CN108165654B - SSR marker SP _ SSR04 closely linked with spinach males and application thereof - Google Patents
SSR marker SP _ SSR04 closely linked with spinach males and application thereof Download PDFInfo
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Abstract
The invention discloses an SSR marker SP _ SSR04 closely linked with spinach males and application thereof in spinach gender identification, belonging to the technical field of molecular biology. The technical scheme provided by the invention has the key points that: the SSR marker SP _ SSR04 closely linked with spinach males is obtained by amplifying the following specific primer pair, the nucleotide sequence of the SSR marker SP _ SSR04 is shown as SEQ ID NO.1 in a sequence table, a specific strip of 176bp is amplified in a spinach male strain, no amplification product is generated in a spinach female strain, and the specific primer pair is as follows: an upstream primer: 5'-TTGATTGCCTCAAGGTGTTG-3', respectively; a downstream primer: 5'-TGGGTTCTTAGCCACTTTTTG-3' are provided. The molecular marker provided by the invention can be used for carrying out early sex identification on spinach cultivars, can also be applied to the early sex identification work of spinach wild species, screens female and male spinach plants in advance, greatly improves the breeding efficiency, and has important value in spinach production practice and breeding.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SSR marker SP _ SSR04 closely linked with spinach males and application thereof in spinach gender identification.
Background
Spinach (B)Spinacia oleraceaL.) alias Potentilla discolor and Red root vegetable, are first and second-year-old herbaceous plants of the genus Spinacia of the family Rinaceae with green leaves as the main product organs. Spinach is rich in nutrition, contains various vitamins, has strong cold resistance, short production period, high multiple cropping index and higher yield and output value, and is popular with producers and consumers. At present, the vegetable is generally cultivated in all parts of China, is one of main green leaf vegetables in spring, autumn and winter, and is also one of main export vegetables in China. According to the statistics of Food and Agricultural Organization (FAO) 2014 in the United nations, the annual yield of spinach in China is about 22.1 million tons, which accounts for 90.9 percent of the total yield of spinach in the world, and is the biggest spinach planting country and consumption country in the world. In 2014, the market scale of spinach seeds in China is 11.55 billion yuan, the spinach seeds are imported at 570.0t and the spinach seeds are exported at 60.2 t. The market scale of spinach seeds in China is predicted to reach 19 by 202075 billion yuan, the demand for spinach seeds is enormous. Since the beginning of the 21 st century, excellent spinach varieties cultivated by foreign seed companies such as san nice, Xianzheng da, Bantian and the like are successively and massively popularized and sold to China, and occupy most of seed markets of China. According to the report of the yellow bell (2013), the spinach production in China needs about 6800t of conventional seeds and about 1800t of hybrid seeds every year, the hybrid seeds for production in the market at present mainly come from companies such as san niese, Chengdang, Tiantian, Longjing and the like, and the spinach cultivated autonomously in China has few first-generation hybrids and limited popularization area. Therefore, it is necessary to improve the variety and quality of spinach varieties in China.
The molecular marker assisted breeding utilizes the characteristic that a molecular marker is tightly linked with a gene determining the target character, can detect the existence of the target gene by detecting the molecular marker, achieves the aim of selecting the target character, and has the advantages of rapidness, accuracy and no interference of environmental conditions. The method is an important technical means in breeding work, not only makes up for the defect of low accuracy of the traditional selection technology in crop breeding, but also accelerates the breeding process. An important link in molecular marker assisted breeding is to screen effective molecular markers closely linked with traits. Spinach has not only heterogynic plants but also isogynic plants, which are very good resources for commercial breeding, but spinach has almost no difference in the form of the male and female plants in the vegetative reproduction stage. Therefore, there is an urgent need to find molecular markers that are closely linked to sex and are suitable for most varieties for sex determination of spinach at an early stage. Akamatsu et al (1998) developed molecular markers T11A and V20A linked with spinach X/Y gene, which can be used for identifying spinach male and female plants. Onodera et al (2011) finally identified 10 AFLP markers closely linked to spinach gender determination site X/Y based on analysis of thousands of AFLP markers. Liudan et al (2015) converted the SRAP markers to SCAR markers S5.7 and S9.5 co-segregating with the Y gene. Kudoh et al (2017) developed 8 male-specific molecular markers based on the conversion of AFLP to SCAR markers and the design of primers on BAC end sequences. Except T11A and V20A, the markers are not universal, only can be applied to certain specific varieties, and sex-linked molecular markers developed by national scientific research institutions are very few, so that the markers are not favorable for protecting intellectual property of spinach varieties. According to the method, on the basis of sequencing spinach XX and YY plants by using a PacBio third-generation sequencing technology, bioinformatics software is used for analyzing male specific Contigs on a Y chromosome to obtain an SSR marker, and the marker has the characteristics of stability and suitability for different varieties and can be widely applied to molecular marker assisted breeding of spinach.
