CN112143830A - Molecular marker of rice sword leaf width regulation gene NAL1 and application thereof - Google Patents
Molecular marker of rice sword leaf width regulation gene NAL1 and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker capable of identifying rice sword leaf width gene NAL1, a positive primer and a negative primer thereof, and application of the molecular marker in screening and breeding of rice leaf width characters. The marker has no genetic exchange through sequence difference design, and has high accuracy; the kit is directly used for agarose gel electrophoresis detection, and is simpler and more convenient; the marker is a codominant marker, homozygous and heterozygous individuals can be detected, the breeding period is shortened, and the breeding efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of plant propagation, particularly relates to the technical field of agricultural biology, and particularly relates to a molecular marker of a rice flag leaf width regulation gene NAL1 and application thereof.
Background
Rice is the staple food of nearly half of the population all over the world and is also the most important food crop in China. The rice flag leaf is the most important functional leaf and is an important place for photosynthesis. The wide sword leaves have important significance for accumulating more photosynthetic products in the rice, so that the proper increase of the width of the sword leaves has a positive effect on the increase of the yield of the rice. A large number of researches show that the width of the flag leaf of the rice is simultaneously regulated by dual factors of an internal genetic mechanism and external environmental factors. Therefore, the understanding of the genetic rule of the rice sword leaf width has important significance for improving the rice yield by improving the sword leaf width.
In recent years, with the development of genetic and molecular biological techniques, genes NAL1(Qi et al, 2008), NARROW LEAF 7(NAL7) (Fujino et al, 2008), NAL9(Li et al, 2013), etc., which control the LEAF width of rice, have been cloned. Among them, studies have shown that the introduction of NAL1 allele of japonica rice nipponica into indica rice 9311 can improve traits such as leaf width, photosynthetic efficiency and yield of indica rice (Zhang et al, 2014). The width of the sword leaf of rice is a typical quantitative character and is controlled by multiple genes. Molecular marker assisted selection is a modern breeding technology which utilizes a marker closely linked with a leaf width gene or an intragenic functional marker to select target traits by combining genotype and phenotype identification in later generations. The method not only can greatly shorten the breeding period and improve the breeding efficiency, but also saves a large amount of labor and material cost. The NAL1 wide sword leaf allele is mainly derived from part of japonica rice varieties, and indica rice basically does not contain the NAL1 wide sword leaf allele, so that the method has great application value in the aspects of improving the width of rice sword leaves, improving the rice photosynthetic efficiency and improving the rice yield, and the development of the functional marker of the NAL1 gene has important significance for fully utilizing the gene, further improving the width of the rice sword leaves and improving the rice yield.
Disclosure of Invention
Based on the above, the invention aims to provide a molecular marker of rice leaf width gene NAL1, and positive and negative primers and application thereof. The specific technical scheme is as follows:
a molecular marker of rice sword leaf width gene NAL1 is NAL1-Rsa I, 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 embodiments, the molecular marker is located on chromosome 4 of the rice genome.
The invention also provides application of the molecular marker of the rice sword leaf width gene NAL1, which comprises the following specific steps:
the molecular marker is applied to the identification of the genotype of the rice leaf width regulation gene NAL1 and/or the auxiliary selective breeding of wide-leaf rice.
The invention also provides a method for identifying the rice xiphoid leaf width regulation gene NAL1 by using the molecular marker, and the specific technical scheme is as follows:
a method for identifying a rice xiphoid leaf width regulation gene NAL1 by using molecular markers comprises the following steps:
1) extracting the genomic DNA of a test rice sample;
2) 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;
3) and (3) carrying out enzyme digestion on the PCR amplification product by using restriction enzyme Rsa I.
In some of the examples, the cleavage product obtained in step 3) is detected by electrophoresis on a 1% agarose gel, and if a single band of 399bp exists, the sample is the 97B allele type of the variety Zhenshan; if the 265bp and 134bp bands exist, the genotype of the detection sample is represented as IRAT109 allele; if the three banding patterns of 399bp, 265bp and 134bp are contained simultaneously, the detection sample is represented as a NAL1 heterozygous genotype.
