 amino acid residues and is positioned on chloroplasts. D27 protein is a member involved in strigolactone (strigolactone, SL) biosynthesis, the latter involved in control of rice tillering. In the D27 mutant, the 4 th exon of the D27 gene is deleted by 4bp, and a stop codon is formed in advance. d27 mutant strain is dwarfed and tillering number is increased as much as 3 times of wild type. In situ hybridization showed that D27 was expressed mainly in young leaves, axillary buds, inflorescence primordia, lateral roots and coronal roots, especially in the aerial top of main stems and in vascular bundle cells of young leaves and nodes and internodes. By genetic comparison analysis with other similar multi-tiller mutants, the d27 d10 double mutant phenotype was found to be similar to d10, whereas d10 was the mutant due to deletion of the MAX4/RMS1 homologous protein. d27 mutant can restore the wild phenotype by GR24 (strigolactone analogue). The results show that the D27 protein participates in the MAX/RMS/D pathway and is a novel member participating in the biosynthesis of strigolactone. The polar transport of auxin was enhanced after the D27 mutation, no strigolactone was detected in the root secretions, resulting in the inhibition of tillering bud outgrowth being relieved, thus exhibiting a multi-tillering phenotype (Lin et al, 2009). In general, the more tillers, the more the plant trees and ears of the crop, and the higher the yield of the crop. However, excessive tillering can also cause the cluster of crops to be depressed, the ventilation and light transmission to be poor, and the like, thereby affecting the growth and the yield of crops. Therefore, it is necessary to develop linkage markers for improving the tiller number and the individual plant yield of rice, which can be used for assisting in selecting and improving the tiller number and the individual plant yield of rice, further improve the tiller number and the individual plant yield of rice, and has important significance for improving the individual plant yield of grain and ensuring the grain safety.
Disclosure of Invention
Based on the above, the invention aims to provide a molecular marker closely linked with rice tillering number and single plant yield regulation and control, and a forward primer and a reverse primer and application thereof. The specific technical scheme is as follows:
the molecular marker is a molecular marker D27_218 of the tiller number and the single plant yield regulating gene D27, and the nucleotide sequence of a primer for amplifying the molecular marker is shown as SEQ ID NO.1 and SEQ ID NO. 2.
In some of these embodiments, the molecular marker is located on chromosome 11 of the rice genome.
In some of these embodiments, the size of the molecular markers is 362bp and/or 144bp.
In some of these embodiments, the nucleotide sequence of the molecular marker is SEQ ID No.3 and/or SEQ ID No.4.
The invention also provides a method for obtaining the molecular marker related to the tiller number of rice and the yield characteristic of a single plant, which comprises the following steps:
A method for obtaining a molecular marker related to the tiller number of rice and the yield characteristics of a single plant comprises the following steps:
respectively taking genome DNA of rice varieties Zhenshan 97B and IRAT109 as templates, and adopting primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 for PCR amplification;
And separating the PCR amplified product by electrophoresis to obtain an amplified fragment with the size of 362bp or an amplified fragment with the size of 144bp, namely the target molecular marker.
The invention also provides application of the molecular marker of the rice tillering number and single plant yield regulating gene D27, which comprises the following specific steps:
the molecular marker of the rice on chromosome 11 is applied to the identification of the tiller number and the single plant yield of the rice and/or the auxiliary selective breeding for improving the tiller number and the single plant yield of the rice.
The invention also provides a method for identifying the rice tillering number and single plant yield regulating gene D27 by using the molecular marker, which comprises the following specific technical scheme:
a method for identifying rice tillering number and individual yield characteristics by using molecular markers, comprising the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
In some of the embodiments, detecting the PCR product obtained in the step 2) through 1% agarose gel electrophoresis, if a band of 144bp exists, the sample is represented as a tiller number and a single plant yield higher variety Zhenshan 97B allele type; if 362bp bands exist, the genotype of the detected sample is the tiller number and the IRAT109 allele of the variety with lower single plant yield.
In some of these embodiments, the PCR amplified amplification system comprises: taq enzyme, template DNA, dNTPs, and primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
In some embodiments, the PCR amplification is performed by a PCR reaction procedure comprising: pre-denaturation at 94℃for 4-5min, denaturation at 94.+ -. 1℃for 30s, denaturation at 55.+ -. 0.5℃for 15-25s and denaturation at 72℃for 15s, 36 cycles, and extension at 72℃for 4min. The invention also provides a molecular marker assisted selective breeding method for improving the tiller number and the single plant yield of rice, which comprises the following steps:
A molecular marker assisted selective breeding method for improving tiller number and single plant yield of rice comprises the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) And (3) carrying out PCR amplification on the genome extracted in the step (1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product.
