21 Molecular Marker Primer Combinations for the Identification of Actinidia chinensis var. deliciosa and Their Applications

Abstract

The present invention belongs to the technical fields of molecular biology and genetic breeding, and discloses 21 molecular marker primer combinations for the identification of Actinidia deliciosa and their applications. Using the 21 pairs of primers provided by the present invention to amplify the DNA of 16 common Actinidia deliciosa, bands of different sizes can be specifically amplified; according to the sizes and numbers of different bands, Actinidia deliciosa such as 'Jade', 'Morning Light', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate', 'Yuxiang', etc. can be distinguished.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to a combination of 21 molecular marker primers for the identification of Actinidia chinensis var. deliciosa and applications thereof. Background Art

[0002] The planting areas of kiwifruit in China are mainly distributed in the southern provinces of Sichuan, Guizhou, Hubei, etc. and the northern provinces of Shaanxi and Henan. Over the past 40-odd years, the exploration, utilization and innovative breeding of wild germplasm resources in China have promoted the rapid development of the kiwifruit industry. A total of 156 new varieties have been cultivated, among which 8 varieties have an orchard planting area of over 100,000 mu: 'Hongyang', 'Xuxiang', 'Cuixiang', 'Donghong', 'Guichang', 'Jinyan', 'Miliang No. 1' and 'Jintao'. According to the pulp colors of cultivated kiwifruit varieties, the proportions of the planting areas of green-fleshed, yellow-fleshed and red-fleshed kiwifruit are 49.87%, 15.80% and 32.56% respectively. Among them, green-fleshed kiwifruit accounts for nearly half of the total area.

[0003] Most of the green-fleshed kiwifruits belong to the Actinidia deliciosa species. In the breeding of new kiwifruit varieties, the center selected the "Qinmei" variety in the 1980s of the last century, which became the variety with the largest popularization area among the first-generation kiwifruit varieties in China and won more than 10 awards. 'Jade', 'Ruiyu' and 'Tianyu' are all high-quality Actinidia deliciosa varieties selected by the Shaanxi Rural Science and Technology Development Center and Shaanxi Berry Kiwifruit Research Institute Co., Ltd. The fruit of 'Jade' is long cylindrical, with a uniform fruit shape. The average single fruit weight is 95 grams, and the maximum fruit weight is 138 grams. The pulp is green, with abundant juice, tender texture, sweet and sour taste, containing 15.3% - 17.6% soluble solids and 158.7 mg / 100 g of vitamin C. The fruit is resistant to storage and transportation, has a long shelf life, and can be stored at room temperature for about 30 days. The average single fruit weight of 'Ruiyu' kiwifruit is 90 g, and the maximum single fruit weight is 128 g; the peel is brown, covered with golden-brown stiff hairs, the pulp is green, delicate and juicy, with a sweet flavor; it contains 18.3% - 21.6% soluble solids, 20% - 25% dry matter content, 174.3 mg / 100 g of vitamin C, 0.83% titratable acid, and 12.09% soluble sugar. The ripening period at room temperature is 25 - 30 days, and it can be stored in cold storage for about 5 months. The tree vigor of 'Ruiyu' is strong, it bears fruit early and has a high yield. The yield in the full fruiting period is more than 2000 kg / mu. 'Tianyu' is a new Actinidia deliciosa variety selected by using 'Cuixiang' as the female parent and pollinating with the pollen of 'Actinidia eriantha' male plant (male parent) for interspecific cross breeding. The fruit is cylindrical, with an average single fruit weight of 90 grams and a maximum fruit weight of 130 grams. The pulp is green, delicate, juicy, with a strong flavor, sweet and sour taste, high sugar content, containing 17.3% - 19.6% soluble solids and 0.75% - 1.42% total acid. The comprehensive performance is: strong tree vigor, early fruiting, high and stable yield, resistant to canker disease, wide adaptability; beautiful and neat fruit shape, strong flavor, sweet and sour taste, resistant to storage and transportation. It is a new medium-maturing Actinidia deliciosa variety with high quality, high yield and disease resistance.

[0004] The purity and authenticity of varieties directly affect the yield and quality of crops. The essence of germplasm purity and authenticity identification is the identification of genotypes. Currently, the identification methods mainly include morphological method, biochemical method and molecular marker method. However, the first two methods are easily affected by the environment and the material itself, while the molecular marker identification method is not affected by external conditions and has higher accuracy compared with the first two methods. In the kiwifruit industry, there are relatively many chaotic and miscellaneous varieties, and field test identification is difficult to meet the requirements of timeliness and accuracy, and it is time-consuming and laborious.

[0005] Developing molecular identification markers suitable for Actinidia deliciosa varieties is beneficial to clearly define patented varieties at the molecular level, which is conducive to the identification and variety protection of kiwifruit varieties. Summary of the Invention

[0006] The purpose of the present invention is to provide a combination of 21 molecular marker primers for Actinidia deliciosa identification by using molecular marker technology.

[0007] Another object of the present invention is to provide the application of the molecular marker primer combination for the identification of Actinidia deliciosa in kiwifruit breeding.

[0008] To achieve the above object, the present invention takes the following technical measures:

[0009] Obtaining the molecular marker primer combination for the identification of Actinidia deliciosa: The applicant downloads the latest version of the Actinidia V3 whole-genome sequence from the kiwifruit genome website (http: / / kiwifruitgenome.org / ). SSR markers are developed throughout the whole genome by SSR Finder software. According to the gene structure annotation results, gene loci are selected to design SSR marker primers. Using the designed and synthesized primers, PCR amplification is carried out on 16 Actinidia deliciosa varieties, and 21 molecular markers are screened for the identification of Actinidia deliciosa. The specific primers are shown in Example 1 of the specific implementation manner.

