CN114717354B - Molecular marker combination, primer set, kit, identification method and application for identifying asparagus super-male plants - Google Patents
Molecular marker combination, primer set, kit, identification method and application for identifying asparagus super-male plants Download PDFInfo
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Abstract
The invention relates to a molecular marker combination for identifying an asparagus super-male plant, a primer group, a kit, an identification method and application, and belongs to the technical field of plant molecular genetics. The invention provides a molecular marker combination for identifying an asparagus super-male strain, which comprises an asparagus Y chromosome male specific gene SOFF fragment and an asparagus X chromosome specific region gene WIP2/NTT fragment. The molecular marker combination can be used for predicting the sex of the asparagus, particularly identifying the super-male plant, provides a simple and stable molecular marker for identifying the super-male plant of the asparagus, and has important application value in the aspect of auxiliary breeding of the total male molecular marker of the asparagus.
Description
Technical Field
The invention relates to the technical field of plant molecular genetics, and further relates to a plant sex identification technology based on molecular markers, in particular to a molecular marker combination, a primer group, a kit, an identification method and application for identifying an asparagus super-male plant.
Background
Asparagus (Asparagus officinalis L.) is a perennial herb plant of Asparagus genus of Asparagus family, its young stem is tender and delicious, nutritious, the flavor is unique, it is a high-grade vegetable with higher medicinal, nutritive and health-care value, sell the whole world, enjoy the reputation of "king of vegetable".
Asparagus are typically hermaphroditic plants with XY-type sex-determining systems. There are 4 sex phenotypes in natural populations of asparagus, female, male, super male and amphoteric strains, respectively, and their sex chromosome compositions are XX, XY, YY and XY, respectively. The amphiprotic plant can develop normal amphiprotic flowers, so that self-flower or cross-pollination, namely self-copulation, can be carried out, and is commonly used for breeding research of asparagus; the super-male plant can grow normally, has the same shape and physiological characteristics as the normal male plant, but the offspring hybridized with the female plant is male, so the super-male plant has important significance for asparagus breeding. In the actual cultivation process, under the same conditions, the male asparagus grows fast, has long service life and strong resistance, and the yield is about 25% higher than that of the female asparagus, so that the total male variety consisting of all male plants currently takes the dominant role of the global asparagus seed market. The asparagus total male breeding technology shows the advantages which cannot be achieved by other breeding methods, the key point of total male breeding is to screen out super-male plants with extremely small proportion in the population as male parents, but the sex cannot be judged from the plant phenotype in the young seedling stage of asparagus, and even after flowers are opened, only male plants and female plants can be judged, and the male plants and the super-male plants cannot be distinguished, so that the screening work of the super-male plants is very difficult. In the traditional screening, male plants or super-male plants and female plants are subjected to single-plant hybridization by adopting a genetic method, and the sex expression of the F1 offspring strain is seen, if the sex ratio of the F1 female and male is about 1:1, the male parent is a male plant; if F1 is male, the male parent is super-male. Although the method can identify the male strain and the super-male strain, a large amount of hybridization is needed, and the time and effort are consumed for 2-3 years.
The modern molecular marker technology can assist in breeding asparagus, improve the efficiency of screening super-male plants and greatly shorten the breeding period. However, most of the existing asparagus molecular markers are molecular markers for identifying male and female, the application of the existing asparagus molecular markers is limited to the identification and differentiation of female and male sexes, and only 2 cases of identification of supermale plants are reported at present. In 1998, reacon-Buttner et al screened 3 markers linked to sex genes using non-radioactive AFLP technique and BSA method, all 3 markers were no amplified bands in female, amplified bands in male, and amplified bands in super-male were brighter than in general male, and could be used as co-dominant markers for distinguishing male from super-male. Gebler et al (2007) found that 1 RAPD primer amplification band of about 700bp was closely linked to the asparagus sex gene: plants that appear strong bands are female plants, plants that appear weak bands are male plants, and the potential for no such bands are supermale plants. Therefore, although molecular markers capable of distinguishing asparagus stamen from superstamen exist at present, the molecular markers have great defects, and the only difference in strip brightness is that the type of the strip intensity is difficult to judge in actual production and cannot be directly applied to marker selection, and the other markers are unstable. Meanwhile, since none of the molecular markers developed by the former is based on sex-linked genes, none of the molecular markers is a functional marker coseparated from sex phenotype, and certain false positives and errors often exist in detection results.
