CN115976055A - Corn dwarf gene and molecular marker thereof - Google Patents
Corn dwarf gene and molecular marker thereof Download PDFInfo
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Abstract
The invention discloses a maize dwarf gene, the nucleotide sequence of which is shown as SEQ ID No.1, and discloses a protein coded by the dwarf gene shown as SEQ ID No. 2. Also discloses CAPS molecular markers of the dwarf gene, and the molecular markers can be used for early auxiliary selection of the dwarf gene. The invention also discloses a corn dwarf related gene with a nucleotide sequence shown as SEQ ID No. 7. The corn dwarf gene has obvious effect of reducing the height of the corn plants, is not linked with bad characters, widens the dwarf germplasm basis of the corn, and provides a new approach for dwarf breeding; secondly, the dwarf gene is controlled by a single recessive gene, the breeding method is simple, and auxiliary selection can be carried out through CAPS markers, so that the breeding speed is accelerated; in addition, the invention provides a maize dwarf related gene, more dwarf genes can be created by means of gene editing, and the number of the dwarf genes is greatly expanded.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a corn dwarf gene; also relates to a molecular marker of the dwarf gene.
Background
Corn is an important crop and feed dual-purpose crop and is also an important industrial raw material. With the improvement of living standard of people, the corn yield is far from meeting the increasing demand, so the improvement of the corn yield is always the aim of the corn breeder to strive for. Generally, increasing planting density is an important way for improving the yield per unit of corn, but too high planting density often leads to the reduction of the diameter of the plant stem and the increase of the plant height and the ear height, and when the corn encounters strong wind and rainstorm from the jointing stage to the mature stage, the field lodging rate is obviously increased, thereby causing serious yield loss. Compared with the high-stalk corn, the low-stalk corn has strong lodging resistance, stronger fertilizer resistance and tightness resistance, and is beneficial to improving the yield per unit; meanwhile, the dwarf corn plants are compact and beneficial to mechanized planting and harvesting, so the dwarf breeding is an important way for improving the planting density of the corn and further improving the unit yield of the corn.
The genes for controlling the maize dwarf comprise polygene and monogene, the dwarf traits controlled by polygene are difficult to apply in breeding because of being greatly influenced by the environment, and the dwarf traits controlled by monogene are suitable for applying in maize dwarf breeding because of simple heredity and no influence by the environment. At least 60 maize dwarf genes have been found, wherein most of the dwarf systems controlled by single genes are recessive genes, such as br1, br2, bv1, ct2, d1, d2, d3, d5, d6, d12, mi, na1, na2, na3, py1, py2, rd1, rd2, yd, clt-1, td1, wrp1, dm1, d2003, and the like; only a few of D8, D9, dt, D8-1023, D10, D11, etc. were dominant genes. However, the dwarf trait controlled by most single genes is closely linked with the poor trait gene, so that the application in breeding is difficult. At present, the widely applied dwarf gene in the corn dwarf breeding is br2 gene, but the br2 genotype corn also has some unfavorable traits which are difficult to overcome, such as short plant internodes, dense overlapping leaves, uncoordinated male and female parts, bad pollination, poor explanation and the like (Chaishui Chinese breed, 12 th 2014), so that the application of the br2 gene is limited, and a new corn dwarf gene resource needs to be searched urgently.
The corn relative wild species has abundant genetic diversity due to long-term natural evolution, and can also have dwarf genes, but due to reproductive isolation, the corn relative wild species and the cultivated corn cannot usually fruit in hybridization, so that the excellent characters of the corn relative wild species are difficult to transfer into the cultivated corn.
MTF-1 (Tripsazea critical T.2n = 76) is a corn allopolyhexaploid (Suyue, shuangchuan university Master thesis, 2009) which is bred by Sichuan university of agriculture and contains complete chromosomes of corn, tripsacum and tetraploid perennial corn, the female ear of the corn allopolyploid is fertile, and the corn, tripsacum or tetraploid perennial corn is used as a male parent for pollination, so that a small number of offspring can be generated, and the problem of reproductive isolation among related genera of the corn is solved. Therefore, by taking MTF-1 as a bridge material, the excellent characters in wild resources can be transferred to the corn.
