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CN-121975975-A - CAPS mark for identifying soybean leaf type and application thereof

CN121975975ACN 121975975 ACN121975975 ACN 121975975ACN-121975975-A

Abstract

The invention relates to the technical field of soybean genetic breeding and molecular marking, in particular to a CAPS marker for identifying soybean leaf types and application thereof. The invention solves the problems that soybean leaf pattern identification in the prior art depends on field phenotype observation and has low efficiency. The invention is used for identifying CAPS markers of soybean leaf types, wherein the CAPS markers target G/C variation SNP loci at chromosome 38947579 of soybean, and the SNP loci are numbered SGM20_38947579. The CAPS mark is applied to soybean molecular breeding. The CAPS mark for detecting the soybean leaf profile and the application thereof provided by the invention realize early, rapid and accurate identification of the soybean leaf profile and provide an efficient tool for directional breeding of the soybean leaf profile.

Inventors

  • ZHANG HENGYOU
  • WANG LE
  • ZHANG YUAN
  • WEN XIAOTING
  • LI YAN

Assignees

  • 中国科学院东北地理与农业生态研究所
  • 南京农业大学

Dates

Publication Date
20260505
Application Date
20260325

Claims (9)

  1. 1. CAPS marker for identifying soybean leaf patterns, wherein the CAPS marker targets a G/C variant SNP site at chromosome 38947579 of soybean, the SNP site number sgm20_38947579.
  2. 2. A primer for identifying the CAPS tag of claim 1, wherein the primer sequence is: forward primer F5'-TCCCCCACTACTACACCTTCAC-3' with the sequence shown in SEQ ID NO. 1, The sequence of the reverse primer R5'-GGTAACAAACCACGGGCAGT-3' is shown as SEQ ID NO. 2; The corresponding endonuclease is BstYI.
  3. 3. The method for identifying soybean leaf patterns by CAPS markers according to claim 1, wherein the identification method is performed according to the following steps: 1. Extracting genomic DNA of soybean to be identified; 2. performing PCR amplification by using the genomic DNA extracted in the first step as a template and using the primer sequence of claim 2 to obtain an amplification product of 368 bp; 3. Performing enzyme digestion treatment on the amplified product of the step two by adopting BstYI endonuclease; 4. And (3) performing agarose gel electrophoresis detection on the enzyme-digested product, wherein if the electrophoresis result shows that the sequences of the two bands of 244bp and 130bp are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, the soybean to be identified is oval wide-leaf soybean, and if the electrophoresis result shows that the single band of 368bp is shown as SEQ ID NO.5, the soybean to be identified is narrow-leaf soybean.
  4. 4. The method for identifying soybean leaf types by CAPS markers according to claim 3, wherein the genomic DNA of the soybean leaf to be identified is extracted in the first step by using a conventional CTAB method or a kit method.
  5. 5. A method for identifying soybean leaf patterns by CAPS markers according to claim 3, wherein the PCR reaction system in the second step comprises the steps of pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 15s, and extension at 72 ℃ for 15s for 30 cycles, and final extension at 72 ℃ for 5min and preservation at 4 ℃.
  6. 6. The method for identifying soybean leaf patterns by CAPS markers according to claim 3, wherein the digestion conditions in the step three are that the digestion is performed in a water bath at 60 ℃ for 1 hour.
  7. 7. The method for identifying soybean leaf patterns by CAPS marking according to claim 3, wherein the agarose gel electrophoresis detection gel concentration in the step three is 1%.
  8. 8. Use of the CAPS marker of claim 1 in soybean molecular breeding.
  9. 9. The use according to claim 8, characterized in that soybean of the leaf type of interest is identified as parental breeding by CAPS markers.

