CN107805672B

CN107805672B - Method for identifying authenticity of Indian pumpkin and Chinese pumpkin hybrid stock varieties

Info

Publication number: CN107805672B
Application number: CN201610837480.6A
Authority: CN
Inventors: 宋慧; 张香琴; 王迎儿; 黄芸萍
Original assignee: Ningbo Academy of Agricultural Sciences
Current assignee: Ningbo Academy of Agricultural Sciences
Priority date: 2016-09-09
Filing date: 2016-09-09
Publication date: 2021-03-12
Anticipated expiration: 2036-09-09
Also published as: CN107805672A

Abstract

The invention relates to a method for identifying the authenticity of a variety of a hybrid stock of Indian pumpkin and Chinese pumpkin, belonging to the field of biology. The identification of variety genuineness is increasingly important to maintaining the interests of producers and breeders. The variety of the hybrid pumpkin stocks in the seal is similar in appearance, the authenticity is discriminated by using morphological indexes, the consumed time is long, and the reliability is poor, so that the method for identifying the authenticity of the variety by using the molecular marker becomes urgent. The technical scheme is that 5 pairs of SSR primers (SEQ ID NO.1-SEQ ID NO.10) are utilized to perform PCR amplification on three stock varieties and a contrast material, 9 generated difference sites are assigned with values of '1' and '0', and a variety fingerprint is constructed for authenticity identification. The method disclosed by the invention can be used for identifying and protecting the variety of the hybrid pumpkin rootstock, namely Yongyu No.7 ', Yongyu No.8 ' and Yongyu No.10 ', more accurately and more effectively by a more sensitive identification means and lower time and labor cost.

Description

Method for identifying authenticity of Indian pumpkin and Chinese pumpkin hybrid stock varieties

One, the technical field

The invention belongs to the field of biology, and particularly relates to an identification method for authenticity of Yongzhen series Indian pumpkin multiplied by Chinese pumpkin hybrid stock varieties 'Yongzhen No. 7', 'Yongzhen No. 8' and 'Yongzhen No. 10'.

Second, background Art

Watermelon fusarium wilt causes continuous cropping obstacles which commonly occur in melon planting areas and are a great problem in melon production. According to statistics, the incidence rate of watermelon fusarium wilt per year can reach 10-30%, and huge economic loss is caused by serious plots and even dead production. At present, because of the restriction of land resources and the intractable and poor crop rotation and chemical prevention and control effects of pathogenic bacteria of the blight, and the problems of environmental pollution, food safety and the like, grafting is an effective means for overcoming continuous cropping obstacles and the blight in watermelon production.

The pumpkin is one of the oldest ancient crops cultivated by human, has various varieties and rich variation, is not only a health-care colorful vegetable on a human dining table, but also an important stock resource for overcoming continuous cropping obstacles in melon production. The pumpkin seeds which are common in production mainly comprise Chinese pumpkin (Cucurbita moschata), Indian pumpkin (Cucurbita maxima), American pumpkin (Cucurbita pepo) and the like. These species of pumpkin vary in origin and ecological adaptability. The Indian pumpkin originates from the dry zone of south America Peru, Boliviana and northern Argentina, and virus diseases and powdery mildew are seriously developed under the condition of high temperature. The Chinese pumpkin originates from Mexico and Central and south America, has a long cultivation history in China, generally shows heat resistance, barren resistance and disease resistance, and has wide adaptability. Through interspecific distant hybridization, the advantages of different parents can be polymerized, new phenotypic variation is formed, and abundant germplasm resources are provided for stock breeding. Yongzhen No.7 ', Yongzhen No.8 ' and Yongzhen No.10 ' are a series of Indian pumpkin multiplied by Chinese pumpkin hybrid stock varieties which are independently bred aiming at watermelon grafting cultivation by Ningbo city agricultural science research institute. The series has the resistance of the Chinese pumpkin and the grafting compatibility of the Indian pumpkin, has no adverse effect on the quality of the watermelon, and is popular with melon farmers.

With the popularization of cross pumpkin stock varieties in channel series imprinting, the identification of variety authenticity is increasingly important to the benefit of maintenance producers and breeders. However, the identification of the stock varieties is carried out by conventional methods such as plant morphological identification. The methods are long in time consumption, easy to be influenced by environment and poor in reliability, and the method is difficult to identify the truth in the face of the hybrid pumpkin stock materials in the imprints with various varieties and similar appearance phenotypes.

