Method for comprehensively evaluating cold resistance of porphyra yezoensis

Abstract

The invention provides a cold resistance comprehensive evaluation method for porphyra yezoensis, and belongs to the technical field of biological breeding. Four phenotypic indexes related to the cold resistance character are obtained through screening experiments, the four phenotypic indexes are the leaf area of the porphyra yezoensis thallus, the maximum light quantum efficiency Fv / Fm, the photosynthetic pigment chlorophyll a and allophycocyanin respectively, and the four phenotypic indexes can not only meet sustainable determination in a lossless state, but also can be used for detecting the cold resistance character of the porphyra yezoensis. And the cold resistance evaluation model constructed based on the few phenotypic indexes can achieve the purpose of accurately, efficiently and comprehensively evaluating the cold resistance of the porphyra yezoensis. Experimental results show that the method provided by the invention is relatively consistent with the cold resistance of an actual porphyra yezoensis variety or strain, the cold resistance grade of a to-be-detected sample can be accurately identified, the blank of cold resistance evaluation of the porphyra yezoensis is filled, and a technical basis is provided for subsequent screening, identification and breeding of a cold-resistant porphyra yezoensis culture variety.

Description

Technical Field

[0001] The invention belongs to the technical field of biological breeding, and particularly relates to a comprehensive evaluation method for cold resistance of Porphyra yezoensis. Background Art

[0002] Porphyra yezoensis is an important marine economic crop. During its cultivation, it will be affected by low temperature stress due to environmental and climate changes, which will trigger physiological and biochemical responses, affect the growth and development of algae, and then cause damage to yield and quality. For example, Porphyra is an important large economic seaweed in my country, and its main cultivation areas are in coastal areas such as Jiangsu and Shandong in my country. In recent years, my country's laver industry has shown a trend of "southern vegetables and northern cultivation". Affected by the cold winter temperatures in the north, and the lower the annual average temperature with increasing latitude, the monthly average sea surface temperature is as low as about 1°C, which is not conducive to the growth of laver. Breeding low-temperature resistant and excellent germplasm is an effective means to solve low temperature damage and improve the growth rate and quality of algae.

[0003] The evaluation of low temperature tolerance of terrestrial crops and vegetables and flowers (such as rice, cucumber, corn, and peanut) is relatively systematic and in-depth, and different methods are used in cold tolerance or low temperature tolerance identification and index screening. However, for Porphyra yezoensis, which has a relatively simple morphological structure and grows and reproduces in water bodies, the acquisition of phenotypic traits is more complicated and cumbersome than that of terrestrial crops. It also has its own particularities in the identification and evaluation of low temperature tolerance traits, and cannot be simply applied to terrestrial plants. At present, there is a lack of rapid and accurate low temperature tolerance evaluation methods for Porphyra yezoensis. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a comprehensive evaluation method for the cold resistance of Porphyra yezoensis, to construct a prediction model for comprehensively evaluating the cold-resistant Porphyra yezoensis varieties or strains based on sustainably measured cold resistance phenotypic indicators, and to achieve the purpose of accurate and efficient evaluation.

[0005] The present invention provides a comprehensive evaluation method for cold resistance of Porphyra yezoensis, comprising the following steps:

[0006] The phenotypic indexes of Porphyra yezoensis cultured under low temperature stress of 1.5-2.5°C and cultured under suitable temperature of 9.5-10.5°C were measured to obtain phenotypic index data; the phenotypic indexes included leaf area of ​​the thallus of Porphyra yezoensis, maximum light quantum efficiency Fv / Fm, photosynthetic pigment chlorophyll a and allophycocyanin;

[0007] According to the phenotypic index data of Porphyra yezoensis, the cold tolerance coefficient CTC was calculated according to Formula 1;

[0008]

[0009] In formula I, X L It is the phenotypic index data measured under low temperature stress of 1.5-2.5℃. NIt is the phenotypic index data measured under the optimum temperature of 9.5-10.5℃;

[0010] Substitute the cold tolerance coefficients of different phenotypic indicators of Porphyra yezoensis into the cold tolerance comprehensive evaluation formula 2 to calculate and obtain the D value of the tested Porphyra yezoensis;

[0011] D = 0.387 × CTC Area +0.122×CTC Chl a +0.242×CTC Fv / Fm +0.066×CTC APC -0.086 Formula 2

