CN-121994954-A - Method for screening drought-enduring alfalfa varieties

CN121994954ACN 121994954 ACN121994954 ACN 121994954ACN-121994954-A

Abstract

The invention discloses a method for screening drought-tolerant alfalfa varieties, which relates to the technical field of pasture breeding, and comprises the specific steps of uniformly processing seeds and planting and culturing, carrying out drought stress treatment in a four-leaf period, collecting leaf samples in a later stress period, carrying out liquid nitrogen freezing preservation, obtaining metabolite data by using a phase chromatography mass spectrometry technology, constructing characteristic metabolite fingerprint spectrums, screening drought-tolerant alfalfa varieties and materials by combining a cosine similarity algorithm, forming a complete technical path from the seeds to the varieties and material identification screening, and realizing efficient screening and breeding of the drought-tolerant varieties.

Inventors

WANG GUODONG
GU XIAN
MA XUEJUN
GUO BIN
LIU WENYU

Assignees

甘肃省农业科学院畜草与绿色农业研究所(甘肃省农业科学院农业质量标准与检测技术研究所)

Dates

Publication Date: 20260508
Application Date: 20260204

Claims (10)

1. A method for screening drought-tolerant alfalfa varieties, which is characterized by comprising the following specific steps: S1, seed treatment and planting, namely selecting seeds of different alfalfa varieties, soaking, sterilizing and accelerating germination by using an ethanol solution, transplanting the seeds into a uniformly-proportioned sterilization matrix after the seeds are exposed to white, enabling the planting quantity of each pot to be consistent, and placing all plants in the same artificial climate box for culture, so as to maintain the initial water content of soil to be stable; s2, drought stress treatment, namely starting drought stress treatment when plants are cultivated to a four-leaf period, stopping watering, adopting a soil moisture tachometer to detect the soil moisture content every day, and keeping the parameters of the cultivation environment stable during the stress period; s3, collecting and storing samples, namely collecting leaf samples of all varieties on the 7 th day of drought stress, taking plants with consistent growth of each variety as repeated samples, immediately putting the collected plants into liquid nitrogen for freezing, and then transferring the collected plants to an ultralow temperature refrigerator for storage; S4, extracting and analyzing the metabolites, namely extracting the metabolites in the frozen leaf samples, centrifuging, taking supernatant, drying and re-dissolving, filtering by a filter membrane, and performing non-targeted metabonomics analysis by adopting a liquid chromatography-mass spectrometry technology to obtain the metabolite data of each variety; s5, fingerprint construction and screening, namely constructing a characteristic metabolite fingerprint of drought-tolerant alfalfa based on metabolite analysis results, determining key drought-tolerant metabolites, calculating the matching degree of metabolite maps of all varieties and the fingerprint by adopting a cosine similarity algorithm, and screening out drought-tolerant alfalfa varieties meeting a threshold value.
2. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the S1, seed treatment and planting, disinfection and germination accelerating are carried out by soaking in 75% ethanol solution for 30 seconds, then washing with sterile water, after residual ethanol is removed, uniformly spreading the seeds in a culture dish paved with 2 to 3 layers of wet filter paper, and then, germination accelerating in a climatic chamber with a relative humidity of 58 to 62% at 24 to 26 ℃ for 16 hours and 16 hours, and a small amount of sterile water is added daily to keep the filter paper moist until the seeds are whitened.
3. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the S2 drought stress treatment, the regular watering of seedlings growing to the four-leaf stage is stopped during the drought stress treatment, a small amount of water is supplemented by drip irrigation, the soil water content is maintained at 28-32%, the temperature, the illumination and the relative humidity are kept consistent with those before the drought stress during the stress period, and the soil water content is detected and recorded by a soil water content tachometer every day.
4. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the S3, sample collection and preservation, the specific operation of collecting leaf samples of each variety is that 3 seedlings which are consistent in growth state, free of plant diseases and insect pests and free of mechanical damage are selected for each variety, 2-3 functional leaves which are fully unfolded are collected at the top of each plant, 0.5g to 0.7g of each plant are collected, the same variety of leaf is mixed into 1 sample, the total fresh weight of each sample is controlled at 2g, sterile scissors are used during collection, 75% ethanol solution is used for wiping the scissors for sterilization after each variety is collected, the leaf stalks and the leaf veins are removed after collection, and the leaf stalks and the leaf veins are placed into a sterile centrifuge tube after surface moisture is sucked by sterile filter paper.
5. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in S3, the specific operation of extracting metabolites in frozen leaf samples is that the frozen leaf samples are taken out, ground into powder in a liquid nitrogen environment for no more than 5 minutes, methanol-chloroform-water mixed extract is added into the powder, the powder is fully contacted with the extract by vortex oscillation, the supernatant is centrifugally taken out to a new sterile centrifuge tube after standing for 12 hours, and the supernatant is re-dissolved by methanol-water solution after being dried and filtered to obtain the metabolite extract.
6. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the step S4, metabolite extraction and analysis, a liquid chromatography-mass spectrometry combined technology is an analysis technology combining high-efficiency separation capacity of liquid chromatography with high-sensitivity detection capacity of mass spectrometry, a re-dissolved and filtered metabolite extract is separated by liquid chromatography, different metabolites flow out of a chromatographic column in sequence according to distribution differences of the metabolites between stationary phases and mobile phases, the separated metabolite components are continuously led into mass spectrometry, metabolite molecules are converted into charged ions by adopting an electrospray ion source, and mass-charge ratios of the ions are detected by a mass analyzer, so that characteristic signals of each metabolite are obtained.
7. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the step S4, metabolite extraction and analysis, non-targeted metabonomics analysis is non-biased qualitative and quantitative analysis of all detectable metabolites in samples, wherein characteristic signal data acquired by liquid chromatography-mass spectrometry is preprocessed, including identification and matching of signal peaks, correction of retention time differences among different samples, normalization of signal intensity to eliminate errors, and finally metabolite information of all samples is integrated to generate a data set containing metabolite varieties and relative contents.
8. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the step of S5, fingerprint construction and screening, when characteristic metabolite fingerprints of drought-tolerant alfalfa are constructed, firstly metabolite data of the drought-tolerant alfalfa varieties are collected, screening is carried out on the data, metabolite information corresponding to interference signals is removed, metabolite related data with specificity are reserved, the screened metabolite data are classified and sorted, characteristic detection signals corresponding to all metabolites are recorded at the same time, and finally the determined metabolite types, relative content ranges and the characteristic detection signals are integrated and arranged according to a unified standard format to form a fixed pattern form, so that the construction of the drought-tolerant alfalfa characteristic metabolite fingerprints is completed.
9. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the step S5, the mathematical expression of the cosine similarity algorithm in the fingerprint construction and screening is: Wherein, the Representing the matching degree of the metabolite pattern of the alfalfa varieties to be screened and the characteristic metabolite fingerprint pattern; Represents the total number of key drought tolerant metabolites determined; Representing the relative content of the i-th key drought-enduring metabolite in the metabolite map of the alfalfa variety to be screened; representing the relative content of the ith key drought-enduring metabolite in the characteristic metabolite fingerprint.
10. The method for screening drought-tolerant alfalfa varieties according to claim 1, wherein in the S5, the specific process of screening drought-tolerant alfalfa varieties meeting a threshold is that a preset matching degree threshold is 0.8, the matching degree calculation result of metabolite patterns of alfalfa varieties to be screened and characteristic metabolite fingerprint patterns is compared with the matching degree threshold, when the matching degree of alfalfa varieties to be screened is greater than or equal to 0.8, the initially determined drought-tolerant alfalfa varieties meeting the condition are initially determined, then the initially determined varieties are repeatedly verified, metabolite data are obtained again through metabolite extraction and analysis steps, the matching degree is recalculated, and when the matching degree after repeated verification is still greater than or equal to 0.8, the varieties are finally determined to be drought-tolerant alfalfa varieties meeting the threshold.