The new species of the rootstock, namely Yongyu No.7, Yongyu No.8 and Yongyu No.10, are only subjected to field morphological purity identification so far, a convenient and effective variety authenticity identification method is lacked, and the variety protection is far from being lacked.

Third, the invention

Aiming at the problems, the invention provides a method for identifying the authenticity of hybrid pumpkin rootstock varieties 'Yongyu No. 7', 'Yongyu No. 8' and 'Yongyu No. 10' in Yongyu series imprinting by using a DNA fingerprint.

In order to achieve the purpose of the invention, the inventor provides the following technical scheme: extracting young leaf DNA of 'Yongzhen No. 7', 'Yongzhen No. 8' and 'Yongzhen No. 10' and comparison varieties thereof by adopting a CTAB method; performing PCR amplification by using 5 pairs of SSR primers (the corresponding relation between the primer names and the sequence numbers in the sequence table is shown in table 1, and the specific sequences of the primers are shown in the sequence table finally attached to the specification); and (3) carrying out high-resolution agarose electrophoresis separation on the amplification product, dyeing with ethidium bromide, and detecting polymorphism on an ultraviolet fluoroscope. The polymorphism of the amplification product reflects the polymorphism of the genome. The difference allelic sites, the amplified band is marked as '1', and the band at the same position is not marked as '0'; the differential amplification fragments are converted into character strings consisting of 1 and 0, namely, a DNA fingerprint is formed to distinguish 3 copies of the hybrid pumpkin rootstock varieties and controls thereof.

The invention has the advantages that: compared with conventional morphological identification, the method has the advantages of accurate and reliable identification result, short time consumption, no environmental influence, simple operation, capability of identifying a plurality of varieties simultaneously, convenience and rapidness.

Description of the drawings

FIG. 1 shows the amplification of SSR primers N11/12, N41/42, N51/52, N121/122 and N155/156 in Conn No.7, Conn No.8, Conn No.10 and

control materials

5558, 5559, MingXiu and Jinjiatian in the present invention.

In FIG. 1, the three left pictures are from top to bottom the amplifications of SSR primers N11/12, N41/42 and N51/52 in ` Yongzhen No.7 `, ` Yongzhen No.8 `, ` Yongzhen No.10 ` and

reference materials

5558, 5559, ` Mingxu ` and ` Jinjiatian `, respectively; the two images on the right are from top to bottom the amplifications of SSR primers N121/122 and N155/156 in ` Langcheng No.7 `, ` Langcheng No.8 `, ` Langcheng No.10 ` and

control materials

5558, 5559, ` MingXiu ` and ` Jinjiatian `, respectively. The two groups of picture samples are arranged from left to right in sequence as DNA Marker, Yongzhen No.7, Yongzhen No.8, Yongzhen No.10, 5558, 5559, MingXiu and Jinjiatian. The left side of the picture is marked with DNAmarker standard molecular weight, and the right side is marked with the name of the primer and the size of the segment of the differential site.

Fifth, detailed description of the invention

1. Sampling: seeding No.7 Yongzhen, No.8 Yongzhen and No.10 Yongzhen, collecting 1g of tender leaves when the plant grows to 5-6 true leaves, and storing at-80 deg.C.

2. DNA extraction: fresh leaf total genomic DNA was extracted by the CTAB method (see: Murray HG, Thompson WF. Rapid isolation of high weight DNA. nucleic acids Res, 1980, 8: 4321).

3. And (3) PCR amplification: the mixing system was 15. mu.l (Fazio G, Staub JE, Stevens MR. genetic mapping and QTL analysis of Horticultural traits in cumber L.) using recombinant expressed lines, the same Genet, 2003, 107: 864. sup. 874), including 1.5. mu.l dNTP (1.25mM), 1.5. mu.l 10 XBuffer, 1.5. mu.l MgCl₂(25mM), 2.0ul of primers (5 mM; 1.0ul of each primer of SSR), 5ul of DNA (10ng/ul), 0.05ul of Taq (1U), and 3.45ul of ultrapure water. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 1 min; denaturation at 93 deg.C for 1min, annealing at 40 deg.C for 1min, and extension at 72 deg.C for 2min (40 cycles); extension at 72 ℃ for 1 min.