[0012] In Formula II, CTC Area represents the cold tolerance coefficient of the leaf area of ​​the Porphyra yezoensis thallus to be tested;

[0013] CTC Chl a It represents the cold tolerance coefficient of the photosynthetic pigment chlorophyll a in the thallus of Porphyra yezoensis;

[0014] CTC Fv / Fm The cold tolerance coefficient representing the maximum light quantum efficiency Fv / Fm of the thallus of Porphyra yezoensis;

[0015] CTC APC It represents the cold tolerance coefficient of allophycocyanin in the thallus of Porphyra yezoensis;

[0016] The cold tolerance of the Porphyra yezoensis is judged according to the D value of the Porphyra yezoensis: the larger the D value is, the stronger the cold tolerance of the Porphyra yezoensis is.

[0017] Preferably, the light intensity of the low temperature stress culture or the optimal temperature culture of Porphyra yezoensis is 47 to 53 μmol·photons·m -2 ·s -1 ; The light-dark cycle of the low temperature stress culture or the suitable temperature culture of Porphyra yezoensis is 12h:12h;

[0018] The culture solution for low temperature stress culture or suitable temperature culture of Porphyra yezoensis is a PES culture solution with a mass percentage of 2%;

[0019] The time for low temperature stress culture or suitable temperature culture of Porphyra yezoensis is 10 to 20 days.

[0020] Preferably, the length of the Porphyra yezoensis thallus is 2 to 3 cm.

[0021] Preferably, the method for determining the leaf area of ​​the thallus of Porphyra yezoensis comprises obtaining an RGB image of the thallus of Porphyra yezoensis, and processing and analyzing the image using Image J software to obtain the thallus area.

[0022] Preferably, the method for determining the maximum light quantum efficiency Fv / Fm comprises determining it using a chlorophyll fluorescence imager.

[0023] Preferably, the method for determining the photosynthetic pigment chlorophyll a or allophycocyanin includes obtaining a hyperspectral image of a thallus using a hyperspectral imager; extracting hyperspectral data from the hyperspectral image, preprocessing using The Unscrambler X10.4 software, and then substituting the preprocessed data into a fitting model in MATLAB R2019b software to calculate the content of the photosynthetic pigment chlorophyll a or allophycocyanin.

[0024] Preferably, the fitting model of allophycocyanin includes a support vector regression model; and the preprocessing method is completed by standardized grouping combined with standard normal vector.

[0025] Preferably, the fitting model of the photosynthetic pigment chlorophyll a includes a partial least squares regression model; the preprocessing method includes standardized grouping and combined standardization.

[0026] Preferably, the cold tolerance of Porphyra yezoensis is divided into five levels according to the D value:

[0027] When the D value is ≥0.69, level I is strong resistance type;

[0028] When 0.59≤D value<0.69, level II is tolerant;

[0029] When 0.45≤D value<0.59, Grade III is moderately tolerant;

[0030] When 0.25≤D value<0.45, level IV is moderately sensitive;

[0031] When the D value is <0.25, level V is sensitive.

[0032] The present invention provides a comprehensive evaluation method for cold tolerance of Porphyra yezoensis. The present invention obtains four phenotypic indicators related to cold tolerance through screening experiments, namely, leaf area of ​​thallus of Porphyra yezoensis, maximum light quantum efficiency Fv / Fm, photosynthetic pigment chlorophyll a and allophycocyanin. These four phenotypic indicators can not only meet the sustainability measurement under non-destructive conditions, but also the cold tolerance evaluation model constructed based on these few phenotypic indicators can achieve the purpose of accurately and efficiently comprehensively evaluating the cold tolerance of Porphyra yezoensis. The experimental results show that the method provided by the present invention is relatively consistent with the cold tolerance of actual Porphyra yezoensis varieties or strains, and can also accurately identify the cold tolerance level of the sample to be tested, filling the gap in the cold tolerance evaluation of Porphyra yezoensis, and providing a technical basis for the subsequent screening, identification and breeding of cold-tolerant Porphyra yezoensis cultivation varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The distribution histograms of various traits and the results of correlation analysis under low temperature conditions;

[0034] Figure 2 This is the result of the cold tolerance classification of Porphyra yezoensis;