In some of these embodiments, the PCR product obtained in step 2) is detected by 1% agarose gel electrophoresis, if there is a band of 144bp, indicating that the sample is a tiller number and a higher yield of individuals variety of the allelic type; if 362bp bands exist, the genotype of the detected sample is tillering number and single plant yield less variety alleles.
The invention also provides a kit for identifying the tiller number and the yield characteristics of a single plant of rice, which comprises the following specific technical scheme:
A kit for identifying tiller number and individual yield characteristics of rice comprising: the nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer is used for amplifying a molecular marker closely linked with the rice tillering number and the single plant yield regulation, and the molecular marker is D27_218.
Based on the technical scheme, the invention has the following beneficial effects:
Molecular marker assisted selection is a modern breeding technology for selecting target traits by utilizing a marker closely linked with a gene for regulating and controlling tillering number and single plant yield or an intragenic functional marker and combining genotype and phenotype identification in offspring.
The molecular marker capable of identifying the rice tillering number and the single plant yield regulation gene D27 and the forward and reverse primers thereof can be applied to improved screening and breeding of the rice tillering number and the single plant yield, and the effective spike number in unit area can be improved by properly increasing the rice tillering number in improved breeding of the rice through the linkage molecular marker, so that the tillering number and the single plant yield are improved simultaneously, and the single plant yield of the rice in unit area is improved. The marker has no genetic exchange through sequence difference design, and has high accuracy; the method is directly used for 1% agarose gel electrophoresis detection, and is simple and convenient; the breeding method using the molecular marker for auxiliary selection can shorten the breeding period, improve the breeding efficiency, greatly shorten the breeding period, improve the breeding efficiency and save a great amount of labor and material costs.
Drawings
FIG. 1 is a diagram showing the electrophoresis detection of 10 rice varieties by the molecular marker D27_218 of the present invention.
FIG. 2 shows the verification results of D27_218 molecular markers in different population tiller numbers and individual plant yield performance evaluations in the invention.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention is described in detail below by way of examples:
Example 1 development of molecular markers for the D27 Gene of the Rice tillering number and Single plant yield control Gene
(1) Test material: cultivars with high tillering number and single plant yield: zhenshan 97B; tillering number and single plant yield are less: IRAT109.
(2) The extraction method of rice genome DNA comprises the following steps: in a mortar, 10mg of rice leaves are taken, a proper amount of liquid nitrogen is added, the rice leaves are ground into powder, 400 mu L of 1.5 xCTAB (1.5% CTAB,75mmol/L Tris-HCl,15mmol/L EDTA,1.05mol/L NaCl, pH=8.0) is added to grind into homogenate, 400 mu L of 1.5 xCTAB is added to absorb the grinding fluid into a 1.5ml centrifuge tube, 550 mu L of chloroform is added to mix uniformly, the mixture is centrifuged for 10min at 12000r/min, the supernatant is taken to another centrifuge tube, pre-cooled equal volume isopropanol is added, the mixture is centrifuged for 5min at 12000r/min, the supernatant is removed, the precipitate is dried, and finally 200 mu L of ddH 2 O is added to dissolve the precipitate.
(3) Development of a gene molecular marker d27_218: d27 is located on chromosome 11 and is registered in the japanese reference genome as Os11g0587000.
(4) According to the nucleotide sequence difference analysis of the gene of the variety with fewer tillers and single plant yield and the variety with more yield, a primer D27_218 is designed, and the forward and reverse primer sequences are as follows:
D27_218F：5’-CAGAAAGATGTGAGTGGAAACCA-3’(SEQ ID NO.1)；
D27_218R：5’-CCTGATGTAGTGTGGAGAATGG-3’(SEQ ID NO.2)。
(5) The functional marker D27_218 is utilized to carry out PCR amplification on the DNA of the rice variety Zhenshan 97B and IRAT109 to be tested, and the PCR amplification system is as follows: mu.L of 20 ng/. Mu.L of the rice genomic DNA template, 10. Mu.L of 2X TAQ MASTER mix (Nanjinozan Biotechnology Co., ltd.) and 1. Mu.L of each of 10. Mu.M of the front and rear primers were supplemented with dd H 2 O to 20. Mu.L. Pre-denaturation at 94℃for 4-5min, denaturation at 94.+ -. 1℃for 30s, denaturation at 55.+ -. 0.5℃for 15-25s and denaturation at 72℃for 15s, 36 cycles, and extension at 72℃for 4min.