Description

Method for screening drought-enduring alfalfa varieties Technical Field The invention relates to the technical field of pasture breeding, in particular to a method for screening drought-enduring alfalfa varieties. Background Alfalfa is taken as high-quality perennial leguminous forage grass and occupies an important place in animal husbandry production, the yield and the quality of the alfalfa directly influence the development quality of the animal husbandry industry, the drought and semiarid areas in China are wide in area, water resource shortage becomes a key factor for restricting the large-scale planting of the alfalfa, drought stress can lead to the growth resistance and the yield reduction of the alfalfa, even quality degradation and seriously influence the stability of forage grass supply, therefore, the alfalfa variety with strong drought resistance is screened and cultivated, the method has important practical significance for improving the agricultural production benefit in the drought area and guaranteeing the sustainable development of the animal husbandry, along with the continuous progress of agricultural technology, the molecular biology and the metabonomics technology are increasingly widely applied in the field of crop variety screening, more accurate technical support can be provided for variety drought tolerance evaluation by analyzing the metabolic mechanism of crops, the urgent development of the drought tolerance of the alfalfa variety is met, and the sustainable development of the alfalfa industry under drought environment is promoted. The traditional alfalfa drought-resistant variety screening methods are mainly dependent on plant external morphology observation and few physiological index measurement, have obvious limitations, the morphological observation mainly focuses on external performances such as plant growth conditions, leaf withering degree and the like, are easily influenced by environmental fluctuation and human judgment difference, lack of objective unified evaluation standards, result in strong subjectivity and insufficient accuracy of screening results, the physiological index measurement is often only aimed at few known drought-resistant related indexes, the whole metabolic response of plants in response to drought stress cannot be comprehensively captured, the essential characteristics of variety drought resistance are difficult to reflect, the screening results are not comprehensive and reliable, in addition, the traditional methods are complicated in operation flow, long in screening period, require a large amount of manpower and material resources, are difficult to adapt to the requirements of large-scale and batch variety screening, high-efficiency variety evaluation basis cannot be rapidly provided for breeding work, and the progress of cultivation and popularization of drought-resistant alfalfa varieties is restricted. Disclosure of Invention The invention aims to make up the defects of the prior art and provides a method for screening drought-tolerant alfalfa varieties, which is characterized in that seeds are uniformly treated and planted, leaf samples are collected after drought stress is started, metabolites are extracted and analyzed by a liquid chromatography-mass spectrometry combined technology to construct characteristic metabolite fingerprint, non-targeted metabonomics is adopted, and varieties are screened by combining a cosine similarity algorithm, so that the practical application value is high, alfalfa varieties with strong drought tolerance can be screened rapidly and accurately, the agricultural productivity is improved, and the method is favorable for agricultural sustainable development especially in drought or water resource deficient areas. The invention provides a method for screening drought-enduring alfalfa varieties, which aims to solve the technical problems and comprises the following specific steps: S1, seed treatment and planting, namely selecting seeds of different alfalfa varieties, soaking, sterilizing and accelerating germination by using an ethanol solution, transplanting the seeds into a uniformly-proportioned sterilization matrix after the seeds are exposed to white, enabling the planting quantity of each pot to be consistent, and placing all plants in the same artificial climate box for culture, so as to maintain the initial water content of soil to be stable; S2, drought stress treatment, namely starting drought stress treatment when plants are cultivated to a four-leaf period, stopping watering, adopting a soil moisture tachometer to detect the soil moisture content every day, and keeping the cultivation environment parameters stable during stress, wherein the four-leaf period is the period when alfalfa seedlings grow out of fourth true leaves; s3, collecting and storing samples, namely collecting leaf samples of all varieties on the 7 th day of drought stress, taking plants with consistent growth of each variety as repea