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CN-122012773-A - Early-stage screening method for low-browning germplasm resources of pecan based on transcription factor expression quantity

CN122012773ACN 122012773 ACN122012773 ACN 122012773ACN-122012773-A

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to a method for early screening low-browning germplasm resources of pecan based on the expression level of transcription factors, and in particular provides a molecular marker related to pecan germplasm browning, wherein the molecular marker is a transcription factor AsbHLH and/or AsMYB gene or encoded protein thereof, the nucleotide sequence of the AsbHLH68 gene is shown as SEQ ID NO. 5 or a sequence which has more than 90% of homology and the same function with the nucleotide sequence, the nucleotide sequence of the AsMYB gene is shown as SEQ ID NO. 6 or a sequence which has more than 90% of homology and the same function with the nucleotide sequence. The technical scheme of the invention reduces the breeding cost and improves the breeding efficiency, namely, the invention can be used for conducting large-scale investigation on seedlings in the nursery stage, and most of poor single plants with high browning risks can be removed before transplanting and forestation.

Inventors

  • LI DAN
  • SUN SHENGJIE
  • ZHANG YINLONG
  • Liu Jiaole
  • ZONG DAN
  • Zhao Tangjia
  • SHI RUI
  • HE XIAHONG

Assignees

  • 西南林业大学

Dates

Publication Date
20260512
Application Date
20260128

Claims (9)

  1. 1. The molecular marker related to the brown stain of the pecan germplasm is characterized in that the molecular marker is a transcription factor AsbHLH and/or AsMYB gene or a coded protein thereof, the nucleotide sequence of the AsbHLH gene is shown in SEQ ID NO. 5 or a sequence which has more than 90% homology and has the same function, the nucleotide sequence of the AsMYB gene is shown in SEQ ID NO. 6 or a sequence which has more than 90% homology and has the same function.
  2. 2. A product for detecting a molecular marker according to claim 1, wherein the product comprises a reagent, a kit or a gene chip, and the product detects the expression level of the molecular marker.
  3. 3. The application of the molecular marker of claim 1 or the product of claim 2 in the auxiliary selective breeding of the brown stain resistant pecan germplasm molecular marker, which is characterized in that the expression quantity of the transcription factors AsbHLH and/or AsMYB232 genes or the coded proteins thereof is positively correlated with the brown stain property of the pecan, and the lower the expression quantity is, the more brown stain resistant the germplasm.
  4. 4. The method for screening the brown stain resistant coral germplasm is characterized by comprising the following steps of: (1) Collecting a tissue sample of a walnut plant to be tested; (2) Detecting the expression level of the transcription factors AsbHLH and/or AsMYB232 according to claim 1 in a sample; (3) Comparing the detection result with a control threshold value, and selecting plants with the expression quantity of the transcription factors significantly lower than the control threshold value, and judging as browning-resistant candidate germplasm.
  5. 5. The method according to claim 4, wherein in step (1), the tissue sample is a mature coral plant leaf, and the isolated leaf of the plant is subjected to mechanical damage treatment before detection and incubated at room temperature for 0.5 to 2 hours to induce expression of the transcription factor.
  6. 6. The method according to claim 4 or 5, wherein the expression level of the transcription factors AsbHLH and/or AsMYB232 is detected in step (2) by real-time fluorescent quantitative PCR.
  7. 7. The method of claim 6, wherein the specific primer pair sequences for detection AsbHLH and AsMYB and for detection AsbHLH and SEQ ID NO. 2 are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 4, respectively.
  8. 8. The method according to claim 4, wherein the control threshold is an average level of transcription factor expression of a high browning pecan plant or population as the control threshold, and the Gao Hebian pecan judgment is that the pecan is immediately browned to a high browning pecan within 30S of splitting of the pecan kernel.
  9. 9. The method according to claim 4, wherein the screening criteria is specifically that the expression level of AsbHLH and/or AsMYB232 gene is calculated by using AsActin as reference gene, and browning-resistant germplasm is judged when the relative expression level of AsbHLH in the plant under test is not higher than 2.113 and/or the relative expression level of AsMYB232 is not higher than 3.651.

