Mulberry germplasm resource salt tolerance evaluation and salt-tolerant germplasm screening method

Abstract

The invention discloses a mulberry germplasm resource salt tolerance evaluation and salt-tolerant germplasm screening method, and belongs to the technical field of germplasm resource evaluation and screening. The invention discloses a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm. The method comprises the following steps: sampling and storing test materials, preparing explants, disinfecting, culturing, identifying salt tolerance, evaluating the salt tolerance by adopting a membership function and clustering analysis, and comprehensively analyzing the salt tolerance of winter buds of mulberries. According to the method, the salt tolerance of mulberry planting resources collected in different regions is identified, the bud germination state index and the relative strong bud growth amount of mulberry winter buds are taken as evaluation indexes for identifying the salt tolerance of a large number of screened mulberry materials, comprehensive evaluation of the salt tolerance is performed by adopting a membership function and clustering analysis method, and different salt-tolerant germplasms are screened out; and excellent germplasm resources are provided for subsequent salt-tolerant gene mining, salt-tolerant variety breeding and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of germplasm resource evaluation and screening, and specifically relates to a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasms. Background Art

[0002] Mulberry (Morus L.) is a perennial deciduous tree or shrub of the genus Morus in the Moraceae family. It is widely distributed in my country and has significant nutritional, medicinal, economic and social benefits. Mulberry is cold-resistant, salt-resistant, high-temperature-resistant, drought-resistant and barren, and has strong adaptability. It is one of the valuable genetic resources that can withstand abiotic stresses. Its role in ecological governance such as afforestation of barren hills, sand control, and soil erosion control is becoming increasingly prominent. It has become one of the important pioneer tree species in vegetation restoration and reconstruction projects in fragile ecological areas. In recent years, the problem of soil salinization has become increasingly serious and has become an important factor restricting the global environment and agricultural development. According to statistics, irrigated land and arable land around the world are harmed by salinization to varying degrees, and there is a trend of increasing year by year, which poses a serious threat to the sustainability of global crop production and food security. In order to alleviate the impact of soil salinization, people have taken various measures to improve and utilize salinized land. Among them, afforestation is one of the biological measures to improve saline-alkali land. It can not only improve the environment and inhibit soil salinization, but also directly utilize saline-alkali land to produce forest fruits and improve the production capacity of saline-alkali land. Therefore, screening of genetic germplasm resources with strong salt tolerance is the basis for breeding salt-tolerant varieties, which will be of great significance for the cultivation of salt-tolerant mulberry trees and the utilization of salinized land.

[0003] At present, the research on salt tolerance of mulberry trees by domestic and foreign scholars mainly focuses on field identification or soil culture methods in the laboratory. Chen et al., a member of the research team, used different concentrations of NaCl to screen the genetic variation offspring of 14 F1 hybrids of mulberry trees for salt tolerance in the early stage, and found that 0.9% NaCl was the most suitable concentration for evaluating salt tolerance, and screened out the best hybrid combination of Anzhen × Xinghai Neishuangyou parents for salt tolerance. Liu Xueqin et al. used 12g / L NaCl and 400g / L PEG6000 to simulate salt and drought environments, and screened out the salt tolerance of 13 F1 generations of mulberry hybrid combinations, and screened out Guisangyou 62, which is suitable for coastal tidal flat saline-alkali land, Guisangyou 12 and Cesha-Za 6, which are suitable for perennial drought and severe salinization areas. Zhang Heyu et al. evaluated the salt tolerance of 12 mulberry trees and found that Huasang and Baisang were materials with strong salt tolerance. At present, studies have shown that tissue culture is one of the most effective ways to identify plant salt tolerance, while there are few reports on the identification of salt tolerance using mulberry winter bud tissue culture. Foreign scholars have screened salt-tolerant mulberry germplasm materials of genotypes such as C776, S1, SV1, S1635, C763, G2, G3, G4, S34, and S13 by using tissue culture method on mulberry axillary buds in vitro. Zhang Heyu et al. found that Ruisang, 9001, and Husang 197 had strong salt tolerance through salt tolerance culture of winter buds of different varieties of mulberry, and this result was basically consistent with the results of field salt tolerance identification. Therefore, the in vitro winter bud tissue culture method can be applied to the identification and evaluation of salt tolerance of mulberry varieties or strains. The study found that the appropriate NaCl concentration can effectively show the response of mulberry winter buds to salt stress, ensuring the reliability and validity of the test results. At present, the comprehensive evaluation of salt tolerance of germplasm resources mainly adopts correlation analysis, principal component analysis, membership function analysis, cluster analysis and other methods, which can effectively analyze the differences in salt tolerance of different germplasm resources. Summary of the invention