Through retrieval, the research of utilizing the corn allopolyploid and the report of finding the corn dwarf gene are not found.
Disclosure of Invention
The inventor unexpectedly discovers that dwarf plants appear in offspring when the corn allopolyploid MTF-1 is used for carrying out backcross breeding, and further researches show that the dwarf characters can be inherited and are controlled by single recessive genes; further, the dwarf trait is not linked with the undesirable trait, and the method has wide application prospect in breeding and is realized on the basis of the unexpected discovery.
Aiming at the problem of the lack of excellent corn dwarf gene resources, the invention aims to provide a novel corn dwarf gene.
The invention also aims to provide the application of the corn dwarf gene.
The third purpose of the invention is to provide the CAPS marker of the maize dwarf gene.
The fourth purpose of the invention is to provide the application of the maize dwarf related gene in cultivating maize dwarf varieties.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a corn dwarf gene is named as: d024, and the nucleotide sequence of the maize dwarf gene is shown as SEQ ID No. 1.
The maize dwarf gene is located on a maize No.3 chromosome.
The invention also provides a protein coded by the maize dwarf gene, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention also provides application of the corn dwarf gene or protein in cultivating dwarf corn varieties.
The invention also provides an expression vector containing the maize dwarf gene.
The invention also provides a plant containing the maize dwarf gene or the expression vector.
The invention also provides the application of the expression vector or the plant in cultivating dwarf corn varieties.
The invention also provides a CAPS marker of the maize dwarf gene, wherein the CAPS marker is a DNA molecular fragment; the size of the DNA molecular fragment is 230bp; the nucleotide sequence of the DNA molecule fragment is shown as SEQ ID No. 3.
The CAPS marker of the maize dwarf gene is obtained according to the following method: using the dwarf maize genome DNA with the dwarf gene as a substrate and using nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers to carry out PCR amplification to obtain a PCR amplification product (shown as SEQ ID NO. 6) with the size of 656 bp; then carrying out enzyme digestion on the obtained PCR amplification product by BtsCI enzyme, and carrying out gel electrophoresis to obtain enzyme digestion products with the sizes of 426bp and 230bp; wherein the 230bp enzyme digestion product is peculiar to the dwarf gene; wherein the primer sequence is as follows:
CAPS-3F：5’-GCCCAGGACAGTAGGAAGA-3’(SEQ ID No.4)；
CAPS-3R：5’-ACTCGCTGAGATACTTGACG-3’(SEQ ID No.5)。
the invention also provides application of the CAPS marker in auxiliary selection of the maize dwarf gene.
The invention also provides application of the CAPS marker in cultivation of dwarf corn varieties.
The invention also provides a method for cultivating dwarf maize varieties by using the CAPS markers, which comprises the following steps:
(1) Taking the variety with the maize dwarf gene as one of the parents, and taking another selfing line with good comprehensive agronomic characters as the parent for hybridization or backcross to obtain a hybridization or backcross progeny;
(2) Extracting leaf DNA in the seedling stage of the filial generation of the cross or backcross; then, taking the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers to carry out PCR amplification to obtain a PCR amplification product; carrying out enzyme digestion on the obtained PCR amplification product by BtsC I enzyme, and then carrying out gel electrophoresis on the enzyme digestion product; if the enzyme digestion product contains a strip with the size of 230bp, the descendant carries the dwarf gene and is reserved; if the obtained enzyme digestion product has no 230bp band, the material does not have the dwarf gene and is eliminated;
(3) Selfing the filial generation with the dwarf maize gene selected in the step (2); selecting progeny with plant height 12-50% lower than that of the original inbred line from the inbred progeny; continuously selfing for 4-5 generations, simultaneously performing molecular marker-assisted selection on selfed progeny according to the method in the step (2), selecting progeny containing 230bp bands, and breeding into a new dwarf maize selfing line; or using the progeny containing 230bp bands as non-recurrent parent, using the original inbred line as recurrent parent to continue backcross, repeating the step (2) on the backcross progeny, selecting the progeny containing 230bp bands, carrying out continuous backcross for 4-5 generations, then selfing for 1 generation, and selecting the progeny with the plant height lower than 12-50% of that of the original inbred line, namely the newly-bred dwarf maize variety.
