CN113403417B - SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof - Google Patents
SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular genetic breeding, in particular to an SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof, wherein the sequence of an SSR molecular marker AerM01 primer is shown as SEQ.ID No.1 and SEQ.ID No.2, and the nucleotide sequence of an amplified male plant specific fragment is shown as SEQ.ID No. 3; the invention directly utilizes published genome to compare difference sequences, omits the work of constructing kiwi hybridization groups, has the advantages of rapidness, simplicity and low cost, and can be expanded to any group, thereby having universality; the SSR molecular marker AerM01 is mainly aimed at actinidia arguta populations, and the SSR molecular marker AerM01 has specificity.
Description
Technical Field
The invention relates to the technical field of molecular genetic breeding, in particular to an SSR molecular marker AerM01 (AerM 01 is named for distinguishing other SSR molecular markers, aerM is three-letter abbreviation of Latin name Actinidia eriantha of actinidia arguta, M is the acronym of molecular marker) for sex identification of actinidia arguta and application thereof.
Background
The kiwi fruit contains organic matters such as kiwi fruit alkali, proteolytic enzyme, tannin pectin, saccharides and the like, trace elements such as calcium, potassium, selenium, zinc, germanium and the like, 17 amino acids required by a human body, and also contains rich vitamin C, grape acid, fructose, citric acid, malic acid and fat. The kiwi fruit is a large-scale fallen leaf wood vine plant of hermaphroditic strain, the male-female separation ratio of kiwi fruit filial generation is about 1:1, in view of the sex function of the hermaphroditic strain of the kiwi fruit, a small amount of pollination male plants are generally configured in the kiwi fruit garden to ensure the orchard yield and the fruit quality, the male-female strain of the kiwi fruit is distinguished from the morphological structure difference of kiwi fruit flowers after the kiwi fruit flowers bloom, and inconvenience and waste are brought to the breeding and planting of the kiwi fruit.
SSR (Simple Sequence Repeats) is a recently developed molecular marker technology based on specific primer PCR, also called microsatellite DNA (MicrosatelliteDNA), and is a series of repeated sequences of up to several tens of nucleotides consisting of several nucleotides (typically 1-6) as a repeated unit. The sequences flanking each SSR are typically relatively conserved single copy sequences, and SSR markers have the following advantages over other molecular markers: (1) The number is abundant, the whole genome is covered, and the disclosed polymorphism is high; (2) The characteristic of multiple alleles provides high information; (3) inherited in a mendelian manner, being co-dominant; (4) Each site is determined by the sequence of the designed primers, so that different laboratories can exchange with each other to develop the primers cooperatively. However, the existing kiwi fruit SSR molecular markers depend on hybridization groups, have long period and large manpower and capital investment, and the selected potential SSR markers are limited only to the sex determination interval screened, are limited in number, are only applicable to Chinese kiwi fruits and cannot be used for actinidia arguta.
In view of the above drawbacks, the present inventors have finally achieved the present invention through long-time studies and practices.
Disclosure of Invention
The invention aims to solve the problem that the kiwi fruit SSR molecular marker is not suitable for actinidia arguta groups, and provides an SSR molecular marker AerM01 for sexing of actinidia arguta and application thereof.
In order to achieve the aim, the invention discloses an SSR molecular marker AerM01 for sex identification of actinidia arguta, wherein the sequence of an SSR molecular marker AerM01 primer is shown as SEQ.ID No.1 and SEQ.ID No.2, and the nucleotide sequence of an amplified male strain specific fragment is shown as SEQ.ID No. 3.
The invention also discloses a method for obtaining the SSR molecular marker AerM01 primer for sex identification of actinidia arguta, which comprises the following steps:
s1: utilizing the genome sequenced by male and female individuals of actinidia arguta to analyze and compare genome level data, and screening sequence fragments with specificity in the male and female individuals;
s2: identifying SSR sites by using the screened specific sequences, and screening to obtain potential SSR molecular markers;
s3: and (3) synthesizing an upstream primer and a downstream primer of a potential SSR molecular marker, extracting genome DNA of actinidia arguta, performing conventional PCR and agarose gel electrophoresis experiments, and screening to obtain an SSR molecular marker AerM01 which can amplify a clear band in a male plant and does not have any amplified band in a female plant.
The potential SSR molecular marker in the step S2 has a perfect structure type with single repeated motifs arranged in series, and the upstream and downstream of the SSR molecular marker locus has specific flanking sequences, namely, no homologous sequence exists in the other genome, so that the designed upstream and downstream primers can not be expanded to any strip.