[0004] The purpose of the present invention is to provide a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0006] A method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm comprises the following steps:

[0007] (1) Sampling and storage of test materials: The appropriate sampling period is after the mulberry trees have passed the deep dormancy period and before the sap flows. In order to ensure the consistency of the winter bud development status of different germplasm materials, sampling should not be carried out after the winter buds sprout. The sampling trees in Chengde, Hebei Province were not cut down in the summer in the previous year to allow the mulberry buds to complete the flower bud differentiation process. Cut one-year-old strong branches with full wood, plump winter buds, no dead branches, and no diseases and insect pests in each region, cut off about 1 / 3 of the lower part of the branch and discard it, cut the rest into small sections of 25-30 cm, put them in self-sealing bags, and store them at 0-2℃ for later use;

[0008] (2) Explant preparation: Hold the branch upside down and use a scalpel to cut off the leaf scar along the upper edge, deep into the wood. After cutting off all the leaf scars on the branch, hold the branch upright and use a scalpel to remove the secondary buds and the scales below the bud. Then use tweezers to gently peel off the outer scales to expose the first layer of young leaves. Cut off the bud intact, place it in a clean culture dish, cover the dish, and sterilize it within 2 hours.

[0009] (3) Disinfection: Different winter bud materials were placed in small copper wire baskets, soaked in sterile water for 10 min, and then removed from the baskets, drained and sterilized in 70% ethanol for 20 s, rinsed with sterile water 2-3 times, and then sterilized in 0.1% mercuric chloride solution for 6 min, rinsed with sterile water 4 times, and then placed on sterile gauze. After no water droplets remained in the baskets and the winter bud materials in the baskets, inoculation could be carried out;

[0010] (4) Start culture: Inoculate different winter bud materials into the pre-culture medium, with 45-50 explants inoculated in each material and no more than 6 explants in each culture bottle, and culture for 7 days;

[0011] (5) Salt tolerance identification: Place the winter buds of the test materials on a 1 / 2MS solid culture medium containing 150mmol / L NaCl or distilled water (control), and place them in an incubator at a temperature of (25±2)℃, a light intensity of 2000-3000lx, and a day and night light cycle of 14h / 10h. Place 5 winter buds in each bottle, and every 3 bottles are used as 1 treatment, and repeat 3 times. After culturing for 14 days, count the number of dead, atrophic, maintained, debudded, and leafed winter bud explants to calculate the germination state index; select the strong buds and place them in a new control and a sterilized culture medium containing 1 / 2MS containing NaCl. After 14 days of culture, measure the weight of the winter bud explants and calculate the relative strong bud growth. Determine the germination state index of the 6 germplasm materials on the 14th day after the first transfer and the relative strong bud growth on the 14th day after the second transfer under different salt stress concentrations;

[0012] (6) Using membership function and cluster analysis to evaluate salt tolerance: Calculate the membership function values ​​of the two indicators of germination state index and relative strong bud growth in step (5) to obtain the membership function values ​​of the two indicators, then average the membership function values ​​of the two indicators to obtain the membership function average value, and classify the salt tolerance according to the size of the membership function value. At the same time, perform cluster analysis according to the size of the average membership function value;

[0013] (7) Comprehensive analysis of salt tolerance of mulberry winter buds.

[0014] Furthermore, the test materials in step (1) are taken from southern salt-tolerant materials, ancient mulberry single plant materials along the coast of Hebei and Tianjin, wild mulberry single plant materials along the coast of Hebei and Tianjin, and materials preserved in the planting resource garden of the Sericulture Research Institute.

