1. An 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 No1 and SEQ.ID No2, and the nucleotide sequence of a male strain specific fragment obtained through amplification is shown as SEQ.ID No 3.

2. A method for obtaining the SSR molecular marker AerM01 for sex identification of actinidia arguta as claimed in claim 1, comprising the following steps:

s1: utilizing a genome sequenced by male and female actinidia lanchoe plant individuals to perform data analysis and comparison of genome level, and screening sequence fragments with specificity in the male and female actinidia lanchoe plant individuals;

s2: identifying SSR loci by using the screened specific sequences, and screening to obtain a potential SSR molecular marker AerM 01;

s3: synthesizing upstream and downstream primers of potential SSR molecular markers, extracting genomic DNA of actinidia arguta, performing conventional PCR and agarose gel electrophoresis experiments, and screening to obtain the SSR molecular marker AerM01 which can amplify a clear strip in a male plant but does not have any amplified strip in a female plant.

3. The method for obtaining SSR molecular marker AerM01 for sex identification of actinidia lanuginosa according to claim 2, wherein the potential SSR molecular marker in step S2 has a perfect structural type with a tandem arrangement of single repeat motifs, and specific flanking sequences are provided upstream and downstream of SSR molecular marker sites.

4. The use of the amplification primer of the SSR molecular marker AerM01 for sex identification of Actinidia arguta according to claim 1 in sex identification of Actinidia arguta (Actinidia inerantha) 'Huate (White)' and 'companion (Blank)' varieties.

Background

The kiwi fruit contains organic substances such as actinidine, proteolytic enzyme, tannin pectin and saccharides, trace elements such as calcium, potassium, selenium, zinc and germanium, 17 amino acids required by human body, and also contains abundant vitamin C, glucose, fructose, citric acid, malic acid and fat. The kiwi fruit is a large deciduous woody vine of a male-female variant plant, the male-female separation ratio of the filial generation of the kiwi fruit is about 1:1, a small number of pollination male plants are usually configured in a kiwi fruit orchard to ensure the orchard yield and fruit quality in view of the sex function of the male-female variant plant of the kiwi fruit, the identification of the male-female variant plant of the kiwi fruit needs to distinguish the male-female variant plant of the kiwi fruit from the morphological structure difference of the kiwi fruit flowers after the kiwi fruit blooms, and inconvenience and waste are brought to the breeding and planting of the kiwi fruit.

SSR (simple Sequence repeats) markers are a molecular marking technology based on specific primer PCR developed in recent years, also called microsatellite DNA (microspatellite DNA), and are series-connected repetitive sequences which are composed of several nucleotides (generally 1-6) as repetitive units and have the length of dozens of nucleotides. Sequences flanking each SSR are generally relatively conserved single copy sequences, and SSR markers have the following advantages over other molecular markers: (1) the quantity is rich, the whole genome is covered, and the disclosed polymorphism is high; (2) the gene has the characteristics of multiple alleles, and the provided information amount is high; (3) inherited in mendelian fashion, co-dominant; (4) each site is determined by the designed primer sequence, so that different laboratories can conveniently communicate with each other to develop the primers. However, the existing SSR molecular markers of kiwifruits depend on hybrid groups, the period is long, the manpower and capital investment is large, the selected potential SSR markers can only be limited in the selected sex determination interval, the number is limited, the SSR molecular markers are only suitable for Chinese kiwifruits, and the SSR molecular markers cannot be used for wild kiwifruits.

In view of the above-mentioned drawbacks, the inventors of the present invention have finally obtained the present invention through a long period of research and practice.

Disclosure of Invention

The invention aims to solve the problem that no SSR molecular marker of kiwifruit suitable for actinidia arguta populations exists, and provides an SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof.

In order to realize the purpose, the invention discloses an SSR molecular marker AerM01 for identifying sex of actinidia arguta, wherein the sequences of primers of the SSR molecular marker AerM01 are shown as SEQ.ID No1 and SEQ.ID No2, and the nucleotide sequence of the amplified male strain specific fragment is shown as SEQ.ID No 3.