Disclosure of Invention
The invention solves the technical problem of providing an SSR marker SP _ SSR04 closely linked with spinach males and application thereof in spinach gender identification. Firstly, performing whole genome sequencing on XX and YY genomes of spinach by using a PacBio sequencing technology, and performing comparative genome analysis on the obtained whole genome sequence to obtain a male specific sequence; and then assembling the obtained male specific sequences by utilizing SOAPdenovo2 software to obtain male specific contigs, analyzing the obtained male specific contigs by utilizing RepeatMasker software, designing primers according to sequences at two ends of the SSR sequences, carrying out male and female identification on spinach cultivars by utilizing the designed primers, and finally carrying out male and female identification on the spinach cultivars and wild species.
The invention adopts the following technical scheme for solving the technical problems, and the SSR marker SP _ SSR04 closely linked with spinach males is characterized in that: the SSR marker SP _ SSR04 is obtained by amplifying the following specific primer pairs, the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 in a sequence table, 176bp specific bands are amplified in male spinach strains, no amplification products exist in female spinach strains, and the specific primer pairs are as follows:
an upstream primer: 5'-TTGATTGCCTCAAGGTGTTG-3', respectively;
a downstream primer: 5'-TGGGTTCTTAGCCACTTTTTG-3' are provided.
The invention discloses application of an SSR marker SP _ SSR04 closely linked with spinach males in spinach gender identification, which is characterized by comprising the following specific steps: firstly, extracting the genome DNA of spinach to be detected, and carrying out PCR amplification by using the genome DNA of the spinach to be detected as a template and using a specific primer pair, wherein the PCR amplification reaction system is as follows: 10 XPCR Buffer 2 uL, 2.5mM dNTP mix 1 uL, 10 uM/. mu.L upstream primer 1.25 uL, 10 uM/. mu.L downstream primer 1.25 uL, 5U/. mu.L Taq enzyme 0.2 uL, 100 and 200ng template DNA 1 uL and sterile water 13.3 uL, the PCR amplification reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 2min for 30 cycles; finally, extension is carried out for 7min at 72 ℃; and detecting the amplification product by agarose gel electrophoresis, wherein if a specific band of 176bp is amplified, the amplification product is male, and if the amplification product is not amplified, the amplification product is female.
Further preferably, the spinach to be detected is a spinach cultivar or a spinach wild species.
The molecular marker for identifying the spinach gender provided by the invention overcomes the problem of low breeding efficiency caused by that the male and female spinach plants can be distinguished only in the bolting stage in the prior art, and realizes the technical effect of identifying the spinach gender in the seedling stage. By using the specific primer pair provided by the invention to detect the genomic DNA of the spinach to be detected by adopting a PCR method and amplifying the SSR marker SP _ SSR04, when an amplification product has a specific strip of 176bp in agarose gel electrophoresis, the spinach to be detected can be judged to be male, if the specific strip does not exist, the spinach to be detected is female, the spinach can be accurately identified for sex, the spinach can be used for screening plants, and the breeding efficiency of the spinach is greatly improved. Compared with other marker molecules found at home and abroad, the SSR marker SP _ SSR04 is closely linked with spinach males and is positioned on a Y chromosome, so that a foundation is laid for cloning and sequencing of spinach sex determination genes, and the spinach sex determination gene sequence has very important value in spinach production practice and breeding.
Drawings
FIG. 1 is an agarose gel electrophoresis of SP _ SSR04 labeled spinach cultivar gender detection, M is marker, C is negative control;
FIG. 2 is an agarose gel electrophoresis of SP _ SSR04 labeled spinach in different cultivation and wild species sex detection, where M is marker (6 bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively), and the electrophoresis lanes are electrophoresis lanesSpinacia oleracea Cornell ID #273、Spinacia oleracea Chung Pu Lao、Spinacia oleracea Cornell ID #174、Spinacia oleracea DA JIAN YE BO CAI、Spinacia turkestanica SPI 155/83、Spinacia turkestanica1.1 andSpinacia tetrandraSPI 153/89, C was a negative control.
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Example 1
Acquisition and verification of SSR marker SP _ SSR04 closely linked to spinach male
1. Whole genome sequencing of spinach XX and YY genomes by PacBio sequencing technology
Spinach germplasm resources are screened, and spinach cultivar Cornell #9 population has female plants, male plants and hermaphrodite plants. The female plant is XX plant, the hybridization (XY x XY → YY: XY: XX) between the male and female isoplants is utilized to obtain YY plant, and the whole genome sequencing is carried out on the XX and YY genome of the spinach by utilizing the PacBio sequencing technology.
2. Comparing the obtained whole genome sequence and analyzing the genome to obtain male specific sequence
And performing comparative genome analysis by using bioinformatics analysis software to obtain a male specific sequence.
3. Assembling the obtained male specific sequence by utilizing SOAPdenovo2 software to obtain male specific contigs
4. Analyzing the obtained male specific contigs by using RepeatMasker software, and designing primers according to two end sequences of SSR sequences in the male specific contigs
5. Extraction of spinach cultivar Conell #9 genomic DNA
Extracting genome DNA of single spinach plants by a CTAB method, determining female genome and male genome according to flower organs, and equivalently mixing 10 single plant DNAs to construct a female genome pool and a male genome pool.