In some of these embodiments, the PCR amplified amplification system comprises: taq enzyme, template DNA, dNTP, primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
In some embodiments, the PCR reaction procedure for the PCR amplification is: pre-denaturing at 95 + -1 deg.C for 4-6min, performing 33-37 cycles at 98 + -1 deg.C for 28-32s, 54 + -1 deg.C for 28-32s, and 72 + -1 deg.C for 0.8-1.2min, and extending at 72 + -1 deg.C for 4-6 min.
The invention also provides a method for the molecular marker to be used for the auxiliary selective breeding of the broad-leaf rice, and the specific technical scheme is as follows:
a method for using molecular markers for auxiliary selective breeding of broad-leaf rice comprises the following steps:
1) extracting the genomic DNA of a test rice sample;
2) 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;
3) and (3) carrying out enzyme digestion on the PCR amplification product by using restriction enzyme Rsa I.
In some embodiments, the enzyme digestion product obtained in step 3) is detected by 1% agarose gel electrophoresis, and if the band has 265bp and 134bp, the genotype of the detected sample is IRAT109 allele; or if the three banding patterns of 399bp, 265bp and 134bp are contained simultaneously, the detection sample is represented as a NAL1 heterozygous genotype; is a wide-leaf rice variety.
The invention also provides a kit for identifying the rice sword leaf width regulation gene NAL1, and the specific technical scheme is as follows:
a kit for identifying a rice leaf width regulation gene NAL1 comprises: the primers with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 are used for amplifying the molecular marker of the rice leaf width gene NAL1, and the molecular marker is NAL1-Rsa I.
Based on the technical scheme, the invention has the following beneficial effects:
the invention relates to a molecular marker capable of identifying rice sword leaf width gene NAL1, a positive primer and a negative primer thereof, and the molecular marker can be applied to screening and breeding of rice leaf width characters. The marker has no genetic exchange through sequence difference design, and has high accuracy; the kit is directly used for agarose gel electrophoresis detection, and is simpler and more convenient; the marker is a codominant marker, homozygous and heterozygous individuals can be detected, the breeding period is shortened, and the breeding efficiency is improved.
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FIG. 1 is the electrophoresis detection chart of the molecular marker NAL1-Rsa I on 10 rice varieties.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. 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 is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
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. As used herein, the term "and/or" 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 marker of Rice Jianye Wide Gene NAL1
(1) Test materials: variety of Sword leaf: IRAT 109; variety of Sword leaf: zhenshan 97B.
(2) The extraction method of the rice genome DNA comprises the following steps: taking 10mg rice leaves in a mortar, adding a proper amount of liquid nitrogen, grinding the rice leaves into powder, adding 400 mu L of 1.5 xCTAB (1.5% CTAB,75mmol/L Tris-HCl,15mmol/L EDTA,1.05mol/L NaCl, Ph being 8.0), grinding into homogenate, adding 400 mu L of 1.5 xCTAB, sucking the grinding liquid into a 1.5ml centrifuge tube, adding 550 mu L chloroform, mixing uniformly, centrifuging at 12000r/min for 10min, taking the supernatant into another centrifuge tube, adding precooled isopropanol with the same volume, centrifuging at 12000r/min for 5min, removing the supernatant, drying the precipitate, and finally adding 200 mu L ddH2Dissolving O to obtain the final product.
(3) Development of Gene molecular marker NAL1-Rsa I: NAL1 is located on chromosome 4 and is included in the japanese fine reference genome under the accession number AL 662950.2.
(4) Based on the nucleotide sequence differences, primers NAL1-Rsa I were designed using Primer Premier 5.0 software, with the forward and reverse Primer sequences:
NAL1-Rsa I F：5’-TGCTACTCCACATCTTGTTGTTTA-3’(SEQ ID NO.1)；
NAL1-Rsa I R：5’-GTTGGATTGATGGAGAACAAGGA-3’(SEQ ID NO.2)。
(5) performing PCR amplification on the tested rice varieties IRAT109 and Zhenshan 97B by using functional markers NAL1-Rsa I, wherein the PCR amplification system is as follows: mu.L of 20 ng/. mu.L rice genome DNA template, 1. mu.L of 10. mu.L of 2 XTAQA Master mix (Biotech, Inc. of Nanjing Nodezam), 1. mu.L of each of 10. mu.M primers, and dd H2O to 20. mu.L. The PCR reaction program is: pre-variation at 95 DEG CSex for 5min, then according to 98 degrees 30s, 54 degrees 30s, 72 degrees 1min 35 cycles, finally 72 degrees 5min extension.