The invention also provides application of the corn dwarf related gene with the nucleotide sequence shown as SEQ ID No.7 in cultivating dwarf corn varieties.
The invention also provides a breeding method for cultivating the dwarf corn variety by using the maize dwarf related gene, which comprises the steps of carrying out gene editing on the maize dwarf related gene with a nucleotide sequence shown as SEQ ID No.7 in a maize inbred line by using a CRISPR-Cas9 gene editing system, and selecting a plant with the plant height 12-50% lower than that of the original variety in the transgenic plant, namely the dwarf corn variety; wherein the nucleotide sequence of sgRNA of the CRISPR-Cas9 gene editing system is shown as SEQ ID No.8 and SEQ ID No. 9;
sgRNA1：5’-cagcggccacccgaagctgccgg-3’(SEQ ID No.8)，
sgRNA2：5’-cggccacggaaccagctgcaggg-3’(SEQ ID No.9)。
the maize inbred line in the breeding method is an excellent maize inbred line which is already popularized and applied in production or is planned to be popularized and applied in production; such as B73, zheng 58, chang 7-2, ye 478, PH4CV, etc.
The invention also provides a breeding method of the dwarf corn hybrid, which comprises the step of hybridizing the dwarf corn inbred lines with different genetic backgrounds cultured by the method into parents to obtain the dwarf corn hybrid.
Compared with the prior art, the invention has the advantages and beneficial effects that: (1) The invention provides a new maize dwarf gene, widens the dwarf germplasm genetic basis of maize, enriches the genetic diversity of the maize dwarf gene and lays the germplasm basis for cultivating the novel dwarf maize. (2) The dwarf gene is controlled by a single recessive gene, the inheritance is simple, the linkage with bad characters is avoided, the breeding method is simple, and the breeding speed is high. (3) the dwarf gene of the invention has good effect. The dwarf gene variety bred by the invention has 12-50% lower plant height than the original variety, obvious plant height reduction effect, strong lodging resistance and close planting resistance, and can be used for increasing planting density and improving the unit yield of corn. (4) The CAPS marker of the maize dwarf gene provided by the invention can be used for early selection of the dwarf gene in the seedling stage, and particularly for the character controlled by a recessive gene, the CAPS marker can reduce the number of hybridization or backcross generations, greatly shorten the breeding period, improve the breeding efficiency and greatly reduce the breeding cost. (5) The corn dwarf related gene provided by the invention provides a new way for creating more dwarf genes, can greatly expand the number of the dwarf genes and provides more choices for corn dwarf breeding.
Drawings
FIG. 1 is a photograph showing the comparison of plant heights of the dwarf variety dMTPB73 and the original variety B73; wherein 1 is dMTPB73;2 is B73.
FIG. 2 is an electrophoresis diagram of molecular marker Chr3-3214320 polymorphism amplification; wherein P1 is dMTPB73; p2 is B73; f 1 Hybridization F for dMTPB73 and B73 1 Generation; 1 to 39 each represent F 2 And separating 39 dwarf single plants in the colony.
FIG. 3 is a CAPS marker identification electropherogram; wherein M is Marker; 1. 2 and 3 are homozygous dwarf gene plants; 4. 5 and 6 are homozygous normal long-stalked plants; 7. 8 and 9 are heterozygous tall plants.