(7) The PCR product was applied to 8. Mu.L of a 1% polyacrylamide gel electrophoresis. As shown in FIG. 1, lane 10 shows a variety Zhenshan 97B with high tiller number and single plant yield, and the band size is 144bp. Lane 9 shows the PCR product of IRAT109, with a band size of 362bp. From fig. 1, it can be seen that the electrophoresis bands are clear, and the difference is obvious.
(8) Electrophoresis cutting gel recycling/sequencing. The gel was recovered using a gel recovery kit (centrifugal column, catalog number: EG 101-02) from Beijing full gold biotechnology Co., ltd.) and sequencing was performed by Shanghai Seisakusho Heng Biotech Co. The sequencing results are shown in Table 1.
Table 1: base comparison of Zhenshan 97B and corresponding molecular marker locus of IRAT109
Zhenshan 97B: (SEQ ID NO. 3)
CAGAAAGATGTGAGTGGAAACCAAGTACTATTTCATGGACGAAAATTATAA
TGCACGGGTTCCTCTAAAAGAATGTATTTTAAAACTCTAACTGATATAACTC
ATAAATATATGTCAGAAAACCATTCTCCACACTACATCAGGIRAT109(SEQ ID NO.4)
CAGAAAGATGTGAGTGGAAACCAAGTACTATTTCATGGACGAAAATTATAA
TGCACGGATTCCTCTAAAAGAATTTATTTTAAAACTCTAACTGATATAACTC
TCGATCCACTATCAGTTATTAGGGGAAATATTCTATTTGTCACTAAGTATTTT
GAGGAAAATAGAATTCTATATATGTATATGGCTGTCTATGCACTGTCTCATCC
ACCCAATGTGCTAAAAGATTTGAGCAGGGACATTGAAATTATTAGAGATGG
CAATTTTTAAAGTTTGCATGACTATCGAGGCATATGGGACATATAGGACAGT
GCCTAACTCATAAATATATGTCAGAAAACCATTCTCCACACTACATCAGG example 2: parent polymorphism detection of D27_218 molecular marker
(1) Genomic DNA of the rice variety to be tested was extracted in the same manner as in example 1. The rice varieties tested are respectively as follows: dry land recovery 15, shanghai dry land 15, zhongyen No. 3, shanghai dry land 3, CICA, IAC1,
9311、IAC1246。
(2) PCR amplification was performed as in example 1.
(3) Electrophoresis was performed as in example 1. The results are shown in FIG. 1.
Where M represents Marker. Lanes 1 to 10 are arranged from left to right except lane M, and correspond to rice variety Zuohui 15 (tiller number is 12.5), shanghai 15 (tiller number is 13.8), shanghai 3 (tiller number is 7.9), shanghai 3 (tiller number is 10.4), CICA4 (tiller number is 6.0), IAC1 (tiller number is 9.4), 9311 (tiller number is 14.2), IAC1246 (tiller number is 14.0), IRAT109 (tiller number is 11.9) and Zhenhan 97B (tiller number is 17.5).
As can be seen, the sizes of electrophoresis bands of varieties of Hanhui 15 (lane 1), hui 15 (lane 2), 9311 (lane 7), IAC1246 (lane 8) and Zhenshan 97B (lane 10) containing alleles with a large tiller number are 144bp. In the case of the less tillering allele species, the sizes of the electrophoresis bands of drought No. 3 (lane 3), hu drought No. 3 (lane 4), CICA (lane 5), IAC1 (lane 6) and IRAT109 (lane 9) were 362bp.
Example 3: verification and application of D27_218 molecular marker
(1) The expression verification of the tillering number of the Zhenshan 97B/IRAT109 recombinant inbred line group: genomic DNA of a recombinant inbred line population (F 10, 200 lines) of a Zhenhan 97B variety with more rice tillers and a variety IRAT109 with less tillers is subjected to PCR amplification by using a D27 gene molecular marker D27_218, electrophoresis detection is carried out on 1% agarose gel of PCR products, 98 lines respectively contain the allele type (144 bp band) of the Zhenhan 97B and 88 lines contain the allele type (362 bp band) consistent with the IRAT109, and the tillering number identification result shows that the tillering number of the lines containing the Zhenhan 97B allele type is extremely larger than that of the lines containing the IRAT109 allele type (P=4.55E-05) (shown in A in figure).