The invention also discloses an obtaining method of the SSR molecular marker AerM01 primer for identifying the sex of the actinidia arguta, which comprises the following steps:

s1: utilizing a genome sequenced by male and female actinidia lanchoe plant individuals to perform data analysis and comparison of genome level, and screening sequence fragments with specificity in the male and female actinidia lanchoe plant individuals;

s2: identifying SSR loci by using the screened specific sequences, and screening to obtain potential SSR molecular markers;

s3: synthesizing upstream and downstream primers of potential SSR molecular markers, extracting genomic DNA of actinidia arguta, performing conventional PCR and agarose gel electrophoresis experiments, and screening to obtain the SSR molecular marker AerM01 which can amplify a clear strip in a male plant but does not have any amplified strip in a female plant.

The potential SSR molecular marker in the step S2 has a perfect structural type with a single repeated motif in tandem arrangement, and specific flanking sequences are arranged at the upstream and downstream of the SSR molecular marker site, namely, homologous sequences do not exist in another genome, so that the designed upstream and downstream primers cannot be extended to any band.

The invention also discloses application of the amplification primer of the SSR molecular marker AerM01 for identifying the sex of the male and female Actinidia arguta in sex identification of male and female Actinidia arguta in hybrid groups of Actinidia chinensis (White) and' companion (Blank) varieties.

Compared with the prior art, the invention has the beneficial effects that:

1. the existing SSR molecular markers of the kiwi fruits are obtained by screening Chinese kiwi fruit hybrid populations, and experiments prove that the SSR molecular markers cannot be applied to wild kiwi fruit populations, so that the SSR molecular markers mainly aim at the wild kiwi fruit populations and have specificity;

2. the invention directly utilizes published genome to compare the difference sequence, saves the work of constructing kiwi fruit hybrid population, has the advantages of rapidness, simplicity and convenience and low cost, and can be expanded to any object population with genome sequence;

3. the invention can systematically identify the sequence difference of male and female individuals on the genome level through genome comparative analysis, including any specific sequence segments scattered on different chromosomes, which is more comprehensive than a single region positioned by utilizing the traditional hybridization population, thereby providing more potential SSR molecular markers.

Drawings

FIG. 1 is a graph of the amplification bands of SSR molecular marker AerM01 in male and female actinidia lanuginosa plants;

FIG. 2 is a graph of the amplified bands of SSR molecular marker AerM01 in other male and female kiwi fruit strains.

Detailed Description

The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.

First, obtaining of SSR molecular marker AerM01

Test site: china Anhui province, Hefei city, Anhui agriculture university; test time: 2020 to

Materials: the material used in the experiment was a hybrid population of Actinidia eriantha 'White' and 'banko' varieties. The actinidia arguta is a unique actinidia arguta seed in China, the kiwi fruit tastes sour and sweet, the fragrance is strong, the content of vitamin C is extremely high, the adaptability is wide, the stress resistance is strong, and meanwhile, the actinidia arguta root is commonly used for treating diseases such as hepatitis, gastric cancer, nasopharyngeal cancer and the like in the traditional Chinese medicine.

1. Obtaining sequencing genome sequences of male and female actinidia lanchoe plant individuals 'Huate' and 'mate'. Wherein, the 'Huatt' genome of the actinidia arguta is published in 2019, and is downloaded from an international genomic database KGD of the actinidia arguta, and the website is as follows: http:// kiwifruitgene.org/; the actinidia arguta 'passenger' genome is sequenced and published in 2020, wherein a segment of sequence (2000 bp sequences respectively at the upstream and downstream) containing the SSR locus with the length of 4013bp is shown as SEQ.ID No 4.

2. Identification of SSR sites was performed on ` Walter ` and ` Beading ` genomic sequences, respectively, using MISA software. In this procedure, using a more stringent parameter set (10, 6, 5 minimal repeats of repeat motifs comprising one, two, three, four, five, six nucleotides, respectively), only the perfect SSR site type with tandem arrangement of single repeat motifs was retained for molecular marker screening.

3. Based on each identified SSR locus information, acquiring 200bp flanking sequences of upstream and downstream in respective genome sequences; then, the obtained flanking sequence is cross-aligned with another genome sequence (E value is less than or equal to 1E-5, homology is more than or equal to 80), and any aligned flanking sequence is considered as a non-specific site and is removed; finally, only SSR loci which are not aligned and have upstream and downstream flanking sequences are reserved as specific SSR loci.

4. Based on the upstream and downstream flanking sequences of the specific SSR locus, the software Primer3 is utilized to carry out batch design of the upstream and downstream primers of the locus, and the e-PCR software is utilized to identify the specificity in the genome sequence; non-specific primers were removed and only specific primers were retained for subsequent experimental validation.