FIG. 4 is a photograph of a transgenic plant Zmd024 in which the wild type gene is knocked out.
Detailed Description
The invention will be further explained and illustrated with reference to specific examples, but the invention is not limited thereto.
Example 1: test for cultivating dwarf corn variety by using corn allopolyploid MTF-1
The method comprises the following steps:
(1) In spring 2013, when the local temperature is raised to more than 10 ℃ in a Sichuan agriculture university breeding base, performing stump separation, cuttage or other asexual propagation on MTF-1 (Tripsazea Creammaize T.2n =76; suyueGui, master academic paper of Sichuan agriculture university, 2009) to obtain seedlings, and performing single-plant planting according to the plant row spacing of 2m multiplied by 2 m; meanwhile, a maize inbred line B73 (a maize inbred line disclosed in the united states) was sown in batches, planted at plant row spacing of 30cm × 70cm, with 20 plants per batch, at 3-day intervals per batch;
(2) Bagging MTF-1 female ears before spinning, cutting filaments to 2-3 cm after spinning, pollinating the MTF-1 female ears with B73 pollen, pollinating again every 2 days after the first pollination, pollinating for 3 times, marking on the paper bags after each pollination, harvesting single plants when mature, and obtaining F 1 Seed generation;
(3) In 2013 winter, F 1 The generation seeds are planted in a Yunnan Xishuangbanna breeding test base of Sichuan agricultural university according to the plant spacing of 30cm and the row spacing of 80 cm; meanwhile, sowing B73 in stages as in the step (1); with F 1 Backcrossing by taking B73 as a male parent to obtain BC 1 F 1 Seed generation;
(4) 2014 spring, and mixing BC obtained in the step (3) 1 F 1 Planting the seeds according to a single plant with the plant-row spacing of 30cm multiplied by 70 cm; selfing in blooming and pollen scattering period to obtain BC 1 F 2 Seed generation;
(5) And 2014 winter, and mixing the BC obtained in the step (4) 1 F 2 Planting the seeds according to a single plant with the plant-row spacing of 30cm multiplied by 70 cm; in the flowering and pollination period, B73 is used as a control, a single plant with the plant height being 12% -50% lower than that of the B73 plant is selected, andb73 backcrossing to obtain BC 2 F 2 Generation;
(6) Repeating the steps (4) to (5) 3 times to obtain BC 5 F 5 Generation;
(7) And obtaining BC 5 F 5 Selfing for 1 time to obtain BC 5 F 6 Generation; and (3) carrying out plant height identification on the selfing progeny according to the method in the step (5), wherein the plant heights are consistent and are not separated, so that a dwarf maize selfing line is obtained, and the dwarf maize selfing line is named as: dMTPB73.
(8) Measuring related agronomic traits such as plant height, spike height, stem node number, tassel length, tassel branch number and the like of dMTPB73 and B73; during measurement, the plants in the side rows with marginal effect are removed, and finally, the average value of the plants is calculated.
TABLE 1 measurement results (cm) of the plant height and related characters of dwarf maize dMTPB73 bred by the invention
| Name of Material | Plant height | High ear position | Number of stem nodes | Length of tassel | Number of tassel branches |
| dMTPB73 | 90.50 | 35.75 | 8.75 | 28.83 | 8.55 |
| B73 | 173.15 | 58.50 | 9.25 | 42.47 | 8.30 |
As can be seen from figure 1 and table 1, the plant height and the ear height of the dwarf maize dMTPB73 bred by the invention are respectively 52.3 percent and 61.1 percent of the original variety B73, namely compared with the original variety B73, the plant height and the ear height of the dMTPB73 are both obviously reduced, and the number of nodes of stems and the number of branches of tassels are not obviously different. The dwarf gene in the corn alloploid MTF-1 has good effects of reducing the plant height and the ear height; secondly, the gene is simple in heredity, and can be transferred into common corn through backcross, so that the method has wide application prospect in the aspect of corn dwarf breeding.