(2) And (3) verifying the tillering quantity of rice germplasm resources: the genomic DNA of 270 parts of rice germplasm resources is subjected to D27 genotype identification by using a D27 gene molecular marker D27_218, and the PCR product is subjected to electrophoresis detection in agarose gel of 1%, wherein 125 parts of the rice germplasm resources are 144bp band type, 141 parts of the rice germplasm resources are 362bp in band size, and the tiller number of the rice germplasm resources containing 144bp band type is extremely larger than that of the rice germplasm resources containing 362bp band type (P=3.48E-07) (shown in B in figure 2).
(3) And (3) single plant yield performance verification of rice germplasm resource groups: the average individual yield of 125 germplasm resources containing 144bp band type is: 11.83g; the average individual yield of 141 germplasm resources containing 362bp band type is: 7.96g. The average individual yield of germplasm resources containing a 144bp band-type was shown to be significantly greater than the average individual yield of germplasm resources containing a 362bp band-type (p=1.01e-16) (shown as C in figure 2). Wherein, D27-in FIG. 2 represents the genotype corresponding to the 362bp band type, and D27-del represents the genotype corresponding to the 144bp band type. Thus, it is shown that the molecular marker D27_218 can be utilized to assist in improving the tiller number and yield of rice.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A molecular marker closely linked with the tiller number of rice and the single plant yield regulation is characterized in that the molecular marker is located on chromosome 11 of a rice genome and named D27_218, and the nucleotide sequence of a primer for amplifying the molecular marker is shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The use of the molecular marker of claim 1 for detecting and/or predicting tiller number and individual yield of rice.
3. The use of the molecular marker of claim 1 in the identification of genotypes of rice tillering number and individual plant yield and/or in assisted selection breeding for regulating rice tillering number and individual plant yield.
4. A method for detecting and/or predicting tiller number and individual yield characteristics of rice, comprising the steps of:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
3) Detecting the amplification product in the step 2) through electrophoresis, and if the amplification product with the size of 144bp exists, indicating that the tested rice contains alleles with high rice tillering number and high single plant yield; if there is an amplification product of 362bp in size, this indicates that the test rice contains low tillering and low individual yield alleles.
5. The method for detecting and/or predicting tiller number and individual yield characteristics of rice according to claim 4, wherein the electrophoresis detection is a 1% agarose gel electrophoresis detection.
6. The method for detecting and/or predicting tiller number and individual yield characteristics of rice according to claim 4 or 5, wherein the amplification system of PCR amplification comprises: taq enzyme, template DNA, dNTPs, and primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
7. The method for detecting and/or predicting tiller number and individual yield characteristics of rice according to claim 6, wherein the PCR reaction procedure of PCR amplification is: pre-denaturation at 94℃for 4-5min, denaturation at 94.+ -. 1℃for 30s, denaturation at 55.+ -. 0.5℃for 15-25s and denaturation at 72℃for 15s, 36 cycles, and extension at 72℃for 4min.
8. A method for assisted selective breeding for improving tiller number and single plant yield of rice by using molecular markers, which is characterized by comprising the following steps:
1) Extracting genome DNA of a rice sample to be tested;
2) Performing PCR amplification on the genome extracted in the step 1) by using primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 to obtain an amplification product;
3) Detecting the amplification product in the step 2) through electrophoresis, and if the amplification product with the size of 144bp exists, the rice to be tested is a variety with high tillering number and high single plant yield; if the amplification product with the size of 362bp exists, the rice to be tested is a variety with low tillering number and low single plant yield.
9. The method for assisting selective breeding according to claim 8, wherein the amplification product of 144bp is an allele of D27 gene of Zhenshan 97B, a variety with higher rice tillering number and single plant yield, and the amplification product of 362bp is an allele of D27 gene of IRAT109, a variety with lower rice tillering number and single plant yield.
10. A kit for identifying tiller number and individual yield characteristics of rice, said kit comprising: the nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer is used for amplifying a molecular marker closely linked with the rice tillering number and the single plant yield regulation, wherein the molecular marker is D27_218 positioned on a rice chromosome 11.
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