The invention also discloses application of the amplification primer of the SSR molecular marker AerM01 for sex identification of actinidia arguta in sex identification of male and female in hybridization populations of actinidia arguta (Actinidia eriantha) 'Huate (White)' and 'company passenger (Blank)'.
Compared with the prior art, the invention has the beneficial effects that:
1. the existing kiwi fruit SSR molecular markers are obtained by screening Chinese goosebeery hybridization groups, and experiments prove that the SSR molecular markers cannot be applied to the actinidia arguta groups, so that the invention is mainly specific to the actinidia arguta groups;
2. the invention directly utilizes published genome to compare difference sequences, omits the work of constructing kiwi hybridization groups, has the advantages of rapidness, simplicity and low cost, and can be expanded to any species group with genome sequences;
3. the invention can systematically identify the sequence differences of male and female individuals on the genome level through genome comparison analysis, including any specific sequence fragments scattered on different chromosomes, which are more comprehensive than single areas positioned by traditional hybridization groups, thus providing more potential SSR molecular markers.
Drawings
FIG. 1 is a schematic diagram of an SSR molecular marker AerM01 amplified in male and female actinidia arguta plants;
fig. 2 is a schematic diagram of an amplified band of SSR molecular marker aerom 01 in other kiwi fruit male and female strains.
Detailed Description
The above and further technical features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.
1. Acquisition of SSR molecular marker AerM01
Test site: the university of Anhui agriculture in the Hefei city of Anhui province of China; test time: 2020, 2020
Materials: the materials used for the experiments were a hybrid population of actinidia arguta (Actinidia eriantha) 'White)' and 'company passenger (Blank)' varieties. The actinidia arguta is a Chinese specific actinidia arguta seed, the fruit taste is sour and sweet, the aroma is rich, the vitamin C content is extremely high, the adaptability is wide, the stress resistance is strong, and meanwhile, the roots of the actinidia arguta are commonly used for treating diseases such as hepatitis, gastric cancer, nasopharyngeal carcinoma and the like in traditional Chinese medicine.
1. Sequencing genome sequences of 'Huate' and 'companion' of male and female individuals of actinidia arguta are obtained. The sequencing of the actinidia arguta 'Huate' genome in 2019 is published, and the genome is downloaded from the actinidia arguta international genome database KGD, and the website is: http:// kiwifriitgenome. Org/; the genome of actinidia arguta 'companion' is sequenced in 2020 to be published, and a section of sequence (2000 bp sequences on the upstream and downstream sides) with the length of 4013bp containing the SSR locus is shown as SEQ ID No. 4.
2. And (3) carrying out SSR locus identification on the genome sequences of 'Huate' and 'companion' by using MISA software. In this procedure, a more stringent parameter set (minimal number of repeats of repeat motifs comprising one, two, three, four, five, six nucleotides, 10, 6, 5, respectively) was used, leaving only perfect SSR site types with a single repeat motif tandem arrangement for molecular marker screening.
3. Based on the information of each identified SSR locus, flanking sequences of 200bp on the upstream and downstream of the identified SSR locus are obtained from the respective genome sequences; then, the obtained flanking sequence is subjected to cross alignment with another genome sequence (E value is less than or equal to 1E-5, homology is more than or equal to 80), and any flanking sequence on the alignment is considered to be a nonspecific site and removed; finally, only SSR sites with no alignment and with both upstream and downstream flanking sequences were retained as specific SSR sites.
4. Based on upstream and downstream flanking sequences of a specific SSR locus, carrying out batch design of upstream and downstream primers of the locus by using Primer3 software, and identifying the specificity of the Primer in a genome sequence by using e-PCR software; the non-specific primers were removed and only specific primers were retained for subsequent experimental verification.
5. Delivering the designed primer to a biological company for primer synthesis; carrying out PCR amplification on leaf DNA of male and female actinidia arguta individuals by using the synthesized primers, and fumbling proper amplification conditions to ensure the definition and stability of amplified bands; screening resulted in a stable amplified band in the male strain, but no amplified band in the female strain, as shown in FIG. 1.
6. The PCR primers obtained by screening are used for molecular cloning and identification of individuals with known sexes and sexes of other representative species of actinidia (including actinidia chinensis, actinidia deliciosa, actinidia arguta and the like), and the specificity of the molecular marker is determined to be applied to actinidia arguta, as shown in figure 2.