[0015] Furthermore, the culture medium for pre-culture in step (4) is: 1 / 2MS+6-BA (2 mg / L)+sucrose (15 g / L)+agar (6 g / L).

[0016] Furthermore, the formula for the germination state index in step (5) is: germination state index (%) = (number of buds removed*1 + number of opened leaves*2 - number of dead leaves*2 - number of atrophy*1) / (total number of buds)*100%; the formula for the relative strong bud growth amount is: relative strong bud growth amount (%) = (fresh weight after the second transfer culture of salt tolerance treatment for 14 days / fresh weight after the second transfer culture of control for 14 days)*100%.

[0017] Furthermore, the raw data of the experiment in step (5) were subjected to variance analysis using Microsoft Excel 2013 software, and the mean of each treatment was calculated.

[0018] Furthermore, the membership value formula of the germination state index and the relative strong bud growth amount in step (6) is:

[0019] Salt tolerance coefficient = measured value under treatment conditions / measured value under control conditions (1)

[0020] Membership function

[0021] Inverse membership function Where U ij The membership function value of the jth index (j=1,2,…,) of the i-th germplasm material. X ij is the salt tolerance coefficient of the jth index of the i-th germplasm material, X min , X maxThey are the minimum and maximum values ​​of the salt tolerance coefficient of the jth index in all the tested germplasm materials. The membership values ​​of the two indicators, germination state index and relative strong bud growth, were calculated. If the measured index was positively correlated with salt tolerance, the membership function formula (2) was used for calculation. If it was negatively correlated, the inverse membership function formula (3) was used for calculation.

[0022] Furthermore, the salt tolerance grading standard in step (6) is as follows: when the average membership function value is 0.9651-0.9844, it is a particularly salt-tolerant planting material; when the average membership function value is 0.7619-0.8274, it is a strongly salt-tolerant germplasm material; when the average membership function value is 0.488-0.7508, it is a moderately salt-tolerant germplasm material; when the average membership function value is 0.1037-0.446, it is a salt-sensitive germplasm material; when the average membership function value is 0.0000-0.217, it is a highly salt-sensitive germplasm material.

[0023] Furthermore, the cluster analysis in step (6) is performed using SPSS 23.0 software, the cluster analysis utilizes the method of linking between groups, the measurement interval uses squared Euclidean distance for cluster analysis, and the distance is recalibrated.

[0024] Beneficial Effects

[0025] The invention provides a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasms. The invention identifies the salt tolerance of mulberry planting resources collected from different regions, uses the germination state index of winter buds of mulberry trees and the relative strong bud growth amount as evaluation indicators for screening the salt tolerance of a large number of mulberry materials, adopts the methods of membership function and cluster analysis to perform comprehensive evaluation of salt tolerance, screens out different salt-tolerant germplasms, and provides excellent germplasm resources for subsequent salt-tolerant gene discovery and salt-tolerant variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a cluster diagram of salt tolerance of in vitro winter bud germination of 63 mulberry germplasm materials; Note: The numerical range in the figure is the salt tolerance ranking range (average membership function value) of various germplasm materials. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0028] The embodiment of the present invention discloses a method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm, comprising the following steps:

[0029] (1) Collection and storage of experimental materials: The appropriate period for sampling is after the mulberry trees have passed the deep dormancy period and before the sap flows. In order to ensure the consistency of the winter bud development status of different germplasm materials, it is forbidden to sample after the winter buds sprout. The sampling trees in Chengde, Hebei Province were not cut down in the summer in the previous year to allow the mulberry buds to complete the flower bud differentiation process. Cut one-year-old strong branches with full wood, full winter buds, no dead branches, and no diseases and insect pests. Cut off about 1 / 3 of the lower part of the branch and discard it. Cut the rest into small sections of 25-30 cm, put them in self-sealing bags, and store them at 0-2℃ for use. The 63 test materials include 6 southern salt-tolerant materials, 7 ancient mulberry single plant materials along the Hebei-Tianjin coast, 10 wild mulberry single plant materials along the Hebei-Tianjin coast, and 40 materials preserved in the planting resource garden of the Sericulture Research Institute. The sources of the materials are detailed in Table 1;

[0030] Table 1 Sources of tested mulberry winter bud materials

[0031]

[0032]

[0033]

[0034] (2) Explant preparation: Hold the branch upside down and use a scalpel to cut off the leaf scar along the upper edge, deep into the wood. After cutting off all the leaf scars on the branch, hold the branch upright and use a scalpel to remove the secondary buds and the scales below the bud. Then use tweezers to gently peel off the outer scales to expose the first layer of young leaves. Cut off the bud intact and place it in a clean culture dish. Cover the dish with a lid for 2 hours.