4. And (3) detecting a PCR product: 2.5% high resolution standard gel agarose (DNA fragment separation range 40-1Kbp) was used. Add 20. mu.l EB (ethidium bromide ) for color development. Add 2. mu.l of Loading Dye to the PCR product, electrophorese for 2-3 hours, voltage 200-. After the electrophoresis was finished, the results were observed by using a gel imaging system.

5. Data collection and analysis: evaluation was performed on the results of 5 pairs of SSR primers N11/12, N41/42, N51/52, N121/122 and N155/156 in ` Langchong No.7 `, ` Langchong No.8 `, ` Langchong No.10 ` and

control materials

5558, 5559, ` MingXiu ` and ` Jinjiatian `, and the result was marked as "1" when a band was amplified from one allele and "0" when no band was amplified at the same site. These differentially amplified fragments were converted into a string consisting of 1 and 0, constituting a variety fingerprint (Table 1). 9 polymorphic sites generated by 5 molecular markers are utilized to construct a fingerprint map of Yongzhen No.7 'of 1-1-0-1-0-1-0-1, a fingerprint map of Yongzhen No. 8' of 0-0-1-1-0-1-0, a fingerprint map of Yongzhen No.10 'of 0-0-0-0-0-0-0-0-0-0, a fingerprint map of 5558 of 1-0-1-0-1-1-0-1-0, a fingerprint map of 5559 of 1-1-0-1-1-0-1-0-1-0, a fingerprint of MingXiu' of 1-1-0-1-1-0-1-0 and Jinjiatian ' the fingerprint spectrum is 0-1-0-1-1-0-1-0-1. The fingerprint maps can effectively distinguish the three varieties, and can also distinguish the three varieties from similar varieties with easily confused forms, thereby playing roles in variety identification and protection.

TABLE 1 names and SSR fingerprints of hybrid pumpkin rootstock varieties and control materials in test prints

Claims

1. A method for identifying authenticity of a cucurbit variety of cucurbit squash x cucurbita moschata hybrids, the rootstock variety comprising: the method comprises the following steps of 'an anvil No. 7' and 'an anvil No. 8':

(1) sampling: sowing Yongzhen No.7, Yongzhen No.8 and their contrast material, collecting 1g of tender leaves when the plant grows to 5-6 true leaves, and storing at-80 deg.C;

(2) DNA extraction: extracting fresh leaf total genome DNA by referring to a CTAB method;

(3) performing PCR amplification by using 5 pairs of SSR primers: the sample mixing system was 15. mu.l, containing 1.5. mu.l of 1.25mM dNTP, 1.5. mu.l of 10 XBuffer，1.5μl 25mM MgCl₂2.0. mu.l of 5mM primer, 5. mu.l of 10ng/ul DNA, 0.05. mu.l of 1U Taq, and 3.45. mu.l of ultrapure water; wherein, 2.0 mul of 5mM primer is added to 1.0 mul of two primers in the SSR primer pair respectively;

the PCR amplification procedure was: pre-denaturation at 94 ℃ for 1 min; denaturation at 93 deg.C for 1min, annealing at 40 deg.C for 1min, extension at 72 deg.C for 2min, and 40 cycles; extending at 72 ℃ for 1 min;

(4) and (3) detecting a PCR product: using 2.5% high resolution standard gel agarose, the DNA fragment separation range is 40-1 Kbp; adding 20 mul EB for color development, adding 2 mul Loading Dye into PCR product, electrophoresis for 2-3 hours, voltage 200-; after electrophoresis is finished, observing the result by using a gel imaging system;

(5) data collection and analysis: assigning the amplification results of 5 pairs of SSR primers N11/12, N41/42, N51/52, N121/122 and N155/156 in ` Yongzhen No.7 `, ` Yongzhen No.8 `andcontrol materials ` Mingxiu ` and ` Jinjiatian `, wherein a strip is marked as '1' amplified by a certain allele and a strip which is not amplified at the same site is marked as '0', and converting the differential amplification fragments into character strings consisting of 1 and 0, namely forming the variety fingerprint shown in the following table:

the 5 pairs of SSR primers comprise: n11/12, N41/42, N51/52, N121/122, N155/156, the sequence of N11/12 is shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of N41/42 is shown as SEQ ID NO.3 and SEQ ID NO.4, the sequence of N51/52 is shown as SEQ ID NO.5 and SEQ ID NO.6, the sequence of N121/122 is shown as SEQ ID NO.7 and SEQ ID NO.8, and the sequence of N155/156 is shown as SEQ ID NO.9 and SEQ ID NO. 10.