In 2017, the genome sequence of asparagus is resolved, meanwhile, the Y chromosome (Harkess et al) is resolved, and in 2020, the X chromosome (Harkess et al) is also resolved, which lays a solid foundation for asparagus genetics research, especially for variety improvement on the molecular level. On the basis, if the gene segment for distinguishing the asparagus male strain from the super-male strain can be determined on the DNA level, the early and accurate identification of the asparagus male strain and the super-male strain can be realized.
Disclosure of Invention
The invention aims to provide a molecular marker combination, a primer group, a kit, an identification method and application for identifying asparagus super-male plants. The molecular marker combination can be used for predicting the sex of the asparagus, particularly identifying the super-male plant, provides a simple and stable molecular marker for identifying the super-male plant of the asparagus, and has important application value in the aspect of auxiliary breeding of the total male molecular marker of the asparagus.
The invention aims to solve the technical problem that when the molecular marker disclosed by the invention is used for identifying the super-male plants and the male plants of asparagus, a high-efficiency PCR primer and PCR amplification conditions are lacked.
The invention aims to solve the technical problem of how to establish a set of practical operation method on the basis of definitely using the molecular markers, the PCR primers and the PCR amplification conditions, so as to realize rapid and accurate identification of the male plants, the super-male plants and the female plants of asparagus.
The invention provides a molecular marker combination for identifying an asparagus super-male strain, which comprises an asparagus Y chromosome male specific gene SOFF fragment and an asparagus X chromosome specific region gene WIP2/NTT fragment, wherein the nucleotide sequence of the asparagus Y chromosome male specific gene SOFF fragment is shown as SEQ ID NO.1, and the nucleotide sequence of the asparagus X chromosome specific region gene WIP2/NTT fragment is shown as SEQ ID NO. 2.
The invention also provides a primer set for identifying the asparagus super-male strain, which comprises a primer pair YSMF and YSMR for amplifying the asparagus Y chromosome male specific gene SOFF fragment, and a primer pair XSMF and XSMR for amplifying the asparagus X chromosome specific region gene WIP2/NTT fragment; the nucleotide sequence of YSMF is shown as SEQ ID NO.3, the nucleotide sequence of YSMR is shown as SEQ ID NO.4, the nucleotide sequence of XSMF is shown as SEQ ID NO.5, and the nucleotide sequence of XSMR is shown as SEQ ID NO. 6.
The invention also provides a kit for identifying the asparagus super-male strain, which comprises the primer set and the reaction reagent according to the technical scheme, wherein the reaction reagent comprises 2X M5 Hiper super-light speed mix with blue dye.
The invention also provides application of the reagent for detecting the molecular marker combination in the technical scheme in identifying the sex of asparagus.
Preferably, the reagent comprises the primer set according to the above technical scheme.
Preferably, the sex includes one or more of a super-male, a female and a male.
The invention also provides a method for identifying the sex of asparagus based on the primer set or the kit according to the technical scheme, which comprises the following steps:
taking asparagus plant materials to be detected, and extracting total DNA of a single plant;
taking the total DNA of the single plant as a template, and carrying out PCR amplification in the same reaction system by utilizing the primer group or the kit according to the technical scheme to obtain an amplification product;
detecting the amplified product by gel electrophoresis;
and observing the electrophoresis result, wherein the single band with the size of 293bp appears as a super-male strain, the single band with the size of 245bp appears as a female strain, and the double bands with the sizes of 245bp and 293bp appear as male strains.
Preferably, the reaction system includes, per 20. Mu.L of the reaction system: 2 XM 5 Hiper super light speed mix with blue dye. Mu.L, primers at a concentration of 10. Mu. Mol/L each 0.5. Mu.L, template DNA at a concentration of 50 to 100 ng/. Mu.L, ddH 1. Mu.L 2 O 7μL。
Preferably, the conditions for PCR amplification include: pre-denaturation at 95 ℃ for 30s; denaturation at 94℃for 30s, annealing at 59℃for 30s, extension at 72℃for 40s, 30 cycles were performed; extending at 72℃for 5min.
Preferably, the conditions for the gel electrophoresis detection include: the amplification products were electrophoresed in 2g/mL agarose gel at 200V for 30min.
The invention provides a molecular marker combination for identifying an asparagus super-male plant. The invention develops functional STS molecular markers of a male-specific sex-determining gene SOFF and an X chromosome specific region gene WIP2/NTT on an asparagus Y chromosome based on nucleotide sequences of the two genes respectively. The 2 molecular markers provided by the invention can be amplified in the same reaction system through PCR experimental conditions, and the super-male plants, the male plants and the female plants of the asparagus can be identified according to the number and the positions of the bands, wherein the super-male plants are the single bands of 293bp, the female plants are the single bands of 245bp, and the male plants are the double bands of 245bp and 293 bp. The two pairs of STS primers can be used for predicting the sex of asparagus, particularly identifying the super-male plants, provide simple and stable molecular markers for identifying the super-male plants of the asparagus, and have important application value in the aspect of auxiliary breeding of the total-male molecular markers of the asparagus.