Description

CAPS mark for identifying soybean leaf type and application thereof Technical Field The invention relates to the technical field of soybean genetic breeding and molecular marking, in particular to a CAPS mark for identifying soybean leaf types and application thereof. Background Soybean leaf profile is one of important agronomic traits, and different leaf profiles (such as oval wide leaf, narrow leaf and the like) have obvious influence on photosynthesis efficiency, ventilation and light transmission conditions, stress resistance and population yield of soybeans. For example, oval leaf soybeans generally have better photosynthetic area and light energy utilization efficiency, while narrow leaf soybeans have unique advantages in ventilation and light transmittance, and are easier to form reasonable group structures under close planting conditions. The traditional soybean leaf pattern identification method mainly relies on field phenotype observation, has long identification period, can not accurately identify the soybean in early growth stage, and severely restricts the soybean breeding efficiency. With the development of molecular biology technology, molecular marker assisted breeding technology is widely applied to crop breeding due to the advantages of rapidness, accuracy, no environmental influence and the like. CAPS (cleaved amplified polymorphic sequence) marker is a molecular marker technology based on PCR and restriction enzyme digestion, has the characteristics of high specificity, good stability, simple operation and the like, and has been applied to the character identification of various crops. At present, specific CAPS markers of soybean leaf types are not reported yet, and lack of efficient and stable molecular identification tools limits the progress of directional breeding of soybean leaf types. Therefore, the soybean leaf type identification molecular marker which is stable, efficient and wide in application range is developed, and has important practical significance for promoting the molecular breeding process related to soybean leaf types. Disclosure of Invention The invention provides a CAPS mark for identifying soybean leaf types and application thereof, aiming at solving the problems that soybean leaf type identification in the prior art depends on field phenotype observation, has low efficiency and is easy to be interfered by environment. The invention is used for identifying CAPS markers of soybean leaf types, wherein the CAPS markers target G/C variation SNP loci at chromosome 38947579 of soybean, and the SNP loci are numbered SGM20_38947579. The invention is used for identifying the CAPS marked primer in claim 1, wherein the primer sequence is as follows: forward primer F5'-TCCCCCACTACTACACCTTCAC-3' with the sequence shown in SEQ ID NO. 1, The sequence of the reverse primer R5'-GGTAACAAACCACGGGCAGT-3' is shown as SEQ ID NO. 2; The corresponding endonuclease is BstYI. The method for identifying soybean leaf types by CAPS markers is characterized by comprising the following steps of: 1. Extracting genomic DNA of soybean to be identified; 2. Taking the genomic DNA extracted in the first step as a template, and carrying out PCR amplification by utilizing the CAPS mark primer sequence to obtain an amplification product of 368 bp; 3. Performing enzyme digestion treatment on the amplified product of the step two by adopting BstYI endonuclease; 4. And (3) performing agarose gel electrophoresis detection on the enzyme-digested product, wherein if the electrophoresis result shows that the sequences of the two bands of 244bp and 130bp are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, the soybean to be identified is oval wide-leaf soybean, and if the electrophoresis result shows that the single band of 368bp is shown as SEQ ID NO.5, the soybean to be identified is narrow-leaf soybean. Furthermore, the genomic DNA of the soybean to be identified is extracted in the first step, and the genomic DNA of the soybean leaves can be extracted by adopting a conventional CTAB method or a kit method (purity and concentration detection are carried out, so that the DNA quality meets the PCR amplification requirement). Further, the reaction program of the PCR reaction system in the second step is 95 ℃ pre-denaturation for 3min, 95 ℃ denaturation for 15s,62 ℃ annealing for 15s and 72 ℃ extension for 15s, 30 cycles are total, 72 ℃ final extension is 5min, and 4 ℃ storage is carried out. And in the third step, the enzyme digestion treatment is carried out under the conditions of water bath enzyme digestion at 60 ℃ for 1h. Further, in the third step, the gel concentration was 1% by agarose gel electrophoresis detection. The CAPS mark is applied to soybean molecular breeding. Further, the application is to identify the soybean with the target leaf type as a parent breeding through CAPS markers. The invention identifies QTL qLS which is obviously related to the leaf type through whole genome association analy