2. Application of SSR molecular marker AerM01
1. Application of SSR molecular marker in actinidia arguta hybridization population
Collecting 20 parts of fresh and tender leaves in a hybridization group of actinidia arguta ('Huate (White)' and 'company (Blank)') of Anhui agricultural university, wherein the leaves comprise 10 parts of female plants and 10 parts of male plant samples; meanwhile, 10 female and 10 male plant samples of a plurality of species (hereinafter referred to as other kiwi fruits) such as actinidia chinensis, actinidia deliciosa, actinidia arguta, and the like were collected. Firstly, respectively extracting genome DNA of each plant, and then carrying out PCR expansion on 20 parts of actinidia arguta and other actinidia arguta genome DNA by adopting molecular marker primers SEQ.ID No.1 and SEQ.ID No. 2.
The PCR amplification system comprises: a10. Mu.L reaction system comprised 0.3. Mu.M each of forward and reverse primers, 0.5mM dNTPs, 1. Mu.L of 10 XTaq buffer (containing MgCl) 2 ) 0.3units Taq polymerase, 100ng DNA template;
the PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72℃for 10min.
The PCR amplification results were: a specific band (SEQ. ID No 3) with 197bp size is amplified in 10 male strain samples of actinidia arguta, but No band is amplified in 10 female strain samples of actinidia arguta; no bands were amplified in the other kiwi fruit 10 male and 10 female samples. Table 1 shows agarose gel electrophoresis results of amplification of the SSR molecular markers in 10 male strains and 10 female strains randomly selected in the actinidia arguta hybrid population; table 2 shows agarose gel electrophoresis results of amplification of 10 male strains and 10 female strains randomly selected from other kiwi fruits by using the SSR molecular markers, wherein Aer001 and Aer011 are male strains and female strains of known actinidia arguta and are used for positive and negative control.
TABLE 1 agarose gel electrophoresis results of amplification of SSR molecular marker AerM01 in 10 Male and 10 female plants randomly selected in the actinidia arguta hybrid population
| Sample numbering | Sex (sex) | Number of amplified bands | Sample numbering | Sex (sex) | Number of amplified bands |
| Aer001 | Male male | 1 | Aer011 | Female | 0 |
| Aer002 | Male male | 1 | Aer012 | Female | 0 |
| Aer003 | Male male | 1 | Aer013 | Female | 0 |
| Aer004 | Male male | 1 | Aer014 | Female | 0 |
| Aer005 | Male male | 1 | Aer015 | Female | 0 |
| Aer006 | Male male | 1 | Aer016 | Female | 0 |
| Aer007 | Male male | 1 | Aer017 | Female | 0 |
| Aer008 | Male male | 1 | Aer018 | Female | 0 |
| Aer009 | Male male | 1 | Aer019 | Female | 0 |
| Aer010 | Male male | 1 | Aer020 | Female | 0 |
TABLE 2 agarose gel electrophoresis results of amplification of SSR molecular marker AerM01 in 10 Male and 10 female plants randomly selected in other kiwi fruits
| Sample numbering | Sex (sex) | Number of amplified bands | Sample numbering | Sex (sex) | Number of amplified bands |
| Aer001 | Male male | 1 | Aer011 | Female | 0 |
| Aot001 | Male male | 0 | Aot011 | Female | 0 |
| Aot002 | Male male | 0 | Aot012 | Female | 0 |
| Aot003 | Male male | 0 | Aot013 | Female | 0 |
| Aot004 | Male male | 0 | Aot014 | Female | 0 |
| Aot005 | Male male | 0 | Aot015 | Female | 0 |
| Aot006 | Male male | 0 | Aot016 | Female | 0 |
| Aot007 | Male male | 0 | Aot017 | Female | 0 |
| Aot008 | Male male | 0 | Aot018 | Female | 0 |
| Aot009 | Male male | 0 | Aot019 | Female | 0 |
| Aot010 | Male male | 0 | Aot020 | Female | 0 |
As can be seen from the results in Table 1 and Table 2, the SSR molecular marker AerM01 can be used for sexing of actinidia arguta, can not be used for sexing of actinidia arguta of other varieties, has specificity, and the method for obtaining the SSR molecular marker AerM01 for sexing of actinidia arguta can be applied to other varieties or species with known genome sequences, and has universality.