(6) The PCR product was digested with restriction enzyme Rsa I (NEB (Beijing) Co., Ltd.): the restriction system was that 2.5uL of 10 XNEBuffer was added to the PCR product, 1. mu.L of restriction enzyme Rsa I (5U/uL) was added, and dd H was supplemented2O to 25. mu.L. Then reacted at 37 ℃ for 2 hours.
(7) And 8 mu.L of the digestion product is loaded on a 1% agarose gel for electrophoresis detection. As shown in FIG. 1, lane 10 is wide-leaf rice variety IRAT109, and two bands appear after enzyme digestion, the band sizes being 265bp and 134bp, respectively. Lane 9 shows the PCR product of IRAT109, which was not digested, and the band size was 399 bp. Lane 8 is the PCR product of Zhenshan 97B after enzyme digestion, and the size of the band is 399 bp. Lane 7 is PCR product of Zhenshan 97B/IRAT109 hybrid F1 after enzyme digestion. From fig. 1, it can be seen that the electrophoretic bands are clear and distinct.
(8) Electrophoretic dicing tape recovery/sequencing. The gel recovery uses a gel recovery kit (centrifugal column type, product catalog number: EG101-02) of Beijing Quankunjin Biotechnology Limited, and sequencing is completed by Shanghai Boshang Biotechnology Limited. The sequencing results are shown in Table 1.
Table 1: base comparison of IRAT109 and Zhenshan 97B
IRAT109：(SEQ ID NO.3)
TGCTACTCCACATCTTGTTGTTTATTCTACTGATTCCATCTTTTCGTTCGGGCCAGGCCAGCTAGCTAGCCGCAAGCGCTGACTGTCTTGATCATTGATTCCTCCTTCCACAATAACTCTAAAAGATTGGAAGTACATTTGCATGATTGATGGTTTTCCCGTCGCTTTCGGCATTCGTTATCTACCTGTCCATTAGCCTTCAGGATCATGCTTTCTGACTTGCTTGTTCTCATTCTTAGGGCCATAACTTCAGCTTCTCCCATCTATAATAGGTTCGCAAACTGTTCAGCACAATGAAGCCTTCGGACGATAAGGCGCAGCTCTCCGGTTTGGCGCAATCAGAAGAATCGTCACTTGATGTGGATCACCAGTCATTTCCTTGTTCTCCATCAATCCAAC
Zhenshan 97B (SEQ ID NO.4)
TGCTACTCCACATCTTGTTGTTTATTCTACTGATTCCATCTTTTCGTTCGGGCCAGGCCAGCTAGCTAGCCGCAAGCGCTGACTGTCTTGATCATTGATTCCTCCTTCCACAATAACTCTAAAAGATTGGAAGCACATTTGCATGATTGATGGTTTTCCCGTCGCTTTCGGCATTCGTTATCTACCTGTCCATTAGCCTTCAGGATCATGCTTTCTGACTTGCTTGTTCTCATTCTTAGGGCCATAACTTCAGCTTCTCCCATCTATAATAGGTTCGCAAACTGTTCAGCACAATGAAGCCTTCGGACGATAAGGCGCAGCTCTCCGGTTTGGCGCAATCAGAAGAATCGTCACTTGATGTGGATCACCAGTCATTTCCTTGTTCTCCATCAATCCAAC
Example 2: parent polymorphism detection of NAL1-Rsa I molecular marker
(1) The test rice variety genome was extracted in the same manner as in example 1. The varieties of the tested rice are respectively as follows: hunan Qing, SILEWAH, Hanhui 15, Yunlong No. 8, Xiushui 123 and Mihui 725.
(2) PCR amplification was performed as in example 1.
(3) The enzyme was cleaved as in example 1.