[0035] Disinfection within hours;

[0036] (3) Disinfection: Different winter bud materials were placed in small copper wire baskets, soaked in sterile water for 10 min, removed from the baskets, drained, transferred to 70% ethanol for disinfection for 20 s, rinsed with sterile water 2-3 times, and then transferred to 0.1%

[0037] Sterilize in mercuric chloride solution for 6 minutes, rinse with sterile water 4 times, then place the basket on sterile gauze, and inoculate after there are no water drops remaining in the basket and the winter bud materials in the basket;

[0038] (4) Cultivation: Different winter bud materials were inoculated into pre-culture medium, 45-50 explants were inoculated into each material, and no more than 6 explants were inoculated into each culture bottle, and cultured for 7 days. The pre-culture medium was: 1 / 2MS+6-

[0039] BA (2 mg / L) + sucrose (15 g / L) + agar (6 g / L);

[0040] (5) Salt tolerance identification: Place the winter buds of the test material on a 1 / 2MS solid culture medium containing 150mmol / L NaCl or distilled water (control), and place it in an incubator at a temperature of (25±2)℃, a light intensity of 2000-3000lx, and a day and night light cycle of 14h / 10h. Place 5 winter buds in each bottle, and every 3 bottles as 1 treatment, and repeat 3 times. After culturing for 14 days, count the number of winter bud explants that are dead, atrophic, maintained, debudded, and have opened leaves, and calculate the germination state index (Formula 1); select the strong buds and place them in a new control medium and a sterilized culture medium containing 1 / 2MS containing NaCl. After 14 days of culture, measure the weight of the winter bud explants and calculate the relative strong bud growth.

[0041] Determine the germination state index of the six germplasm materials on the 14th day after the first transfer and the relative strong bud growth on the 14th day after the second transfer under different salt stress concentrations (Formula 2);

[0042] Germination state index (%) = (number of buds removed * 1 + number of leaves opened * 2 - number of dead * 2 - number of atrophy * 1) / (total number of buds

[0043] *2)*100% (one)

[0044] Relative strong bud growth (%) = (fresh weight of salt-tolerant treatment after the second transfer culture for 14 days / fresh weight of control after the second transfer culture for 14 days) * 100% (b)

[0045] (6) Using membership function and cluster analysis to evaluate salt tolerance: Calculate the membership function values ​​of the germination state index and the relative strong bud growth in step (5) to obtain the membership function values ​​of the two indicators, then average the membership function values ​​of the two indicators to obtain the membership function average value, and classify the salt tolerance according to the size of the membership function value. At the same time, perform cluster analysis according to the size of the average membership function value.

[0046] The membership function formula of the germination state index and the relative strong bud growth amount is:

[0047] Salt tolerance coefficient = measured value under treatment conditions / measured value under control conditions (1)

[0048] Membership function

[0049] Inverse membership function Where U ij The membership function value of the jth index (j=1,2,…,) of the i-th germplasm material. X ij is the salt tolerance coefficient of the jth index of the i-th germplasm material, X min , X maxare the minimum and maximum values ​​of the salt tolerance coefficient of the jth index in all the tested germplasm materials. Calculate the membership values ​​of the two indicators of germination state index and relative strong bud growth. If the measured index is positively correlated with salt tolerance, use the membership function formula (2) for calculation. If it is negatively correlated, use the inverse membership function formula (3) for calculation.

[0050] (7) Comprehensive analysis of salt tolerance of mulberry winter buds.

