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CN-122012793-A - Molecular marker closely linked with wild apple peel color, detection primer and application thereof

CN122012793ACN 122012793 ACN122012793 ACN 122012793ACN-122012793-A

Abstract

The invention discloses a molecular marker closely linked with the color of wild apple peel, a detection primer and application thereof, belonging to the technical field of genetic breeding. The molecular marker is positioned at the locus 31503385-31511919 of the catalpa bungei genome 9 and is an insertion/deletion polymorphism locus of an LTR retrotransposon in an upstream promoter region of an MdMYB1 gene. The invention also designs a detection primer of the molecular marker, and the nucleotide sequences are SEQ ID NO.2 and SEQ ID NO. 3. When the amplified product of the detection primer appeared in the band at 536bp as the insertion genotype (red peel), no band was the deletion genotype (yellow or yellowish green peel). The method can rapidly and accurately judge the peel color of the filial generation of the wild apple seeds, realize early screening of coloring characters, greatly improve the hybridization breeding efficiency of the wild apples, provide key technical support for genetic improvement and directional utilization of wild apple resources, and have wide application prospects.

Inventors

  • YANG YAZHOU
  • Duan Xuanqi
  • SONG YAXIAO
  • LI KAIWEI
  • SHI YANGJIE
  • YANG HUIJUAN
  • LIU LI
  • BAI JUAN
  • ZHAO ZHENGYANG

Assignees

  • 西北农林科技大学

Dates

Publication Date
20260512
Application Date
20260327

Claims (9)

  1. 1. A molecular marker closely linked with the color of wild apple peel is characterized in that the molecular marker is positioned at 31503385-31511919 locus of a catalpa bungei genome 9 chromosome, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the molecular marker is an insertion or deletion polymorphic locus of an LTR retrotransposon in an upstream promoter region of an MdMYB1 gene, wherein the sequence number of the catalpa bungei genome is CM103599.1.
  2. 2. A detection primer for detecting the molecular marker of claim 1, wherein the nucleotide sequences of the detection primer are shown in SEQ ID NO.2 and 3.
  3. 3. A kit comprising the detection primer of claim 2.
  4. 4. The method for detecting the color of the apple peel is characterized by comprising the following steps of: 1) Extracting genome DNA of an apple sample to be detected; 2) Performing PCR amplification by using the genomic DNA as a template and the detection primer according to claim 2; 3) And (3) carrying out agarose gel electrophoresis on the PCR amplified product, and judging the color of apple peel according to the electrophoresis result.
  5. 5. The method according to claim 4, wherein the PCR amplification procedure comprises pre-denaturation at 94℃for 5min, denaturation at 94℃for 15s, renaturation at 60℃for 10s, extension at 72℃for 10s, and cycling for 35 times, and final extension at 72℃for 5min.
  6. 6. The method according to claim 4, wherein when the specific band appears at 536bp in the amplified product, the amplified product is homozygous inserted genotype or heterozygous inserted genotype, the peel color phenotype is red, the amplified product is free of band, the amplified product is homozygous deleted genotype, and the peel color phenotype is yellow or yellowish green.
  7. 7. Use of the molecular marker of claim 1 or the detection primer of claim 2 or the kit of claim 3 for identifying the color of pericarp of wild species filial generation of apple.
  8. 8. Use of a molecular marker according to claim 1 or a detection primer according to claim 2 or a kit according to claim 3 in apple molecular marker assisted cross breeding.
  9. 9. The use according to claim 8, wherein said use comprises the identification and screening of apple peel color traits.

Description

Molecular marker closely linked with wild apple peel color, detection primer and application thereof Technical Field The invention relates to the technical field of molecular marker assisted breeding, in particular to a molecular marker closely linked with the color of wild apple peel, a detection primer and application thereof in assisted improved breeding. Background The color and luster of apple fruits are important quality indexes of apples, and are always the hot spot field of apple molecular biology research. Takos et al (2006) found that MdMYB1 was expressed only in the pericarp of the red apple variety, not in the yellow and green varieties, and that the SNP site in MdMYB1 was coseparated from the pericarp color. After that, numerous researches show that the amplified band is co-separated from the red character according to the specific primer designed by the MdMYB1 gene and the SNP locus of the promoter region thereof, can be used as a molecular marker for screening fruit color, and has the accuracy rate close to 100%. In 2019, cong Peihua group (Zhang et al 2019) found that by analyzing the genome of hanfu apple haploid plants, a retrotransposon of 4102 bp was present upstream of the MdMYB1 promoter in the red variety (designated RedTE), which was deleted in the yellow and green varieties. Genetic shows that the retrotransposon sequence insertion is separated from the color of the pericarp, and can be used for assisting in selecting the color of the pericarp of the filial generation of apples. However, the pericarp of the Aronia melanocarpa was found to be red through research, but RedTE specific primers failed to amplify bands in the Aronia melanocarpa and its hybrid offspring. The Aronia melanocarpa seeds x Swiss hybrid offspring population was analyzed using BSA-seq and found that the color gene was still located at Chr9. Analysis of the MYB1 gene sequence of catalpa bungei shows that the upstream of the gene promoter has no RedTE sequence. Therefore, a specific primer is designed according to the promoter region sequence of the Sorbus pohuashanensis as a molecular marker, and the result shows that the detection result of the marker is completely consistent with the peel color of the hybrid offspring of Sorbus pohuashanensis and Sweetcrete. Currently, in the breeding process of apples, an interspecific hybridization mode is generally adopted, and the genetic gene library of the apples is gradually narrowed due to long-term interspecific hybridization. The cross breeding by using wild apple resources is an important means for solving the narrowing of apple genetic gene libraries. Therefore, the invention develops the promoter sequence variation as a core molecular marker of wild apple peel coloring property by analyzing the expression characteristics and the promoter sequence variation of the mountain ash peel coloring key gene MdMYB 1. The establishment of the marker provides a key technical support for the accurate identification, genetic background analysis and directional utilization of wild apple resources, and also lays a solid foundation for early screening and molecular auxiliary breeding of coloring characters in related fruit tree breeding. It should be noted that the information disclosed in the above background section is only for enhancing understanding of the background of the present disclosure and thus may include information that does not constitute prior art known to those of ordinary skill in the art. Disclosure of Invention The invention aims to provide a molecular marker closely linked with the color of wild apple seed peel, which is helpful for screening and identifying apples with target peel color and developing germplasm resources. In order to achieve the aim, the invention provides a molecular marker closely linked with the color of wild apple pericarp, wherein the molecular marker is positioned at 31503385-31511919 locus of a catalpa bungei genome 9 chromosome, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the molecular marker is an insertion or deletion polymorphic locus of an LTR retrotransposon in an upstream promoter region of an MdMYB1 gene, wherein the sequence number of the catalpa bungei genome is CM103599.1. The invention also provides a detection primer for detecting the molecular marker, and the nucleotide sequence of the detection primer is shown as SEQ ID NO.2 and SEQ ID NO. 3. The invention also provides a kit containing the detection primer. The invention also provides a method for detecting the color of the apple peel, which comprises the following steps: 1) Extracting genome DNA of an apple sample to be detected; 2) Using the genome DNA as a template, and adopting the detection primer to carry out PCR amplification; 3) And (3) carrying out agarose gel electrophoresis on the PCR amplified product, and judging the color of apple peel according to the electrophoresis result. Preferably, the PCR amplification procedure in the