[0035] Figure 3 The results of phenotypic index comparison of 60 Porphyra yezoensis lines under low temperature and moderate temperature conditions were determined. DETAILED DESCRIPTION

[0036] The present invention provides a comprehensive evaluation method for cold resistance of Porphyra yezoensis, comprising the following steps:

[0037] The phenotypic indexes of Porphyra yezoensis cultured under low temperature stress of 1.5-2.5°C and cultured under suitable temperature of 9.5-10.5°C were measured to obtain phenotypic index data; the phenotypic indexes included leaf area of ​​the thallus of Porphyra yezoensis, maximum light quantum efficiency Fv / Fm, photosynthetic pigment chlorophyll a and allophycocyanin;

[0038] According to the phenotypic index data of Porphyra yezoensis, the cold tolerance coefficient CTC was calculated according to Formula 1;

[0039]

[0040] In formula I, X L It is the phenotypic index data measured under low temperature stress of 1.5-2.5℃. N It is the phenotypic index data measured under the optimum temperature of 9.5-10.5℃;

[0041] Substitute the cold tolerance coefficients of different phenotypic indicators of Porphyra yezoensis into the cold tolerance comprehensive evaluation formula 2 to calculate and obtain the D value of the tested Porphyra yezoensis;

[0042] D = 0.387 × CTC Area +0.122×CTC Chl a +0.242×CTC Fv / Fm +0.066×CTC APC -0.086 Formula 2

[0043] In Formula II, CTC Area represents the cold tolerance coefficient of the leaf area of ​​the Porphyra yezoensis thallus to be tested;

[0044] CTC Chl a It represents the cold tolerance coefficient of the photosynthetic pigment chlorophyll a in the thallus of Porphyra yezoensis;

[0045] CTC Fv / Fm The cold tolerance coefficient representing the maximum light quantum efficiency Fv / Fm of the thallus of Porphyra yezoensis;

[0046] CTC APC It represents the cold tolerance coefficient of allophycocyanin in the thallus of Porphyra yezoensis;

[0047] The cold tolerance of the Porphyra yezoensis is determined by the D value of the Porphyra yezoensis: the larger the D value, the stronger the cold tolerance. In the present invention, the light intensity of the low temperature stress culture or the suitable temperature culture of the Porphyra yezoensis is preferably 47 to 53 μmol·photons·m -2 ·s -1 , which can be 50 μmol·photons·m -2 ·s -1 . The light-dark cycle of the low-temperature stress culture or the suitable temperature culture of the Porphyra yezoensis is 12h:12h. The culture solution for the low-temperature stress culture or the suitable temperature culture of the Porphyra yezoensis is a PES culture solution with a mass percentage of 2%; the time for the low-temperature stress culture or the suitable temperature culture of the Porphyra yezoensis is preferably 10 to 20d, can be 12 to 18h, and can also be 15h. The length of the thallus of the Porphyra yezoensis is preferably 2 to 3cm. The temperature of the low-temperature stress culture is preferably 2 to 3°C. The suitable temperature culture is 10 to 12°C.

[0048] In the present invention, the method for determining the leaf area of ​​the thallus of Porphyra yezoensis preferably includes obtaining an RGB image of the thallus of Porphyra yezoensis, and processing and analyzing the image using Image J software to obtain the thallus area. The method for determining the maximum light quantum efficiency Fv / Fm preferably includes determining it using a chlorophyll fluorescence imager. The method for determining the photosynthetic pigment chlorophyll a or allophycocyanin preferably includes obtaining a hyperspectral image of the thallus using a hyperspectral imager; extracting hyperspectral data from the hyperspectral image, applying The Unscrambler X 10.4 software for preprocessing, and then substituting the preprocessed data into the fitting model in MATLAB R2019b software to calculate the content of the photosynthetic pigment chlorophyll a or allophycocyanin. The fitting model of allophycocyanin includes a support vector regression model; the preprocessing method is completed by standardized grouping combined with standard normal vector. The fitting model of the photosynthetic pigment chlorophyll a includes a partial least squares regression model; the preprocessing method includes standardized grouping combined with standardization. The determination method of the photosynthetic pigment chlorophyll a can also be determined by conventional spectrophotometry, i.e., extraction with 80% acetone, determination of absorbance at 663, 645, 630 and 440 nm by a spectrophotometer, and calculation of the pigment content according to a formula. For details, refer to the prior art (Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. JPlant Physiol 144 (3): 307-313.).

