CN-121978068-A - Method for auxiliary selection of high-nutrition wheat by using molecular markers

CN121978068ACN 121978068 ACN121978068 ACN 121978068ACN-121978068-A

Abstract

The invention provides a method for assisting in selecting high-nutrition wheat by using molecular markers, which comprises the following steps of obtaining seeds in a development stage on a wheat plant, introducing fluorescent resonance energy transfer sensing proteins with specific responses to target nutrients into the seeds, performing in-vivo imaging on the seeds introduced with the fluorescent resonance energy transfer sensing proteins to obtain space-time fluorescent signal data representing a dynamic accumulation process of the target nutrients in the seeds, calculating one or more space-time nutrition kinetic parameters of the dynamic accumulation process based on the space-time fluorescent signal data, and screening the wheat plant with excellent dynamic accumulation process characteristics by taking the space-time nutrition kinetic parameters as selection basis. The invention realizes the high-throughput and high-precision quantitative evaluation of the nutrition characteristics of the breeding materials through nondestructive dynamic living monitoring, and provides a new technical approach for accelerating the breeding of high-nutrition crop varieties.

Inventors

ZENG ZHANKUI
LIU ZHIHAO
LIU LIYUAN

Assignees

科蓝农业科技(河南)有限公司

Dates

Publication Date: 20260505
Application Date: 20260130

Claims (10)

1. A method for auxiliary selection of high-nutrition wheat by using molecular markers, which is characterized by comprising the following steps: S1, obtaining seeds in a development stage on a wheat plant, and introducing fluorescence resonance energy transfer sensing proteins with specific responses to target nutrients into the seeds; S2, performing living body imaging on the grains introduced with the fluorescence resonance energy transfer sensing protein to obtain space-time fluorescence signal data representing the dynamic accumulation process of the target nutrients in the grains; S3, calculating one or more space-time nutrition kinetic parameters of the dynamic accumulation process based on the space-time fluorescence signal data; s4, screening out wheat plants with excellent dynamic accumulation process characteristics by taking the space-time nutrition dynamic parameters as selection basis.
2. The method for selecting high-nutrition wheat by using molecular marker as set forth in claim 1, wherein, In step S2, the in vivo imaging of the kernel into which the fluorescent resonance energy transfer sensor protein is introduced to obtain spatiotemporal fluorescence signal data characterizing the dynamic accumulation process of the target nutrients inside the kernel includes: S21, placing the seeds on a microfluidic chip for perfusion culture; s22, changing the components of the perfusion culture solution to apply standardized environmental stress; S23, acquiring space-time fluorescence signal data of the kernels after stress relief.
3. The method for selecting high-nutrition wheat by using molecular marker as set forth in claim 1, further comprising: In step S1, simultaneously with the introduction of the fluorescence resonance energy transfer sensor protein, co-introducing a gene silencing element targeting a specific candidate gene; In step S2, performing in-vivo imaging on the grain into which the fluorescence resonance energy transfer sensing protein and the gene silencing element are introduced to obtain space-time fluorescence signal data representing a dynamic accumulation process of the target nutrient in the grain; In step S3, calculating one or more spatiotemporal nutritional kinetic parameters of the dynamic accumulation process based on spatiotemporal fluorescence signal data of the kernel into which the fluorescence resonance energy transfer sensor protein and the gene silencing element are introduced; In step S4, selecting a wheat plant with excellent dynamic accumulation process characteristics based on the spatiotemporal thermodynamic parameters of the seed material into which the gene silencing element is not introduced and the gene silencing element is introduced.
4. The method for selecting high-nutrition wheat by using molecular marker as set forth in claim 1, further comprising: s5, genotyping the wheat plants screened in the step S4, and carrying out whole genome association analysis on the space-time nutrition kinetic parameters and genotype data so as to identify molecular markers associated with the dynamic accumulation process.
5. The method for selecting high-nutrition wheat with the aid of molecular markers according to claim 1, wherein: in step S1, the fluorescence resonance energy transfer sensor protein is introduced into the seed by a viral vector-mediated or Agrobacterium-mediated transient expression method.
6. The method for selecting high-nutrition wheat with the aid of molecular markers according to claim 1, wherein: the space-time nutrition kinetic parameters comprise at least one of peak flux rate, stress toughness index, space allocation preference degree and accumulation starting time; the peak flux rate is used to characterize a maximum rate at which the target nutrient accumulates within the kernel; The stress toughness index is used for representing the stability and the recovery capacity of the grain in the target nutrient accumulation process when the grain is subjected to environmental stress; The spatial distribution preference is used for representing the distribution ratio of the target nutrient among different tissues in the grain; the accumulation start time is used to characterize the time required for the target nutrients within the kernel to begin to accumulate significantly.
7. The method for selecting high-nutrition wheat by using molecular marker as set forth in claim 6, wherein: The space-time nutrition kinetic parameter is a peak flux rate, and the value of the peak flux rate is determined by the maximum value of the change rate of the fluorescence signal ratio of the fluorescence resonance energy transfer sensing protein to time: Peak flux rate ; In which the number of the components is one, Representing a peak, or maximum, of a certain amount, representing the maximum response of the system, The operator representing the maximum value is selected, Representing a function Variations of (i), i.e. The derivative of the time t is used, Representing a small amount of change in time, Is that Derivative with respect to t, means A rate or slope of change over time.
8. The method for assisted selection of high nutritional wheat using molecular markers according to claim 6, The space-time nutrition kinetic parameter is a stress toughness index, the value of the stress toughness index is jointly determined by the change amplitude of the fluorescence signal ratio of the grain during the period of environmental stress and the recovery rate of the fluorescence signal ratio after stress relief, and the stress toughness index can be calculated by the following formula: ; In which the number of the components is one, For the maximum signal ratio before stress, For the lowest signal ratio during stress, In order to observe the signal ratio at the end of the run, In order for the stress to end up time, For the end time of the observation.
9. The method for assisted selection of high nutritional wheat using molecular markers according to claim 6, The space-time nutrition dynamic parameters are space distribution preference degrees, and the ratio of the fluorescence signal ratio of the target tissue and the reference tissue in the grain is determined by the numerical value of the space distribution preference degrees; it can be determined from the ratio of the average fluorescence signal ratio of the target tissue to the average fluorescence signal ratio of the reference tissue: ; In which the number of the components is one, And Sets of voxels representing the target tissue and the reference tissue respectively, And Respectively the number of voxels thereof, For the ratio of the fluorescence signals of the voxels, Representation of a pair of regions All points in (1) Is a function of (2) Is used in the method of the present invention, Representation of a pair of regions All points in (1) Is a function of (2) Is a sum of (a) and (b).
10. The method for selecting high-nutrition wheat by using molecular marker as set forth in claim 1, wherein, The fluorescent resonance energy transfer sensing protein comprises a first fluorescent protein, a second fluorescent protein and a target nutrient binding domain, wherein the target nutrient binding domain is positioned between the first fluorescent protein and the second fluorescent protein and can specifically bind to the target nutrient; the target nutrient binding domain binds to a target nutrient to cause a change in fluorescence resonance energy transfer efficiency.