Description

Early-stage screening method for low-browning germplasm resources of pecan based on transcription factor expression quantity Technical Field The invention belongs to the technical field of molecular markers, and particularly relates to a method for early screening of pecan low-browning germplasm resources based on transcription factor expression quantity. Background The pecan (Annamocarya sinensis) is a monostable plant of the pecan genus of the juglandaceae family, belongs to a rare endangered tree species with important national protection, and has unique scientific research value and excellent woody oil economic potential. However, kernel browning is a key challenge in restricting industrial utilization of pecan. Studies have shown that browning of the pecan kernel is essentially a substrate-dependent enzymatic oxidation reaction in which, as a result of the accumulation of a large amount of phenolic substances such as flavonoids in the kernel, these substrates, when processed to cause tissue disruption, are contacted with polyphenol oxidase (PPO) or the like and rapidly oxidized to form a blackish-brown quinone or tannin polymer. At present, the breeding of good varieties with low substrate accumulation and browning resistance mainly depends on a traditional phenotype observation method. This method requires that plants must reach reproductive maturity, and browning levels are assessed by disruption of fruit structure and exposure to air. The method has the technical defects of long growth period of the pecks and the high cost, extremely long breeding period and management burden increase due to the fact that a large number of inferior plants are maintained in the field after sowing to the fruiting, irreversibility that the identification process means damage of seeds and seed remaining after identification cannot be carried out on single seeds, detection blind areas that upstream regulatory genes (such as specific transcription factors) which cause substrate accumulation usually have high space-time specificity, and are always in a dormant or low-expression state in vegetative organs (such as leaves) in the seedling stage, so that false negative easily occurs in conventional molecular detection. Therefore, it is highly desirable to establish an early screening system based on a damage induction mechanism, and activate the expression of key regulatory genes by simulating a processing stress environment, so that the metabolic characteristics and browning risks of the kernels after the plant formation can be accurately predicted in a seedling stage, and a breakthrough technical means is provided for shortening the woody plant breeding period. Disclosure of Invention The invention aims to provide a low browning germplasm resource early screening method of pecan based on transcription factor expression quantity to solve the problems in the background technology, and particularly provides the following technical scheme: The invention provides a molecular marker related to pecan germplasm browning, which is a transcription factor AsbHLH and/or AsMYB gene or coded protein thereof, wherein the nucleotide sequence of the AsbHLH gene is shown in SEQ ID NO 5 or a sequence which has more than 90% of homology and has the same function, the nucleotide sequence of the AsMYB gene is shown in SEQ ID NO 6 or a sequence which has more than 90% of homology and has the same function. Further, the invention provides a product for detecting the molecular marker, wherein the product comprises a reagent, a kit or a gene chip, and the product detects the expression level of the molecular marker. Furthermore, the invention provides application of the molecular marker or the product in auxiliary selective breeding of the brown stain resistant pecan germplasm molecular marker, the expression quantity of the transcription factors AsbHLH and/or AsMYB gene or the coded protein thereof is positively correlated with the brown stain property of the pecan, and the lower the expression quantity is, the more brown stain resistant the germplasm is. Further, the invention provides a method for screening brown stain resistant pecan germplasm, which comprises the following steps: (1) Collecting a tissue sample of a walnut plant to be tested; (2) Detecting the expression level of the transcription factors AsbHLH and/or AsMYB232 according to claim 1 in a sample; (3) Comparing the detection result with a control threshold value, and selecting plants with the expression quantity of the transcription factors significantly lower than the control threshold value, and judging as browning-resistant candidate germplasm. Further, in the step (1), the tissue sample is mature pecan leaves, and before detection, the isolated leaves of the tested plants are subjected to mechanical damage treatment and incubated for 0.5-2 hours at room temperature to induce the expression of the transcription factors. Further, in the step (2), the expression level of the trans