Example 2 genetic analysis of the maize dwarf trait of the invention
1. Test materials
(1) The inbred line dMTPB73 of dwarf maize bred in example 1.
(2)、B73。
2. Test method
Carrying out positive and negative hybridization by taking dwarf maize inbred lines dMTPB73 and B73 as parents to obtain positive and negative 2F 1 Combinations (i.e., dMTPB73 × B73 and B73 × dMTPB 73). Planting of all combinations of reciprocal crosses F 1 And in the two-row area, the row length is 2.0m, the row spacing is 0.8m, 7 pits are formed in each row, 2 plants are planted in each pit, and the plant height in the row area is measured in the pollination period. F 1 Selfing and backcrossing at the same time to obtain F 2 The population is backcrossed by taking dMTPB73 as a male parent to obtain a backcross generation BC 1 Group, planting BC respectively 1 Population and F 2 Group, at the blooming and powdering stage, survey F 2 、BC 1 The number of tall and short plants in the population was tested in the chi-square test.
TABLE 2 Quadrature F 1 Generation and backcrossing F 1 Plant height measurement results of generations
As can be seen from Table 2, 2F 1 The combined phenotype is a plant with normal height, and the plant height of the positive and reverse cross combination has no obvious difference, which indicates that the dwarf gene belongs to the control of the nuclear gene and is a recessive gene.
TABLE 3 genetic analysis results of the dwarf trait of the present invention
As can be seen from Table 3, BC was constructed from dMTPB73 and B73 1 The proportion of normal plants to dwarf plants in the population is respectively 28; f 2 The proportion of normal plants to dwarf plants in the segregation population is 1749, and the segregation proportion is 3 according to chi-square test, and the results show that the dwarf trait of the invention is controlled by recessive single gene.
Example 3 Gene mapping of maize dwarf Gene of the invention
Test materials (one): f constructed by dMTPB73 and B73 in example 2 2 Isolating the population.
(II) test method
(1) DNA extraction was performed by CTAB method. At F 2 In the separation population, 30 high-stalk single plants and 30 short-stalk single plants are randomly selected, leaf DNA extraction is respectively carried out by adopting a CTAB method, leaf DNA of each plant is mixed at an equimolar concentration to construct a high-stalk pool and a short-stalk pool, co-dominant polymorphic primer screening is carried out on parent dMTPB73, parent B73, the high-stalk pool and the short-stalk pool by 686 pairs of SSR primers uniformly distributed on 10 chromosomes of corn, and 31 pairs of polymorphic co-dominant primers are selected as a result. 31 pairs of codominant polymorphic primer pairs F 2 Separating 80 dwarf individual plant DNAs in the population for PCR amplification, performing linkage analysis according to polyacrylamide gel electrophoresis band type, and determining the chromosome 3The SSR primer bnlg1144 is linked with the dwarf gene dMTPB73. Therefore, the dwarf gene of the invention is preliminarily positioned on the chromosome 3.
(2) According to the fact that the dwarf gene is positioned on the No.3 chromosome in the step (1), 20 pairs of new SSR primers and InDel molecular markers are designed to carry out molecular marker encryption, then polymorphism analysis is carried out on two parents, a high stalk pool and a dwarf pool, and 4 pairs of markers Chr3-3214320, chr3-3549420, chr3-5847200 and Chr3-9451460 with polymorphism are screened as a result, and F is subjected to linkage marker pair selected by the method 2 613 dwarf single plants in the segregation population are analyzed, and according to the electrophoresis result, the band with the B73 band type is marked as 1, the band with the dMTPB73 band type of the dwarf gene is marked as 2, and the band with the amphiphilic band type is marked as 3. The genetic distance (cM) = (number of individuals of band 3 +2 × number of individuals of band 1)/(2 × number of total individuals) between each molecular marker and the target gene. Drawing a genetic linkage map by using software mapmaker according to the arrangement positions of genetic distance and molecular markers on the chromosome, and using the results (shown in figure 2) that the markers Chr3-3214320, chr3-3549420, chr3-9451460 and Chr3-10465520 on the No.3 chromosome are in parents, high stems, dwarf pools and F3-10465520 2 The segregation population has polymorphism and has linkage relation with the dwarf gene, and the dwarf gene can be determined to be positioned on the No.3 chromosome of the corn.