5. Delivering the designed primer to a biological company for primer synthesis; carrying out PCR amplification on leaf DNA of male and female plants of the actinidia arguta by using synthesized primers, and searching for appropriate amplification conditions to ensure the definition and stability of amplified strips; the molecular marker obtained by screening can stably amplify a clear band in a male strain, but does not amplify any bands in a female strain, as shown in figure 1.

6. The PCR primers obtained by screening are used for molecular cloning and identification of known male and female sex individuals of other representative species (including Chinese kiwifruit, delicious kiwifruit, actinidia arguta and the like) of actinidia, and the molecular marker specificity is determined to be applied to the actinidia arguta, as shown in figure 2.

Second, application of SSR molecular marker AerM01

1. Application of SSR molecular marker in actinidia arguta hybrid population

Collecting 20 fresh and tender leaves of a hybrid population of Actinidia majus ('White' and 'Bankson (Blank)') at the university of agriculture in Anhui, wherein the fresh and tender leaves comprise 10 female plant samples and 10 male plant samples; meanwhile, 10 female and 10 male plant samples of a plurality of species (hereinafter referred to as other kiwifruits) such as Chinese kiwifruit, delicious kiwifruit, actinidia arguta, and the like are collected. Firstly, genome DNA of each plant is respectively extracted, and then 20 parts of actinidia arguta and other actinidia arguta genome DNA are subjected to PCR expansion by adopting molecular marker primers SEQ.ID No1 and SEQ.ID No 2.

The PCR amplification system is as follows: a10. mu.L reaction system included 0.3. mu.M each of the 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 5 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min.

The PCR amplification result is: a specific strip (SEQ. ID No3) with the size of 197bp is amplified in 10 male plant samples of the actinidia arguta, and a strip is not amplified in 10 female plant samples of the actinidia arguta; no bands were amplified in the other kiwi fruit 10 male and 10 female samples. Table 1 shows the results of agarose gel electrophoresis of the SSR molecular marker amplified in 10 male and 10 female plants randomly selected from actinidia arguta hybrid population; table 2 shows the results of agarose gel electrophoresis of the SSR molecular marker amplified in 10 male and 10 female plants randomly selected from other kiwifruits, wherein Aer001 and Aer011 are male and female plants of known Maohua kiwifruit and are used as positive and negative controls.

TABLE 1 agarose gel electrophoresis results of amplification of SSR molecular marker AerM01 in 10 male and 10 female strains randomly selected from actinidia arguta hybrid population

Sample numbering	Sex	Number of amplified bands	Sample numbering	Sex	Number of amplified bands
						Aer001	Male part	1	Aer011	Female part	0
Aer002	Male part	1	Aer012	Female part	0
						Aer003	Male part	1	Aer013	Female part	0
Aer004	Male part	1	Aer014	Female part	0
						Aer005	Male part	1	Aer015	Female part	0
Aer006	Male part	1	Aer016	Female part	0
						Aer007	Male part	1	Aer017	Female part	0
Aer008	Male part	1	Aer018	Female part	0
						Aer009	Male part	1	Aer019	Female part	0
Aer010	Male part	1	Aer020	Female part	0

TABLE 2 agarose gel electrophoresis results of the amplification of SSR molecular marker AerM01 in 10 male and 10 female strains randomly selected from other kiwi fruits

Sample numbering	Sex	Number of amplified bands	Sample numbering	Sex	Number of amplified bands
						Aer001	Male part	1	Aer011	Female part	0
Aot001	Male part	0	Aot011	Female part	0
						Aot002	Male part	0	Aot012	Female part	0
Aot003	Male part	0	Aot013	Female part	0
						Aot004	Male part	0	Aot014	Female part	0
Aot005	Male part	0	Aot015	Female part	0
						Aot006	Male part	0	Aot016	Female part	0
Aot007	Male part	0	Aot017	Female part	0
						Aot008	Male part	0	Aot018	Female part	0
Aot009	Male part	0	Aot019	Female part	0
						Aot010	Male part	0	Aot020	Female part	0

The results in tables 1 and 2 show that the SSR molecular marker AerM01 can be used for sex identification of wild kiwi fruits, can not be used for sex identification of kiwi fruits of other varieties, has specificity, and can be applied to varieties or species with known genome sequences and has universality.

The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. It will be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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