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CN-122012795-A - Method for identifying melon low-temperature weak light phenotype and special primer thereof

CN122012795ACN 122012795 ACN122012795 ACN 122012795ACN-122012795-A

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a method for identifying a melon low-temperature weak light phenotype and a special primer thereof. According to the invention, the leaf DNA of melon breeding materials is amplified through specific molecular marker combination, and whether the materials are low-temperature-resistant weak light materials is judged according to the amplified specific fragments. The technique can preliminarily judge the low-temperature weak light tolerance of the melon material without planting the melon material to a fruiting period and measuring the phenotype and the physiological index of plants, thereby greatly improving the screening efficiency of breeding materials.

Inventors

  • SONG HUI
  • ZANG QUANYU
  • DING WEIHONG
  • MA ERLEI
  • Hao Fangmin
  • YING QUANSHENG

Assignees

  • 宁波市农业科学研究院

Dates

Publication Date
20260512
Application Date
20260331

Claims (8)

  1. 1. A specific primer composition for identifying melon low-temperature weak light phenotype, which is characterized by comprising two specific primer pairs of Mel-Hs-1-F and Mel-Hs-2-R, and Mel-Hs-3-F and Mel-Hs-4-R.
  2. 2. The specific primer composition according to claim 1, wherein the nucleotide sequences corresponding to the primer pair Mel-Hs-1-F, mel-Hs-2-R, mel-Hs-3-F and Mel-Hs-4-R are shown in SEQ ID NO.1-4, respectively.
  3. 3. A kit for identifying a low temperature, low light phenotype of melon, comprising the specific primer composition of claim 1.
  4. 4. A method for identifying a low temperature, low light phenotype of melon comprising the steps of: S1, DNA extraction, namely taking young leaves of muskmelon, and extracting total genome DNA of the leaves by adopting a CTAB method; S2, PCR amplification, namely performing PCR reaction on the total genome DNA by using the specific primer composition of claim 1 to obtain a PCR amplification product; s3, data collection and analysis, namely, electrophoresis is carried out on PCR amplification products after fluorescent staining, and a gel imaging system is utilized to observe results; wherein, if the PCR amplified product fragment is 506 bp, the low-temperature weak light resistant phenotype is indicated, and if the PCR amplified product fragment is 938 bp, the low-temperature weak light resistant phenotype is indicated.
  5. 5. The method according to claim 4, wherein the reaction system of the PCR reaction is 10. Mu.L, comprising 5. Mu.L of 2 XTaq Master mix (Dye), 2. Mu.L of primer (5 mM), 2. Mu.L of DNA (10 ng/. Mu.L) and 1. Mu.L of ultrapure water.
  6. 6. The method of claim 4, wherein the PCR reaction is performed in a pre-denaturation step of 94℃and 2 min, a denaturation step of 94℃and 30: 30 s, an annealing step of 50℃and 30: 30 s, an extension step of 72℃and 10 s and 35 cycles, and a final extension step of 72 ℃.
  7. 7. The method of claim 4, wherein the fluorescent staining of step S3 is staining with 2.5% high resolution standard gel agarose with the addition of 20 μl ethidium bromide.
  8. 8. Use of the specific primer composition of claim 1 or the method of claim 4 for identifying low temperature resistant, low light phenotype melon.

Description

Method for identifying melon low-temperature weak light phenotype and special primer thereof Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a method for identifying a melon low-temperature weak light phenotype and a special primer thereof. Background The cultivation area and yield of the muskmelon in China are the first place in the world. The economic benefit of melon can be greatly improved by early cultivation in spring, and the cultivation method is an important crop rotation. However, in early spring, the outside air temperature is low, the rainy weather is more, melon seedlings in the greenhouse can be in a low-temperature (< 15 ℃) weak light (3000 lux-4000 lux) state for a long time, the growth is slowed down, and physiological injury occurs. Therefore, the breeding of melon varieties with low temperature and weak light resistance is an important target for breeding melons at home and abroad. Molecular markers related to the low-temperature and weak light resistant characteristics of muskmelon are screened for selecting breeding materials, and the molecular markers have important theoretical and practical significance for breeding low-temperature and weak light resistant varieties suitable for facility cultivation. The prior art identifies breeding materials by phenotypic or physiological indicators related to low temperature dim light characteristics. The phenotype indexes comprise leaf SPAD value, root length, root fresh weight and the like, and the physiological indexes comprise root activity, malondialdehyde content, chlorophyll content and the like. The morphological and physiological indexes are used as plant low-temperature weak light characteristic identification indexes, and the problems of complicated measurement operation and unstable measurement results often exist. For example, root system activity and MDA content, plant roots need to be cleaned, the operation is complex, and the data reliability is high under the condition of high root cleanliness. Therefore, the traditional breeding phenotype and physiological data acquisition technology has the defects of high cost, low efficiency and the like. Therefore, there is a need to develop a new method to improve the screening efficiency of melon low temperature and weak light resistant breeding materials. Disclosure of Invention The invention aims to provide a method for identifying melon low-temperature weak light phenotype and a special primer thereof, the special primer pair provided by the invention can specifically detect the melon material with low-temperature resistant weak light phenotype, so that the screening efficiency of melon breeding materials is improved. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a specific primer composition for identifying melon low-temperature weak light phenotype, which comprises two specific primer pairs of Mel-Hs-1-F and Mel-Hs-2-R, and Mel-Hs-3-F and Mel-Hs-4-R. Preferably, the nucleotide sequences corresponding to the primer pair Mel-Hs-1-F, mel-Hs-2-R, mel-Hs-3-F and Mel-Hs-4-R are respectively shown as SEQ ID NO. 1-4. The invention also provides a kit for identifying the low-temperature weak light phenotype of muskmelon, which comprises the specific primer composition. The invention also provides a method for identifying the low-temperature weak light phenotype of the muskmelon, which comprises the following steps: S1, DNA extraction, namely taking young leaves of muskmelon, and extracting total genome DNA of the leaves by adopting a CTAB method; S2, PCR amplification, namely performing PCR reaction on the total genome DNA by using the specific primer composition to obtain a PCR amplification product; s3, data collection and analysis, namely, electrophoresis is carried out on PCR amplification products after fluorescent staining, and a gel imaging system is utilized to observe results; wherein, if the PCR amplified product fragment is 506 bp, the low-temperature weak light resistant phenotype is indicated, and if the PCR amplified product fragment is 938 bp, the low-temperature weak light resistant phenotype is indicated. Preferably, the reaction system of the PCR reaction is 10. Mu.L, including 5. Mu.L of 2 XTaq Master mix (Dye), 2. Mu.L of primer (5 mM), 2. Mu.L of DNA (10 ng/. Mu.L) and 1. Mu.L of ultrapure water. Preferably, the amplification procedure of the PCR reaction is pre-denaturation 94℃at 2 min, denaturation 94℃at 30 s, annealing temperature 50℃at 30 s, extension 72℃at 10s at 35 cycles, and final extension 72 ℃. Preferably, the fluorescent staining described in step S3 is staining with 2.5% high resolution standard gel agarose with the addition of 20. Mu.L ethidium bromide. The invention also provides application of the specific primer composition or the method in identifying low-temperature resistant weak light phenotype melons. The inventi