(3) Designing 64 pairs of new SSR primers and InDel molecular markers on No.3 chromosome of corn, performing polymorphism detection on parents and a height pool to obtain 7 markers with polymorphism (see table 4), and detecting F 2 613 dwarf single plants in the segregation population are analyzed, and a genetic linkage map is drawn according to the electrophoresis result to position the dwarf gene in a 7.2kb interval between molecular markers Chr3-5580580 and InDel-3. The MazeGDB database B73 Reference Gramene 4.0 shows that the localization interval has only 1 gene with the gene number Zm00001d039453, and the inventor names the dwarf gene as follows: d024. the gene is amplified by utilizing a PCR molecular experiment technology and sent to Kangbio-technology limited company for Sanger sequencing analysis, and the nucleotide sequence of the dwarf gene d024 is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. d024The corresponding nucleotide sequence of the B73 wild type gene is shown in SEQ ID No. 7.
TABLE 4 linkage markers for fine positioning screening
| Primer name | Primer sequence F (5 '→ 3') | Primer sequence R (5 '→ 3') |
| Chr3-4716040 | GGGGAGGGACTGCAGAAT | TGGAAAAGGAGAGGTCAGGA |
| Chr3-5410580 | TCTGTCAGTTTGGGCAATCA | TTGGCTTTATTCCCTCATCG |
| Chr3-5580580 | TGACGTGACTGAAATCTGCAT | CAGGCTCACCAGCAAAGACT |
| InDel-3 | GCTAGTCCCTAGACGGCAAAG | ACTTGCCAGCCGACTACCC |
| InDel-4 | GACTGGATCTGGGATACTGGGC | CTCTTGGACTCCCTCTCCACTTC |
| InDel-6 | CCGGACCCTTCCAAAACTCC | TGGGCTTGGTTGAGGACTTG |
| Chr3-5847200 | ATTTTGGACGAGTTGCTTGC | ACCGGTCGATTTTCGCTT |
Compared with the gene sequence of wild type B73, the 825 th base of the d024 exon 4 is mutated from T to C, the 831 th to 836 th bases are deleted, the 866 th base is mutated from T to C, and the 984 th base is mutated from C to A. Resulting in deletion of 278 th glycine residue and 279 th glutamic acid residue in the amino acid sequence of the protein encoded by the gene, and mutation of 289 th amino acid from valine residue (Val) to alanine residue (Ala). It is demonstrated that the gene mutation leads to the generation of dwarf mutants.
Example 4: design of CAPS marker of maize dwarf gene
The method comprises the following steps:
(1) 500bp sequences were cut on both sides of the genome of the exon 4 mutation site of gene d024 (nucleotide sequence SEQ ID No.7, in which the 825 th base was mutated from T to C) and used as templates for Primer synthesis, and 5 pairs of primers were designed using Primer 5 (see Table 5).