[0010] The application of 21 molecular marker primers for the identification of Actinidia deliciosa in kiwifruit breeding includes using the primers of the present invention for kiwifruit breeding, or using the primers of the present invention to identify and distinguish Actinidia deliciosa varieties 'Jade', 'Chenguang', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate', 'Yuxiang' according to the size and number of different bands.

[0011] Compared with the prior art, the advantages of the present invention are as follows:

[0012] 1. Morphological markers are simple and intuitive, but the quantity is small, the polymorphism is poor, and they are easily affected by environmental conditions. Molecular markers are easy to operate and can be detected in various tissues and developmental stages of plants, without being restricted by seasons and environments. The quantity is large and spreads throughout the whole genome. The polymorphism is high, and there are many allelic variations among varieties.

[0013] 2. The primers of the present invention can amplify DNA fragments of different sizes in a series of kiwifruit varieties. The amplification stability of this molecular marker can be used for the identification of Actinidia deliciosa varieties.

[0014] 3. The molecular marker primers provided by the present invention have high specificity and a wide application range.

[0015] 4. The Actinidia deliciosa varieties 'Jade', 'Chenguang', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate', 'Yuxiang' can be distinguished according to the size and number of bands. Specific implementation manner

[0016] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the art; unless otherwise specified, the reagents or materials are all from commercial or public channels.

[0017] Example 1:

[0018] Obtaining 21 molecular marker primers for the identification of Actinidia deliciosa:

[0019] The applicant downloaded the latest version of the Actinidia V3 whole genome sequence from the Actinidia genome website (http: / / kiwifruitgenome.org / ). SSR markers were developed throughout the whole genome by SSR Finder software. According to the gene structure annotation results, gene loci were selected to design SSR marker primers. Using the designed and synthesized primers, PCR amplification was performed on 16 Actinidia deliciosa varieties, and 21 molecular markers were screened for the identification of Actinidia deliciosa as follows:

[0020]

[0021] Example 2:

[0022] Application of 21 molecular marker primer combinations for identifying Actinidia deliciosa in the identification of different varieties:

[0023] (1) DNA samples of 16 different Actinidia deliciosa varieties were extracted respectively according to the CTAB method (the specific variety names are shown in Table 1).

[0024] (2) Use a pipette to aspirate 990 μl of the mixture of HIDI and 10 μl of ROX500 or LIZ500 and add it to a 96-well reaction plate, 10 μl per well.

[0025] (3) Place the 96-well plate in a tabletop centrifuge and centrifuge at 500 g and stop immediately.

[0026] (4) Add the DNA of each sample to the corresponding wells of the 96-well plate, 50 pg for each sample.

[0027] (5) Refer to the following PCR reaction system to add the corresponding reagents to the 96-well plate, seal the 96-well plate with a sealing film, shake it, place the 96-well plate in a tabletop centrifuge and centrifuge at 500 g and stop immediately. Perform the PCR reaction according to the following procedure.

[0028] a. PCR reaction system

[0029] Preparation of a 25 μl system

[0030] Template (μl) 1 Primer F (μl) 0.5 Primer R (μl) 0.5 dNTP 10mM (μl) 0.5 Taq Buffer (μl) 2.5 <![CDATA[25mM MgCl2(μl)]]> 2.0 Taq Enzyme (μl) 5U / μl 0.2 Water (μl) 17.8

[0031] The 21 pairs of primers are shown in Example 1.

[0032] b. PCR reaction conditions

[0033]

[0034] (6) Immediately after the program ends, place the 96-well plate on an ice-water mixture for rapid cooling, and then place the 96-well plate in a tabletop centrifuge and centrifuge at 2000 g and stop immediately.

[0035] (7) Use a 3730xl sequencing analyzer (ABI, USA) to detect the amplified fragments.

[0036] (8) Use Genemapper software to analyze the SSR amplification data, and use STR capillary electrophoresis to detect the amplified fragment peaks. The abscissa is the fragment size, and the ordinate is the fluorescence intensity value.

[0037] (9) Use 21 molecular markers to amplify 16 Actinidia deliciosa varieties. According to the size and number of different bands, distinguish the Actinidia deliciosa varieties 'Jade', 'Chenguang', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate', 'Yuxiang'. The specific amplification results are shown in Table 1. The unit after the numbers in the table is "bp".

[0038] Table 1 Name of kiwifruit varieties and PCR amplification results of primer combinations

[0039]

[0040]

[0041] As shown in Table 1, the 21 pairs of molecular marker primers of the present invention can completely distinguish 16 Actinidia deliciosa varieties.

Claims

1. 21 molecular marker primer combinations for kiwifruit identification: The kiwifruit varieties are 'Jade', 'Chenguang', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate' or 'Yuxiang'.

2. Use of the primer combination according to claim 1 in the identification of kiwifruit varieties, where the kiwifruit varieties are 'Jade', 'Chenguang', 'Cuixiang', 'Guichang', 'Huamei', 'Hayward', 'Jinfu', 'Jinkui', 'Miliang', 'Qinmei', 'Qiuming', 'Ruiyu', 'Tianyu', 'Xuxiang', 'Yate' or 'Yuxiang'; Its application process includes: Amplify the above kiwifruit varieties with the primer combination according to claim 1, and distinguish the above kiwifruit varieties according to the size and number of different bands. The specific amplification results are shown as follows: The unit after the numbers in the table is bp.

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