The foregoing description of the preferred embodiment of the invention is merely illustrative of the invention and is not intended to be limiting. It will be appreciated by persons skilled in the art that many variations, modifications, and even equivalents may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Anhui university of agriculture
<120> SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof
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ttgtgaaagt tgagagc 197
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actatgatga aacgtttagt ccaatggcga aacttacaac aatacgagtc ctacttgcac 60
ttgcagccaa caaacatgga atttgtggca gatggatgtg aacaatgctt ttcttcatgg 120
agagctggat cgagagatct acataatcca accaatgggt tttcagagcc aagatcatct 180
tgaatttgtg tgtaagctgc ggaaagcatt ttacttgatt gaaacaagca cccagggtgt 240
ggtatggtaa gattactgaa tttttaacac aaagtggtta ttcagtaaca cctgcagatt 300
ccaacctgtt ttgtcaaagc caatgaagga aactagctat cgtgctagtg tatgtggatg 360
acttaatcat aaccggtgat gatgagacag aaattcttcg aacgaaggag aatttattag 420
tccgttttca aatgaaggaa cttggacagc tcaaacactt cctttgttca gaggttgatc 480
acacacaaga aggaatattt ctttgtcaac aaaaatattc caaagatttg ttgaagaggt 540
tcggaatact cgaatgcaag ccaatttcga cgccgatgga accaaaattc aaaatgtgtt 600
gcacatgaag gaaaagattt ggaagatgcg acgatgtatc gacaattggt aggcagcctg 660
atttacttaa ccttgacttg acctgacatt tcttatgcag ttagtgtgat gagtcggtac 720
atgcaaaatt caaagaagcc tcatttggaa gtagttcgat gaatactgag atatgtaaat 780
catacaattg actatggtct ttttacaaga aaggtgaaga ctacaagtta gttggatact 840
acgatgctga ctatgcaaga gatcatgaca ccatgagatc aataactggg tacgtgttta 900
agcttggttc cggaataatt tcttggtgta gcaaaagaca gccgacgata tcattatcaa 960
ccactaaagt gaagtatcga gcagcagcaa tggcagctca agaaagtaca tggttgatac 1020
agctaatgaa taatctacat caactattag attatgtagt tccgttatac tatgacaatc 1080
agtcggcaat tcgtttggac gagaatccga tttttcatgc aagaactaag catgttgaag 1140
tgcactatca ttttatcaga gaaaaagttc tgcaagaaaa ttgagatgag acagatcaag 1200
acggatgatc aagttgcgga tttgttcaca aagtctaagt acaggcaagc tcgaaatgtt 1260
tcgtctacag cttgacatag tacagtgaat gagagctgac attgagggga ggttaagaat 1320
taatgtcagt caaaagtcaa atccaagcta tcatacaatg tccaagccca agtcctctct 1380
caagaattta ttgaagccca tagacacttc tcaaacatat aatagatatg ttgtggcaag 1440
tttgttgcaa agcaccttgt gtggatacaa attaattagc cttttattat ttgtgtggta 1500
agaaattaat tagtttaaat aaatgtgtgg tgtgtatttc atatagcttg ctataaatac 1560
ccaagttctc gagccttgta aattattaag gagtaaagaa ctaaataaga ctaaaaagtc 1620
ttatttatca aatattgatg tgaatggttt tgctgaaatg attaaacatt ggtggctgct 1680
agggcagatt atgtgtgcaa ttggattttt gttgctctga acaatttggg tgctgatgtt 1740
ttgcttctgt ttggatcctc ccttgcttga gagttgctta gcatagggtg ctttagattt 1800
ttgccttgtg tttttatgtt tctgtttgta tcttgccaac tttctacgat tttttgggtt 1860
taggtcgcac atgtttggct actgtgtatg tgccatccta gtcgagatag gtatccatag 1920
ccctgatgtt ttcatctaaa tccttattat tatctcgttg taatttttct cgagttaata 1980
atatttttct tttgctgatc aaaaaaaaaa aaagtcttat ttatagttga gtcttgagtg 2040
cttgtgaaag ttgagagctt tttttatatt ttttttagtt taataaaatc ttttgatgtg 2100
tctccaattt ttgagtgaat ttctttttga gatgtaaaat ttagagttgt atattctaaa 2160
ttttttctca acaataagtt tgttttgttt tacatgcaac aaatgagttc attttttctc 2220
ttccttcgtg taatttttgt ttattctgtg gctagctcac taactctgat ttctgtttga 2280
ctctctctcc tttctcaggc aaggtttctg ctgttagttt ggaaattctt tttcaaggtc 2340
aaagtaaaca taggctacgt tggattatag attttgaggg tgaaattgga ctattaatcg 2400
tgtggggccc actttaacta tattttagtg caaaaaacta aaccatgggc taaagtttat 2460
tgggattgtt aatcctctaa catggaggat taagaatccc aacatggagg ttaagaatcc 2520
cattggggtt ggtattacca atccaatccc atgggactat taatctaagg gaattgggat 2580