Table 5 primer List designed for screening CAPS markers
| Name of primer | Primer sequence F (5 '-3') | Primer sequence R (5 '-3') |
| CAPS-1 | GCTCGCCCAGGACAGTAGGA | TAGGTGGCATGGCACAACG |
| CAPS-2 | CAGACAGACAGGCAGACAGA | GCAGATTAGCCTCGTCACCA |
| CAPS-3 | GCCCAGGACAGTAGGAAGA | ACTCGCTGAGATACTTGACG |
| CAPS-4 | TTGGCAGCTGCAGCCTAGTA | ATGATCCCCTGTATCAGCGTG |
| CAPS-5 | GATCATACAGGAGAAGAGGC | CGGGCACTCGCTGAGATACTT |
(2) Separate extraction of F from example 2 2 Separating the leaf DNA of 10 long-stalk single plants and 10 short-stalk single plants of the colony, and screening CAPS specific markers by using the following PCR system and PCR program; the total volume of the PCR system is 25. Mu.L, wherein each of the forward and reverse primers is 1. Mu.L (10. Mu.M), the genomic DNA is 1. Mu.L, the PrimeSTAR Max Premix (2X) polymerase is 12.5. Mu.L, and ddH 2 Make up to 25. Mu.L of O. The PCR reaction program is as follows: pre-deforming at 98 ℃ for 5min; denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, and elongation at 72 ℃ for 10s for 35 cycles; overextension at 72 ℃ for 5min; storing at 12 deg.C.
The result shows that only the CAPS-3 primer pair can specifically amplify a target fragment with a product size containing a mutation site, the nucleotide sequence of the CAPS-3 primer pair is shown as SEQ ID No.4 and SEQ ID No.5, the other primers cannot specifically amplify the target fragment, and the target fragment is sent to Kangbio technologies and Limited company for Sanger sequencing analysis, so that the size of the DNA molecular fragment obtained by PCR amplification in the dwarf plant is 656bp, and the nucleotide sequence is shown as SEQ ID No. 6. The CAPS-3 primer pair is as follows:
CAPS-3F：5′-GCCCAGGACAGTAGGAAGA-3′(SEQ ID NO.4)；
CAPS-3R：5′-ACTCGCTGAGATACTTGACG-3′(SEQ ID NO.5)。
according to the nucleotide sequence of the dwarf gene shown as SEQ ID NO.1, because the 825 th base of the dwarf gene is mutated from T to C, the BtsCI enzyme cutting site of the site is deleted, and the fragment can not be cut by BtsCI enzyme. And the 825 th base of the homozygous high-stalk plant has no variation, and the BtsCI enzyme cutting site of the site has no deletion, so the fragment can be subjected to enzyme cutting segmentation by BtsCI enzyme.
Example 5 CAPS marker verification test of dwarf genes of the invention
(I) test materials
Example 2 in F 2 Separating the leaf DNA of 10 long-stalk single plants and 10 short-stalk single plants of the colony; CAPS-3 primer pairs in example 5.
(II) test method
(1) As in example 2F 2 Separating leaf DNA of 10 high-stalk single plants and 10 short-stalk single plants of the colony as substrates, and performing PCR amplification by using CAPS-3F (SEQ ID No. 4) and CAPS-3R (SEQ ID No. 5) as primers to obtain a PCR amplification product (SEQ ID No. 6); the PCR system and the PCR reaction procedure are described in example 5.
(2) Adding 1 mu L of BtsC I enzyme into the PCR amplification reactant obtained in the step (1), and preserving the heat at 50 ℃ for 15min; and (3) carrying out 1% agarose gel electrophoresis detection on the product after enzyme digestion.
The result (see figure 3) is that the homozygous dwarf plant contains 2 bands of 426bp and 230bp (see SEQ ID No. 3), the homozygous normal long-stalked plant contains 2 bands of 426bp and 172bp, and the heterozygous long-stalked plant contains 3 bands of 426bp, 230bp and 172 bp; wherein the 230bp strip is a specific strip of the dwarf gene and is a CAPS mark of the dwarf gene.
The results show that the CAPS marker can well distinguish normal wild type genotypes from mutant genotypes, the CAPS marker of the dwarf gene d024 is accurate and reliable, and the CAPS marker can be used for early seedling stage screening, so that material resources and labor are saved, breeding cost is saved, and breeding speed is accelerated. In conclusion, the genotype of the maize dwarf gene d024 can be quickly and accurately detected by using the CAPS marker due to the point mutation on the maize dwarf gene d024. And the CAPS marker is further used for auxiliary selection, and the maize dwarf gene d024 can be introduced into a maize strain by a hybridization or backcross method to culture a dwarf maize variety.