Drawings
FIG. 1 shows the amplification results of molecular markers provided by the invention on female, male and supermale individuals of a champion variety; wherein M is DL2000plus molecular weight marker, 1-5 is female strain, 6-10 is male strain, 11-15 is super-male strain;
FIG. 2 shows the verification result of the sex accuracy of asparagus identified by the molecular marker provided by the invention; wherein M is DL2000plus molecular weight marker, and 1-30 are 30 asparagus individual plants.
Detailed Description
The invention provides a molecular marker combination for identifying an asparagus super-male strain, which comprises an asparagus Y chromosome male specific gene SOFF fragment (Y chromosome specific molecular marker YSM) and an asparagus X chromosome specific region gene WIP2/NTT fragment (X chromosome specific molecular marker XSM), wherein the nucleotide sequence of the asparagus Y chromosome male specific gene SOFF fragment is shown as SEQ ID NO. 1: GAAAGCCCCATCGTAAATCATGATTTTTAGGAACTCATCATCATTCTTCATCCAAAAGGTGTCAAGATCTTGATAGCAGGCTCTGAAATCGTGCAGGTTCTTCCTCAGGGCTCGTATGATGTCATGGATCGACACTTGACATCGGATGAGGAAGTTCAGTAGCGCCCTGTGTTTATGACGTTCCATACGAAGGAGATTCAGATCTCCATTGTACCGTGGTCCAATGCTGCACGTTGATGGCTTATGGATCTGTGGGCTAATCCTTTTGAATGTGTCTGGTACGTCGTAGATTG the nucleotide sequence of the asparagus X chromosome specific region gene WIP2/NTT fragment is shown in SEQ ID NO. 2: CCCATGCCCGAAAGACCTAACGTGATCCTTCAAGCTCCTCTTATGCTTAAAATCAGACCCACAAGTACAGTACCAAAGTTTGCCGCAGTTCTTCTCGTGAGTCCTCCAGTCCCCCCTCACTGCGAAGGCCTTGCCACATTTTCGACACATAAACGGCTTGATGCCGTGTTTTCGCTTATAATGAGTTTGTAGCGTTCTGAAGTCTTTTAAGGGTTTCGATCGAGGGTGGTCGATGTTATTTCTGC. Specifically, 2 molecular markers are respectively positioned in the Y chromosome male specific gene SOFF and the X chromosome specific region gene WIP2/NTT of asparagus. The molecular marker combination can solve the technical problem that the identification of the super-male plants and the male plants of the asparagus is inaccurate in the prior art. Meanwhile, the molecular marker combination can be used for identifying female plants, male plants and super-male plants at the same time.
The invention also provides a primer set for identifying the asparagus super-male strain, which comprises a primer pair YSMF and YSMR for amplifying the asparagus Y chromosome male specific gene SOFF fragment, and a primer pair XSMF and XSMR for amplifying the asparagus X chromosome specific region gene WIP2/NTT fragment; the nucleotide sequence of YSMF is shown as SEQ ID NO. 3: 5'-GAAAGCCCCATCGTAAATCA-3', YSMR has the nucleotide sequence shown in SEQ ID NO. 4: the nucleotide sequence of 5'-CAATCTACGACGTACCAGAC-3', XSMF is shown in SEQ ID NO. 5: the nucleotide sequence of 5'-CCCATGCCCGAAAGACCTAAC-3', XSMR is shown in SEQ ID NO. 6: 5'-GCAGAAATAACATCGACCA CC-3'. The two pairs of primers can be amplified in the same reaction system, are used for detecting the male plants and the super male plants of the asparagus, can identify the female plants, provide simple and stable molecular markers for identifying the sex of the asparagus, and have important application value in the aspect of asparagus breeding.
The invention also provides a kit for identifying the asparagus super-male strain, which comprises the primer set and the reaction reagent according to the technical scheme, wherein the reaction reagent comprises 2X M5 Hiper super-light speed mix withblue dye.
The invention also provides application of the reagent for detecting the molecular marker combination in the technical scheme in identifying the sex of asparagus. In the present invention, the reagent preferably comprises the primer set according to the above-mentioned technical scheme. In the present invention, the sex preferably includes one or more of a super-male strain, a female strain and a male strain.