[0051] Results and Analysis

[0052] Response of winter buds of different mulberry planting materials to salt stress

[0053] Under 150mmol / L NaCl stress, the germination state index and relative strong bud growth of 63 mulberry germplasm materials were statistically analyzed and shown in Table 2. The results showed that compared with the relative strong bud growth of each sample, Yingjiaoting No. 1 (0012) from Qinhuangdao, Hebei Province had the highest relative strong bud growth of 91.57%, followed by Taiwan Qingpi (0006) and J3-3 (00062), which were 86.27% and 81.39%, respectively, while Tengsang (0034) from Chengde, Hebei Province had the lowest relative strong bud growth of only 15.58%. The results of germination state index determination showed that Taiwan Qingpi (0006) and Yingjiaoting No. 1 (0012) had the highest germination state index under salt stress, which were 91.11% and 88.89%, respectively, while Tengsang (0034) had the lowest germination state index, which was only 19.79%. Combining the two indicators, it was found that the germination state index and relative strong bud growth of Taiwan Qingpi (0006) and Yingjiaoting No. 1 (0012) were higher, indicating that the germination ability and vitality of the winter buds of both under salt stress were at a high level, thus having better salt tolerance at the bud stage, while the salt tolerance of the winter buds of Tengsang (0034) was the weakest.

[0054] Table 2 Germination characteristics of mulberry winter buds in vitro under salt stress

[0055]

[0056]

[0057]

[0058] Statistical analysis of coefficient of variation of each index of the tested materials

[0059] The statistical analysis of the coefficient of variation of the two indicators of winter buds of 63 mulberry germplasm materials showed (Table 3) that after the winter buds of different mulberry germplasm resources were subjected to salt stress, the average values ​​of the germination state index and the relative strong bud growth were 65.07% and 51.07%, respectively. The variation range of the germination state index was 9.38%-100.00%, and the variation range of the relative strong bud growth was 12.62%-96.22%. Among them, the coefficients of variation of these two indicators were relatively large, which were 25.51 and 35.37, respectively, indicating that there were significant differences in the germination state index and relative strong bud growth of winter buds of different mulberry germplasm resources under salt stress. Both can well reflect the germination state of mulberry winter buds under salt stress and serve as indicators for salt tolerance identification.

[0060] Table 3 Descriptive statistics and variance analysis of salt tolerance indices of 63 mulberry germplasm materials under salt stress

[0061]

[0062] Evaluation of salt tolerance of mulberry germplasm materials at bud stage using membership function and cluster analysis

[0063] In order to comprehensively evaluate the salt tolerance of mulberry germplasm materials at the bud stage, the membership function values ​​of the two indicators, germination state index and relative strong bud growth, were added and averaged to obtain 63 membership values ​​(Table 4, Table 5). At the same time, according to the size of the average membership function value, the inter-group linkage method based on the Euclidean square distance was used for cluster analysis ( Figure 1 ), when the Euclidean square distance was between 3 and 4, the tested mulberry germplasm materials were divided into 5 categories. The mean value of the membership function of the first group was between 0.9651 and 0.9844, with a total of 2 germplasm materials, accounting for 3.2% of the tested materials. These materials had the highest relative values ​​of each trait under salt stress and were special salt-tolerant germplasm materials; the mean value of the membership function of the second group was between 0.7619 and 0.8274, with a total of 10 germplasm materials, accounting for 15.9%, with high relative values ​​of each trait and strong salt-tolerant germplasm materials; the mean value of the membership function of the third group was between 0.9651 and 0.9844, with a total of 2 germplasm materials, accounting for 3.2% of the tested materials. These materials had the highest relative values ​​of each trait under salt stress and were special salt-tolerant germplasm materials; The range is between 0.488 and 0.7508, with a total of 31 germplasm materials, accounting for 49.2%, and the relative values ​​of each trait are medium, belonging to moderately salt-tolerant germplasm materials; the fourth group has a membership function mean range of 0.1037-0.446, with a total of 15 germplasm materials, accounting for 23.8%, and the relative values ​​of each trait are low, which are salt-sensitive germplasm materials; the fifth group has a membership function mean range of 0.0000-0.217, with a total of 5 germplasm materials, accounting for 7.9%, and the relative values ​​of each trait are very low, which are highly salt-sensitive germplasm materials. Among them, the top five mulberry germplasm resources in terms of salt tolerance are 0012, 0006, 0007, 0062, and 0033.