tggattgcta acctatcaaa ttaaaatctg ttattagacg tggccataat gataagcgaa 2640
agtgagaggt tatattttgt gtaattgaca tatcaaagtg gtagaatggt ctctacactg 2700
cacgtaggcc cgtagaaacg aggttttaag aggcgaatac accactataa tgcacccatt 2760
acacatataa aaccccctac ttaagtaggg gctgaaccac ttccacatgt ggactattta 2820
gggactttaa ttcattactc acacttgtgg gaactacggg gaactttttg aactggacta 2880
gacctaatac atcacagtat ataagaatgt ggttaaaacg cactgggaac tttggacaga 2940
tgagacattt ctgggatgca taaagagcac aattaattat gacacttttc tgaggcaata 3000
cgtggtttgg tatctcagca agagagaaaa ttaatgtcta atagcattca ctgaaaacaa 3060
acacatcctt atcttttcaa attgaaataa atgatttccc atctgtccca acagatctag 3120
gcctttgaaa gtgttcgacc cggtgcccag aataaacttc agggggcagt gcgtcatttg 3180
tgagcaggtt ttgtggcacc accccataaa tgtccgacat gtcaacttgg tggtggtgct 3240
ggcggactgg gccagcaacc accccatcgt cattgtccaa taatgggtgc aaccggtcaa 3300
aagtggcatt cgcgaatgat gcagccatta tgacaacggg acccgatgca actagtgcac 3360
caaccacagc cccacccgcg accaggcctt gtgcccccgc taagtaaatg gtcagcccgg 3420
tgacccctgg cggtgcaggt ggaggtaaga tggatccgag gagtgagagt atctcaaagc 3480
gtccatggag tgtcgccatt gcccccatga ggccggttga cgcaaagtaa cattggtgac 3540
gcaaccggtg gcgtttagta cacagatgcc tcgctgcttc ctccttgcaa agttggtcaa 3600
gctcttgctt atgtcacacc cagaggtcac ctccatggca tgagcccgga gagcatttgg 3660
gctgtcccgg tgatgataat gggctgcttg ggcttattct tcgagcccac tggcctgcca 3720
cgagtcctcc gaatggtttc tccctcagtc actgttggca tcgccttggg gactttattc 3780
atactgctat tctctggttc tgaacctttg gactcaatgc aaggaagagt agtaagatca 3840
gccattgatg gagttgttgt ttggcttgca gaggacgaga acatcactaa accttacctg 3900
tctggaaatg agtataaata agcaactcaa cctataaaat tatgatgaaa actaagagaa 3960
aaaatgaggg ttaggacatg gttaagtaac actaaccctg tatttgccta act 4013
Claims (4)
1. The SSR molecular marker AerM01 for sex identification of actinidia arguta is characterized in that the sequence of an SSR molecular marker AerM01 primer is shown as SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of an amplified male strain specific fragment is shown as SEQ ID No. 3.
2. A method for obtaining the SSR molecular marker aerom 01 for sexing of actinidia arguta according to claim 1, comprising the steps of:
s1: utilizing the genome sequenced by male and female individuals of actinidia arguta to analyze and compare genome level data, and screening sequence fragments with specificity in the male and female individuals;
s2: identifying SSR sites by using the screened specific sequences, and screening to obtain a potential SSR molecular marker AerM01;
s3: and (3) synthesizing an upstream primer and a downstream primer of a potential SSR molecular marker, extracting genome DNA of actinidia arguta, performing conventional PCR and agarose gel electrophoresis experiments, and screening to obtain an SSR molecular marker AerM01 which can amplify a clear band in a male plant and does not have any amplified band in a female plant.
3. The method for obtaining the SSR molecular marker AerM01 for sex identification of actinidia arguta according to claim 2, wherein the potential SSR molecular marker in the step S2 has a perfect structure type with single repeated motifs arranged in series, and the upstream and downstream of the SSR molecular marker locus have specific flanking sequences.
4. The amplification primer of SSR molecular marker AerM01 for sex identification of actinidia arguta according to claim 1Actinidia eriantha) Application of 'Huat (White)' and 'company' variety sexing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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