Example 6 verification test of maize dwarf-related genes of the invention
The method comprises the following steps:
(1) The wild-type gene Zm00001d039453 was annotated as cyp45 when queried in the maize GDB database B73 Reference grade 4.0 (https:// mail gdb. Org /).
(2) Designing sgRNAs by using on-line software CRISPR RGEN TOOLS (http:// www.rgenom. Net /), designing two sgRNAs (sgRNA 1 and sgRNA 2) according to a nucleotide sequence SEQ ID No.7 of a B73 wild-type gene cyp45 corresponding to d024,
sgRNA1：5’-cagcggccacccgaagctgccgg-3’(SEQ ID No.8)，
sgRNA2：5’-cggccacggaaccagctgcaggg-3’(SEQ ID No.9)。
(3) The method comprises the following steps of constructing a CRISPR/Cas9 vector by utilizing sgRNA1 and sgRNA2 provided by the inventor by Wuhan Boehringer Biotech limited company, converting the constructed vector into agrobacterium EHA105, and finally genetically converting B73 to obtain a gene knockout mutant, wherein the name is as follows: zmd024.
(4) And (3) planting the progeny of the plant with the knockout gene mutant, and measuring the relative agronomic characters such as plant height after selfing and purifying the stable phenotype.
TABLE 6 comparison of plant height and panicle height measurements (cm) of knockout mutant Zmd024
| Name of Material | Plant height | High ear position |
| Zmd024 | 83.51 | 33.86 |
| B73 | 173.15 | 58.50 |
Results from fig. 4 and table 6, it can be seen that the plant height and ear height of knockout mutant Zmd024 are significantly reduced compared to B73, which indicates that wild-type gene cyp45 is a dwarf-related gene of maize (the nucleotide sequence of which is shown in SEQ ID No. 7), and can be used for maize dwarf breeding by gene editing means.
Claims (10)
1. The maize dwarf gene is characterized in that the nucleotide sequence of the maize dwarf gene is shown as SEQ ID No. 1.
2. The protein coded by the maize dwarf gene as claimed in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.
3. The use of the maize dwarf gene of claim 1 or the protein of claim 2 for breeding dwarf maize varieties.
4. An expression vector containing the maize dwarf gene of claim 1.
5. The CAPS marker of the maize dwarf gene of claim 1, wherein the CAPS marker is a DNA molecular fragment; the size of the DNA molecular fragment is 230bp; the nucleotide sequence of the DNA molecule fragment is shown as SEQ ID No. 3.
6. The use of the CAPS marker of claim 5 for aiding in the selection of maize dwarf genes.
7. The use of the CAPS marker of claim 5 to breed dwarf corn varieties.
8. An application of a corn dwarf related gene with a nucleotide sequence shown as SEQ ID No.7 in cultivating dwarf corn varieties.
9. A breeding method for cultivating dwarf corn varieties by using corn dwarf related genes is characterized by comprising the steps of carrying out gene editing on the corn dwarf related genes with nucleotide sequences shown as SEQ ID No.7 in a corn inbred line by using a CRISPR-Cas9 gene editing system, and then selecting plants with plant heights 12-50% lower than those of original varieties in transgenic plants, namely the dwarf corn varieties; wherein the nucleotide sequence of sgRNA of the CRISPR-Cas9 gene editing system is shown as SEQ ID No.8 and SEQ ID No. 9;
sgRNA1：5’-cagcggccacccgaagctgccgg-3’(SEQ ID No.8)，
sgRNA2：5’-cggccacggaaccagctgcaggg-3’(SEQ ID No.9)。
10. a breeding method according to claim 9, characterized in that the maize inbred line is a superior maize inbred line that has been or is to be promoted for production.
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