[0064] Table 4 Membership values ​​of salt tolerance of winter buds of mulberry germplasm materials

[0065]

[0066]

[0067] Table 5 Classification of salt tolerance of 63 mulberry germplasm resources

[0068] Classification Salt tolerant type Membership value interval quantity Proportion / % I Special salt tolerance 0.9651～0.9844 2 3.2 II Strong salt tolerance 0.7619～0.8274 10 15.9 III Moderate salt tolerance 0.488～0.7508 31 49.2 IV Salt sensitivity 0.1037～0.446 15 23.8 V Salt sensitivity 0.0000～0.217 5 7.9

[0069] Comprehensive analysis of salt tolerance of mulberry winter buds

[0070] Cluster analysis based on salt tolerance at bud stage ( Figure 1 ) and 63 salt tolerance classification lists of mulberry trees (Table 6). Salt tolerance (moderate tolerance) and above salt tolerance resources accounted for 68.3% of the tested resources. Among them, Taiwan Qingpi (0006) from Hangzhou, Zhejiang and Yingjiaoting No. 1 (0012) from Qinhuangdao, Hebei had high values ​​of various traits under salt stress and the best comprehensive salt tolerance, which were special salt-tolerant materials; Yongqing No. 6 (0058), Huanglu Self-cross-B1 (0039), Huanglu (0060), Shengli B1 (0054) and Tengsang (0034) and other 6 The salt tolerance of winter buds of all germplasm resources was poor, and they were highly salt-sensitive germplasm materials. Among them, the membership values ​​of the germination state index and relative strong bud growth of Tengsang (0034) were both 0, and the winter bud salt tolerance was ranked 63, with the worst salt tolerance. At the same time, 7 single-plant materials of ancient mulberry from the coastal areas of Hebei and Tianjin and 10 single-plant materials of wild mulberry from the coastal areas of Hebei and Tianjin were sampled, all of which performed better than moderately salt-tolerant germplasm materials, indicating that the materials from the saline-alkali land around the coastal areas of Hebei and Tianjin have good salt tolerance, which can provide an excellent source of parents for salt-tolerant breeding of mulberry trees.

[0071] Table 6 Classification of salt tolerance of 63 mulberry germplasm materials at the seedling stage

[0072]

[0073] It should be noted that the above embodiments are only some embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm, characterized in that: The following steps are involved: (1) Sampling and storage of test materials: The appropriate sampling period is from the time when mulberry trees have passed the deep dormancy period to before the sap flows. In order to ensure the consistency of the winter bud development of different germplasm materials, sampling should not be carried out after the winter buds sprout. Cut one-year-old strong branches with full wood, full winter buds, no dead branches, and no diseases and insect pests from various regions, cut off about 1 / 3 of the lower part of the branch and discard it, cut the rest into small sections of 25-30 cm, put them in self-sealing bags, and store them at 0-2℃ for later use; (2) Explant preparation: Hold the branch upside down and use a scalpel to cut off the leaf scar along the upper edge, deep into the wood. After cutting off all the leaf scars on the branch, hold the branch upright and use a scalpel to remove the secondary buds and the scales below the bud. Then use tweezers to gently peel off the outer scales to expose the first layer of young leaves. Cut off the bud intact, place it in a clean culture dish, cover the dish, and sterilize it within 2 hours. (3) Disinfection: Different winter bud materials were placed in small copper wire baskets, soaked in sterile water for 10 min, and then removed from the baskets, drained and sterilized in 70% ethanol for 20 s, rinsed with sterile water 2-3 times, and then sterilized in 0.1% mercuric chloride solution for 6 min, rinsed with sterile water 4 times, and then placed on sterile gauze. After no water droplets remained in the baskets and the winter bud materials in the baskets, inoculation could be carried out; (4) Start culture: Inoculate different winter bud materials into the pre-culture medium, with 45-50 explants inoculated in each material and no more than 6 explants in each culture bottle, and culture for 7 days; (5) Salt tolerance identification: Place the winter buds of the test material on 1 / 2MS solid culture medium containing 150mmol / L NaCl or distilled water, and place it in an incubator at 25±2℃, 2000-3000lx of light, and a day and night photoperiod of 14h / 10h. Place 5 winter buds in each bottle, and every 3 bottles as 1 treatment, with 3 replicates. After 14 days of culture, count the number of winter bud explants that are dead, atrophic, maintained, debudded, and have opened leaves, and calculate the germination state index; The strong buds were selected and placed in the new control medium and the sterilized medium containing 1 / 2MS of NaCl. The weight of the winter bud explants was measured after 14 days of culture, and the relative strong bud growth was calculated. The germination state index of the six germplasm materials on the 14th day after the first transfer and the relative strong bud growth on the 14th day after the second transfer were measured under different salt stress concentrations; (6) Using membership function and cluster analysis to evaluate salt tolerance: Calculate the membership function values ​​of the germination state index and the relative strong bud growth in step (5) to obtain the membership function values ​​of the two indicators, then average the membership function values ​​of the two indicators to obtain the membership function average value, and classify the salt tolerance according to the size of the membership function value. At the same time, cluster analysis is performed according to the size of the average membership function value; (7) Comprehensive analysis of salt tolerance of mulberry winter buds.

2. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, characterized in that: The test materials in step (1) are taken from southern salt-tolerant materials, ancient mulberry single plant materials along the Hebei-Tianjin coast, wild mulberry single plant materials along the Hebei-Tianjin coast, and materials preserved in the planting resource garden of the Sericulture Research Institute.

3. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, characterized in that: The culture medium for pre-culture in step (4) is: 1 / 2MS+6-BA+sucrose+agar.

4. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 3, characterized in that: The 6-BA concentration is 2 mg / L, the sucrose concentration is 15 g / L, and the agar dosage is 6 g / L.

5. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, characterized in that: The formula of the germination state index in the step (5) is: germination state index (%) = (number of buds falling off * 1 + number of opened leaves * 2 - number of dead leaves * 2 - number of atrophy * 1) / (total number of buds) * 100%; the formula of the relative strong bud growth amount is: relative strong bud growth amount (%) = (fresh weight after the second transfer culture of salt tolerance treatment for 14 days / fresh weight after the second transfer culture of control for 14 days) * 100%.

6. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, wherein: The raw data of the experiment in step (5) were analyzed for variance using Microsoft Excel 2013 software, and the mean of each treatment was calculated.

7. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, wherein: The membership value formula of the germination state index and the relative strong bud growth amount in step (6) is: Salt tolerance coefficient = measured value under treatment conditions / measured value under control conditions (1) Membership function Inverse membership function Where U ij The membership function value of the jth indicator of the i-th germplasm material. ij is the salt tolerance coefficient of the jth index of the i-th germplasm material, X min , X max They are the minimum and maximum values ​​of the salt tolerance coefficient of the jth index in all the tested germplasm materials. The membership values ​​of the two indicators, germination state index and relative strong bud growth, were calculated. If the measured index was positively correlated with salt tolerance, the membership function formula (2) was used for calculation. If it was negatively correlated, the inverse membership function formula (3) was used for calculation.

8. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, characterized in that: The salt tolerance classification standard in the step (6) is as follows: when the average membership function value is 0.9651-0.9844, it is a particularly salt-tolerant planting material; when the average membership function value is 0.7619-0.8274, it is a strongly salt-tolerant germplasm material; when the average membership function value is 0.488-0.7508, it is a moderately salt-tolerant germplasm material; when the average membership function value is 0.1037-0.446, it is a salt-sensitive germplasm material; when the average membership function value is 0.0000-0.217, it is a highly salt-sensitive germplasm material.

9. The method for evaluating salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm according to claim 1, characterized in that: The cluster analysis in step (6) is performed using SPSS 23.0 software. The cluster analysis uses the method of linking between groups. The measurement interval uses squared Euclidean distance for cluster analysis, and the distance is recalibrated.

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