The invention also provides a method for identifying the sex of asparagus based on the primer set or the kit according to the technical scheme, which comprises the following steps:
taking asparagus plant materials to be detected, and extracting total DNA of a single plant;
taking the total DNA of the single plant as a template, and carrying out PCR amplification in the same reaction system by utilizing the primer group or the kit according to the technical scheme to obtain an amplification product;
detecting the amplified product by gel electrophoresis;
and observing the electrophoresis result, wherein the single band with the size of 293bp appears as a super-male strain, the single band with the size of 245bp appears as a female strain, and the double bands with the sizes of 245bp and 293bp appear as male strains.
In the present invention, the reaction system preferably includes, per 20. Mu.L of the reaction system: 2 XM 5 Hiper super light speed mix with blue dye. Mu.L, primers at a concentration of 10. Mu. Mol/L each 0.5. Mu.L, template DNA at a concentration of 50 to 100 ng/. Mu.L, ddH 1. Mu.L 2 O 7μL。
In the present invention, the conditions for PCR amplification preferably include: pre-denaturation at 95 ℃ for 30s; denaturation at 94℃for 30s, annealing at 59℃for 30s, extension at 72℃for 40s, 30 cycles were performed; extending at 72℃for 5min.
In the present invention, the conditions for the gel electrophoresis detection include: the amplification products were electrophoresed in 2g/mL agarose gel at 200V for 30min. After gel electrophoresis detection, the invention preferably uses a gel imaging system for observation and photographing. The gel electrophoresis detection of the present invention is preferably performed using ethidium bromide staining. The present invention preferably uses DL2000plus markers as molecular weight markers.
The invention uses designed specific primer sequence, takes asparagus individual genome DNA as a template to carry out PCR amplification, electrophoreses and separates amplified products, observes and photographs under a gel imaging system, records that the bands with the size of 293bp appear in super-male individuals, that the bands with the size of 245bp appear in female individuals and that the bands appear in male individuals simultaneously.
The molecular marker combination, the primer set, the kit, the identification method and the application for identifying the asparagus super-male strain are described in further detail below by combining specific embodiments, and the technical scheme of the invention comprises but is not limited to the following embodiments.
Example 1
Primer design
Based on the nucleotide sequences of the asparagus Y chromosome specific region gene and the X chromosome specific region gene, screening the sites with different nucleotide sequences of the Y chromosome specific region gene in the XX female genome and the sites with different nucleotide sequences of the X chromosome specific region gene in the YY super male genome by Blastn comparison, and respectively designing and developing the Y chromosome and X chromosome specific functional molecular markers by combining Primer premier5.0 and Oligo7 software. The two markers are named YSM and XSM, are respectively derived from a Y chromosome specific region gene SOFF and an X chromosome specific region gene WIP2/NTT, and simultaneously define forward and reverse primer sequences and corresponding characteristic bands of the two molecular markers. Specifically, the results are shown in Table 1.
TABLE 1 characterization of specific STS molecular markers for the Y and X chromosomes of asparagus
Example 2
STS molecular marker polymorphism analysis between female, male and super-male asparagus individuals
(1) Test materials
The breeding of the variety "champion" from the Shandong province Weifang city agricultural academy of sciences. Randomly selecting 5 plants of asparagus male plants, super male plants and female plants with known flowering sexes, respectively taking leaves of the 5 plants, and extracting plant genome DNA by adopting a CTAB method.
(2) PCR reaction system
The 20. Mu.L PCR reaction system comprises: 2 XM 5 Hiper super light speed mix with blue dye. Mu.L, primers at a concentration of 10. Mu. Mol/L each 0.5. Mu.L, template DNA at a concentration of 50 to 100 ng/. Mu.L, ddH 1. Mu.L 2 O 7μL；
(3) PCR amplification reaction procedure
Pre-denaturation at 95 ℃ for 30s; denaturation at 94℃for 30s, annealing at 59℃for 30s, extension at 72℃for 40s, 30 cycles were performed; extending at 72 ℃ for 5min;
(4) Agarose gel electrophoresis
The amplified product is electrophoresed for 30min in 2g/mL agarose gel at 200V, and observed and photographed by a gel imaging system.
(5) Amplification results and analysis
The nucleotide sequences of the characteristic bands amplified by the 2 molecular markers are shown in figure 1, the super-male strain amplifies a single band of 293bp, the male strain amplifies two bands of 245bp and 293bp, and the female strain amplifies a single band of 245 bp.
By agarose gel electrophoresis, two markers amplified a 245bp and 293bp band in the male genome, a 293bp single band in the super-male genome and a 245bp single band in the female genome.