Description

Method for auxiliary selection of high-nutrition wheat by using molecular markers Technical Field The invention relates to the technical field of biotechnology and crop genetic improvement, in particular to a method for assisting in selecting high-nutrition wheat by using molecular markers. Background The wheat is used for cultivating crop varieties with high nutritive value, and is an important way for solving the global problem of 'recessive hunger' and guaranteeing human health. Taking wheat as an example, the content of trace nutrients such as zinc, iron and the like in grains directly influences the nutrition intake of human beings. Therefore, the method can accurately and efficiently screen out germplasm resources with excellent nutrient accumulation capacity, and is one of the core targets of modern crop breeding. However, existing breeding selection techniques have inherent limitations in assessing plant nutritional traits. Traditional screening methods rely heavily on chemical analysis of mature kernels, such as by atomic absorption spectrometry or inductively coupled plasma mass spectrometry to determine the final nutrient content. The essence of this approach is endpoint detection, which, while providing a static cumulative total result, completely ignores the dynamic process of nutrients throughout the plant growth and development cycle. Critical kinetic information such as nutrient absorption, distribution among different tissues, and transport efficiency under environmental stress is completely masked. Due to its destructive nature, the detected kernels cannot be used for subsequent reproduction, and breeders can only make an inferred selection based on the detection results of other kernels of the same plant, which undoubtedly introduces potential errors and reduces the accuracy of the selection. With the development of molecular biology technology, although the function of a specific gene can be studied by constructing a stable transgenic strain, the process is long in time consumption and huge in workload, and is difficult to apply to rapid screening and functional verification of multiple candidate genes in a large-scale breeding population. Therefore, the prior art system generally lacks a means capable of nondestructively, high-throughput and high-precision quantification of the nutritional dynamics of living plants. The lack makes the breeding process still a relatively black box type selection process which depends on static end point data, and severely restricts the efficiency and the accuracy of cultivating high-nutrition crop varieties. Disclosure of Invention In view of the above problems, the present invention has been made to provide a method for selecting high-nutrition wheat with the aid of molecular markers, which overcomes the above problems or at least partially solves the above problems, and can solve the problem that the existing method for selecting high-nutrition wheat with the aid of molecular markers mainly relies on static DNA markers, the association with a target trait is easily affected by genetic background and environmental factors, resulting in insufficient selection accuracy, and can also bring about the effect of directly reflecting the actual functional expression and efficiency of genes under a specific genetic background, and avoiding the association uncertainty between the traditional static DNA markers and the final trait due to environmental interaction. Specifically, according to one aspect of the present invention, a method for assisting in selecting high-nutrition wheat using molecular markers comprises the steps of: S1, obtaining seeds in a development stage on a wheat plant, and introducing fluorescence resonance energy transfer sensing proteins with specific responses to target nutrients into the seeds; S2, performing living body imaging on the grains introduced with the fluorescence resonance energy transfer sensing protein to obtain space-time fluorescence signal data representing the dynamic accumulation process of the target nutrients in the grains; S3, calculating one or more space-time nutrition kinetic parameters of the dynamic accumulation process based on the space-time fluorescence signal data; s4, screening out wheat plants with excellent dynamic accumulation process characteristics by taking the space-time nutrition dynamic parameters as selection basis. Preferably, in step S2, the in vivo imaging of the kernel into which the fluorescent resonance energy transfer sensor protein is introduced to obtain spatiotemporal fluorescence signal data characterizing the dynamic accumulation process of the target nutrients inside the kernel includes: S21, placing the seeds on a microfluidic chip for perfusion culture; s22, changing the components of the perfusion culture solution to apply standardized environmental stress; S23, acquiring space-time fluorescence signal data of the kernels after stress relief. Preferably, the method further comprises