6. Verification of SSR marker SP _ SSR04 in spinach cultivar Conell #9
The designed specific primer pair is utilized:
an upstream primer: 5'-TTGATTGCCTCAAGGTGTTG-3', respectively;
a downstream primer: 5'-TGGGTTCTTAGCCACTTTTTG-3' are provided.
The spinach cultivar Conell #9 female and male gene pools are respectively amplified, agarose gel electrophoresis results show that 176bp specific bands appear in male genomes, amplification products do not exist in female genomes, and sequencing results of the amplification products are shown as SEQ ID NO.1 in a sequence table.
The PCR amplification reaction system is as follows: 10 XPCR Buffer (2. mu.L), 2.5mM dNTP mix (1. mu.L), 10. mu.M/. mu.L upstream primer (1.25. mu.L), 10. mu.M/. mu.L downstream primer (1.25. mu.L), 5U/. mu.L Taq enzyme (0.2. mu.L), 100. mu.L 200ng template DNA (1. mu.L) and sterile water (13.3. mu.L).
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 2min for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min.
Example 2
Application of SSR marker SP _ SSR04 closely linked with spinach males in sex identification of spinach cultivars and wild species
1. Extraction of single plant genome DNA of spinach cultivar and wild species
And (3) extracting the genome DNA of the single spinach plant to be detected by adopting a CTAB method.
2. Application of SSR marker SP _ SSR04 in sex identification of spinach cultivars and wild species
The designed specific primer pair is utilized:
an upstream primer: 5'-TTGATTGCCTCAAGGTGTTG-3'
A downstream primer: 5'-TGGGTTCTTAGCCACTTTTTG-3'
And amplifying the single plant genome DNA of the cultivated species and the wild species of the spinach to be detected respectively, wherein male plants are generated when 176bp specific bands appear as agarose gel electrophoresis results, and female plants are generated when amplification products do not exist.
The PCR amplification reaction system is as follows: 10 XPCR Buffer (2. mu.L), 2.5mM dNTP mix (1. mu.L), 10. mu.M/. mu.L upstream primer (1.25. mu.L), 10. mu.M/. mu.L downstream primer (1.25. mu.L), 5U/. mu.L Taq enzyme (0.2. mu.L), 100. mu.L 200ng template DNA (1. mu.L) and sterile water (13.3. mu.L).
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 2min for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
SEQUENCE LISTING
<110> university of south Henan university
<120> SSR marker SP _ SSR04 closely linked with spinach males and application thereof
<130>2018
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ttgattgcctcaaggtgttgcctctatatgtaaattttctagtagtgtgtagttgtaaca 60
ttgtgccagtttgagatgtattttccgccttcgtttttcgacataatggttcatttagtt 120
gtccatcttgttcgagaaatcaagcttcactactacaaaaagtggctaagaaccca 176
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ttgattgcctcaaggtgttg 20
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tgggttcttagccactttttg 21。
Sequence listing
<110> university of south Henan university
<120> SSR marker SP _ SSR04 closely linked with spinach males and application thereof
<130> 2018
<141> 2018-02-27
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ttgattgcct caaggtgttg cctctatatg taaattttct agtagtgtgt agttgtaaca 60
ttgtgccagt ttgagatgta ttttccgcct tcgtttttcg acataatggt tcatttagtt 120
gtccatcttg ttcgagaaat caagcttcac tactacaaaa agtggctaag aaccca 176
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ttgattgcct caaggtgttg 20
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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tgggttctta gccacttttt g 21
Claims (3)
1. An SSR marker SP _ SSR04 closely linked to spinach males, characterized by: the SSR marker SP _ SSR04 is obtained by amplifying the following specific primer pair, the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 in a sequence table, the SSR marker specific primer pair can amplify a specific strip of 176bp in an male spinach strain, no amplification product exists in a female spinach strain, and the specific primer pair is as follows:
an upstream primer: 5'-TTGATTGCCTCAAGGTGTTG-3', respectively;
a downstream primer: 5'-TGGGTTCTTAGCCACTTTTTG-3' are provided.
2. The use of SSR marker SP _ SSR04 closely linked to spinach males in claim 1 in spinach gender identification is characterized by comprising the following steps: firstly, extracting the genomic DNA of spinach to be detected, and carrying out PCR amplification by using the genomic DNA of spinach to be detected as a template and using a specific primer pair shown in claim 1, wherein the PCR amplification reaction system is as follows: 10 XPCR Buffer 2 uL, 2.5mM dNTP mix 1 uL, 10 uM/. mu.L upstream primer 1.25 uL, 10 uM/. mu.L downstream primer 1.25 uL, 5U/. mu.L Taq enzyme 0.2 uL, 100 and 200ng template DNA 1 uL and sterile water 13.3 uL, the PCR amplification reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 2min for 30 cycles; finally, extension is carried out for 7min at 72 ℃; and detecting the amplification product by agarose gel electrophoresis, wherein if a specific band of 176bp is amplified, the amplification product is male, and if the amplification product is not amplified, the amplification product is female.
3. Use of a spinach male tightly linked SSR marker SP _ SSR04 in spinach gender identification as claimed in claim 2, wherein: the spinach to be detected is spinach cultivars or spinach wild species.
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