(4) The electrophoresis was performed as in example 1. The results are shown in FIG. 1.
Wherein 1-10 respectively represent rice varieties Xiangqing (japonica rice, sword leaf width is 1.52mm), SILEWAH (japonica rice, sword leaf width is 2.02mm), Haihu No. 15 (indica rice, sword leaf width is 1.70mm), Yuanluo No. 8 (japonica rice, sword leaf width is 2.40mm), Xiushui 123 (japonica rice, sword leaf width is 1.55mm), Mihui 725 (indica rice, sword leaf width is 1.60mm), Zhenshan 97B/IRAT109 hybrid F1 (sword leaf width is 1.75mm), Zhenshan 97B (indica rice, sword leaf width is 1.60mm), IRAT109 (not enzyme-digested), IRAT109 (japonica rice, sword leaf width is 1.80 mm); and M represents a D2000 Marker.
As can be seen, the size of the electrophoretic band of the species Xiangqing (lane 1), Hanhui 15 (lane 3), Xiushui 123 (lane 5), Mihui 725 (lane 6) containing the narrow-leaf allele is 399 bp; the electrophoresis results of the flag leaf allele SILEWAH (lane 2) and Cloud 8 (lane 4) show two bands with sizes of 265bp and 134bp, respectively.
Example 3: verification and application of NAL1-Rsa I molecular marker
(1) Utilizing NAL1 gene molecular marker NAL1-Rsa I to perform PCR amplification on the genomic DNA of a single hybrid F1 generation of rice maintainer line Zhenshan 97B and wide sword leaf variety IRAT109, then performing enzyme digestion on the PCR product by restriction enzyme Rsa I, performing electrophoresis typing on the enzyme digestion product in 1% agarose gel, detecting three bands of 399bp, 256bp and 134bp in the single hybrid F1 plant, indicating that the NAL1-Rsa I can be used for identifying the hybrid F1, and detecting that the sample is a NAL gene heterozygous genotype.
(2) F of 198 IRAT 109/Zhenshan 97B by using NAL1 gene molecular marker NAL1-Rsa I10PCR amplification is carried out on genome DNA of the generation recombination inbred line, the PCR amplification product is subjected to Rsa I enzyme digestion, the enzyme digestion product is subjected to electrophoresis detection in 1% agarose gel, 106 strains respectively contain Zhenshan 97B allele type (399bp bands), 92 strains contain allele type (containing 256bp and 134bp bands) consistent with IRAT109, and sword leaf width identification results show that sword leaf width of the strains containing the Zhenshan 97B allele type is remarkably smaller than the grain type of the strains containing the IRAT109 allele type (P is less than 1.28E-12).
The gene molecular marker NAL1-Rsa I of NAL1 is utilized to perform NAL1 genotype identification on the genome DNA of 237 rice germplasm resources, the product is subjected to electrophoresis detection in 1% agarose gel, and the result shows that the genome DNA of 210 germplasm resources is 399bp band type, 27 germplasm resources are two bands, the band sizes are 256bp and 134bp respectively, the width of the sword leaf of the germplasm resource containing the two band types (256bp and 134bp) is obviously larger than that of the germplasm resource containing the 399bp band type (P is less than 8.6E-4), and meanwhile, domestic main cultivar parents (Minghui 63, Zhenshan 97B, Baohe 7B, 9311, Huihui No. 3, Mikan 725, Shanghai 7B, C418, Zhong413 and the like) are all 399bp band types.