Example 3
STS molecular marker identification sex accuracy verification
(1) Experimental materials
The strain "champion" selected and bred by the Shandong Weifang agricultural academy of sciences. Randomly selecting 30 asparagus plants with known sexes from the selfing offspring of hermaphrodite plants, and respectively taking the leaves.
(2) DNA extraction, PCR amplification System, PCR amplification reaction procedure, agarose gel electrophoresis, etc., were performed according to the method of example 2. And carrying out PCR amplification on 30 plants by using molecular markers.
(3) Detection results of male, super-male and female strains
And judging the sex of the single plant according to the number and the size of target bands amplified by PCR (polymerase chain reaction) through agarose gel electrophoresis, and rapidly distinguishing male, super-male and female plants. As a result, the super-male plant 2 with 293bp single band was 14 total male plants with 245bp and 293bp double bands, and the female plant 14 total female plants with 245bp single band were shown in FIG. 2. The identification result is consistent with the actual situation, and the identification accuracy is 100%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (9)
1. A molecular marker combination for identifying asparagus super-male plants is characterized by comprising asparagus Y chromosome male specific genesSOFFFragment and asparagus X chromosome specific region geneWIP2/NTTFragment, said asparagus Y chromosome male specific geneSOFFThe nucleotide sequence of the fragment is shown as SEQ ID NO.1, and the asparagus X chromosome specific region geneWIP2/NTTThe nucleotide sequence of the fragment is shown as SEQ ID NO. 2.
2. A primer set for identifying asparagus super-male plants is characterized by comprising the step of amplifying asparagus Y chromosome male specific genesSOFFPrimer pair YSMF and YSMR of fragment and amplification of asparagus X chromosome specific region geneWIP2/NTTPrimer pairs XSMF and XSMR of the fragments; the nucleotide sequence of YSMF is shown as SEQ ID NO.3, the nucleotide sequence of YSMR is shown as SEQ ID NO.4, the nucleotide sequence of XSMF is shown as SEQ ID NO.5, and the nucleotide sequence of XSMR is shown as SEQ ID NO. 6.
3. A kit for identifying an asparagus super-male strain, comprising the primer set of claim 2 and a reaction reagent, wherein the reaction reagent comprises 2x M5 Hiper super-light speed mix with blue dye.
4. Use of a reagent for detecting the combination of molecular markers of claim 1 for identifying sex of asparagus; the sex includes one or more than two of super-male plants, female plants and male plants.
5. The use according to claim 4, wherein the reagent comprises the primer set of claim 2.
6. A method for identifying the sex of asparagus based on the primer set of claim 2 or the kit of claim 3, comprising the steps of:
taking asparagus plant materials to be detected, and extracting total DNA of a single plant;
using the single total DNA as a template, and performing PCR amplification in the same reaction system by using the primer set of claim 2 or the kit of claim 3 to obtain an amplification product;
detecting the amplified product by gel electrophoresis;
as a result of observation, the single band of 293bp, the female band of 245bp and the male band of 245bp and 293bp were observed.
7. The method according to claim 6, wherein the reaction system comprises, per 20. Mu.L of the reaction system: 2 XM 5 Hiper super light speed mix with blue dye. Mu.L, primers at 10. Mu. Mol/L each 0.5. Mu.L, template DNA at 50-100 ng/. Mu.L, ddH 2 O 7 μL。
8. The method of claim 6, wherein the conditions of PCR amplification comprise: 95. pre-denaturation at 30 ℃ s; 94. denaturation at 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 40s, performing 30 cycles; 72. extending at a temperature of 5min.
9. The method of claim 6, wherein the conditions for gel electrophoresis detection comprise: the amplification products were electrophoresed in 2g/mL agarose gel at 200V voltage for 30min.
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| KR100685518B1 (en) * | 2005-09-30 | 2007-03-09 | 대한민국 | Primer for identification of sex in asparagus and method of identification of sex in asparagus |
| WO2016110780A2 (en) * | 2015-01-09 | 2016-07-14 | Limgroup B.V. | Sex determination genes and their use in breeding |
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| KR100685518B1 (en) * | 2005-09-30 | 2007-03-09 | 대한민국 | Primer for identification of sex in asparagus and method of identification of sex in asparagus |
| WO2016110780A2 (en) * | 2015-01-09 | 2016-07-14 | Limgroup B.V. | Sex determination genes and their use in breeding |
| CN107708408A (en) * | 2015-01-09 | 2018-02-16 | 利姆集团有限公司 | Sex determining gene and its purposes in breeding |
| CN107201404A (en) * | 2017-06-15 | 2017-09-26 | 江西省农业科学院蔬菜花卉研究所 | A kind of molecular biology identification method and its application for Asparagus dioecian plant sex |
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