Therefore, the molecular marker NAL1-Rsa I can be used for the identification of the rice flag leaf wide gene NAL1 and/or the auxiliary selection breeding of flag leaf wide rice.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shanghai city agricultural biological gene center
<120> molecular marker of rice sword leaf width regulation gene NAL1 and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gttggattga tggagaacaa gga 23
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<213> Rice (Oryza sativa)
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gctagctagc cgcaagcgct gactgtcttg atcattgatt cctccttcca caataactct 120
aaaagattgg aagtacattt gcatgattga tggttttccc gtcgctttcg gcattcgtta 180
tctacctgtc cattagcctt caggatcatg ctttctgact tgcttgttct cattcttagg 240
gccataactt cagcttctcc catctataat aggttcgcaa actgttcagc acaatgaagc 300
cttcggacga taaggcgcag ctctccggtt tggcgcaatc agaagaatcg tcacttgatg 360
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<213> Rice (Oryza sativa)
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aaaagattgg aagcacattt gcatgattga tggttttccc gtcgctttcg gcattcgtta 180
tctacctgtc cattagcctt caggatcatg ctttctgact tgcttgttct cattcttagg 240
gccataactt cagcttctcc catctataat aggttcgcaa actgttcagc acaatgaagc 300
cttcggacga taaggcgcag ctctccggtt tggcgcaatc agaagaatcg tcacttgatg 360
tggatcacca gtcatttcct tgttctccat caatccaac 399
Claims (10)
1. A molecular marker of rice sword leaf width gene NAL1 is NAL1-Rsa I, 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 rice sword-leaf width gene NAL1 molecular marker as claimed in claim 1, wherein the molecular marker is located on chromosome 4 of rice genome.
3. The application of the molecular marker of claim 1 or 2 in the identification of the genotype of the rice leaf width regulatory gene NAL1 and/or in the auxiliary selective breeding of wide-leaf rice.
4. A method for identifying a rice xiphoid leaf width regulation gene NAL1 by using molecular markers is characterized by comprising the following steps:
1) extracting the genomic DNA of a test rice sample;
2) 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;
3) and (3) carrying out enzyme digestion on the PCR amplification product by using restriction enzyme RsaI.
5. The method for identifying the rice Jianye Width regulatory gene NAL1 by using the molecular marker as claimed in claim 4, wherein the enzyme digestion product obtained in step 3) is detected by 1% agarose gel electrophoresis, and if a single band with 399bp exists, the sample is represented as a Zhenshan 97B allele type; if the 265bp and 134bp bands exist, the genotype of the detection sample is represented as IRAT109 allele; if the three banding patterns of 399bp, 265bp and 134bp are contained simultaneously, the detection sample is represented as a NAL1 heterozygous genotype.
6. The method for identifying the rice xyphoid width regulatory gene NAL1 according to claim 4 or 5, wherein the PCR amplification system comprises: taq enzyme, template DNA, dNTP, primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and PCR buffer.
7. The method for identifying the rice xiphoid width regulatory gene NAL1 according to claim 6, wherein the PCR reaction procedure of the PCR amplification is as follows: pre-denaturing at 95 + -1 deg.C for 4-6min, performing 33-37 cycles at 98 + -1 deg.C for 28-32s, 54 + -1 deg.C for 28-32s, and 72 + -1 deg.C for 0.8-1.2min, and extending at 72 + -1 deg.C for 4-6 min.
8. A method for auxiliary selective breeding of broad-leaf rice by using molecular markers is characterized by comprising the following steps:
1) extracting the genomic DNA of a test rice sample;
2) 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;
3) and (3) carrying out enzyme digestion on the PCR amplification product by using restriction enzyme RsaI.
9. The method for assisted selection breeding according to claim 8, characterized in that the enzyme digestion products obtained in step 3) are detected by 1% agarose gel electrophoresis: if the 265bp and 134bp bands exist, the genotype of the detection sample is represented as IRAT109 allele; or if the three banding patterns of 399bp, 265bp and 134bp are contained simultaneously, the detection sample is represented as a NAL1 heterozygous genotype; is a wide-leaf rice variety.
10. A kit for identifying a rice leaf width regulation gene NAL1 is characterized by comprising: the primers with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 are used for amplifying the molecular marker of the rice leaf width gene NAL1, and the molecular marker is NAL1-Rsa I.
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| MINGLIANG CHEN等: "Fine mapping of a major QTL for flag leaf width in rice, qFLW4,which might be caused by alternative splicing of NAL1", 《PLANT CELL REP》 * |
| 童汉华等: "水稻生育后期剑叶形态和生理特性的QTL定位", 《中国水稻科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736898A (en) * | 2021-08-09 | 2021-12-03 | 上海市农业生物基因中心 | Molecular marker of rice heading stage regulation gene OsGI and application thereof |
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