[0049] In the present invention, it was found through screening that four indicators of the leaf area of ​​the thallus of Porphyra yezoensis, the maximum light quantum efficiency Fv / Fm, the photosynthetic pigment chlorophyll a and allophycocyanin are directly related to the cold resistance trait of Porphyra yezoensis, and these indicators can be sustainably measured in a non-destructive state, thereby ensuring the feasibility and promotion acceptability of the method of the present invention. At the same time, a cold resistance evaluation model is constructed based on the four phenotypic indicators, and the purpose of precision and efficiency is achieved by using fewer phenotypic trait indicators, thereby ensuring the evaluation efficiency and accuracy of the method of the present invention.

[0050] In the present invention, a larger D value indicates that the cold resistance of Porphyra yezoensis is stronger. Specifically, the cold resistance of Porphyra yezoensis is preferably divided into five levels according to the D value:

[0051] When the D value is ≥0.69, level I is strong resistance type;

[0052] When 0.59≤D value<0.69, level II is tolerant;

[0053] When 0.45≤D value<0.59, Grade III is moderately tolerant;

[0054] When 0.25≤D value<0.45, level IV is moderately sensitive;

[0055] When the D value is <0.25, level V is sensitive.

[0056] In the present invention, the evaluation method is consistent with the traditional cold resistance identification result of Porphyra yezoensis, which shows that the evaluation method provided by the present invention is accurate and reliable and has good promotion and application value.

[0057] In an embodiment of the present invention, 60 strains of Porphyra yezoensis were used as test samples and the above method was used to evaluate their cold resistance. The results showed that the D values ​​of the 60 strains of Porphyra yezoensis were 0.18-0.75, and the cold resistance evaluation included 5 levels, specifically including level I strong resistance type, a total of 7 strains; level II tolerance type, a total of 13 strains; level III moderate tolerance type, a total of 19 strains; level IV moderate sensitivity type, a total of 13 strains; level V sensitive type, a total of 8 strains.

[0058] The following is a detailed description of a comprehensive cold tolerance evaluation method for Porphyra yezoensis provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] A method for screening phenotypic indicators related to cold tolerance of Porphyra yezoensis

[0061] 1. Experimental Materials and Culture Methods

[0062] 1.1 Samples to be tested: 60 strains of Porphyra yezoensis.

[0063] 1.2 Culture method: Porphyra yezoensis fronds of the same growth period and 2-3 cm in length were divided into two groups: one group was cultured under 2°C low temperature stress, and the other group was cultured under 10°C suitable temperature conditions with a light intensity of 100±3μmol·photons·m -2 ·s -1 , photoperiod L:D=12h:12h, cultured in 0.1% PES medium for 15 days, with 3 replicates, and the thallus phenotypic traits were measured on the 15th day.

[0064] 2. Phenotypic trait determination

[0065] Homozygous fronds cultured at different temperatures were taken out, and RGB images of each frond were obtained using an RGB camera (Nikon, Japan). The area (area) and aspect ratio (L / W) of each frond were calculated using Image J software (NIH, USA). The calculation formula of the daily growth rate (DGR) is shown in Formula 3:

[0066] DGR = [(A t2 / A t1 ) 1 / t – 1] × 100% Formula 3

[0067] Among them, A t1 is the frond area measured last time, A t2 is the frond area of ​​the last measurement, and t is the time interval between the two measurements (unit: d).

[0068] The thallus was placed in an open chlorophyll fluorescence imaging system (OPEN FluorCam, Beijing), and six chlorophyll fluorescence parameter indicators were selected as the photosynthetic physiological traits of the thallus, including the maximum quantum efficiency (F v / F m ), photon efficiency (Ф PSII ), non-photochemical quenching (NPQ), light-adapted photochemical quenching based on the Puddle model (qP), light-adapted photochemical quenching based on the Lake model (qL) and chlorophyll fluorescence decrease ratio (Rfd).

[0069] Hyperspectral images of thallus were obtained using Specim IQ (Specim, Finland). The images were imported into ENVI 5.3 software (Exiles Visual Information Solutions, USA) to extract hyperspectral data, imported into TheUnscrambler X 10.4 software (CAMO, Norway) for preprocessing, and imported into MATLAB R2019b software (MathWorks, USA). The fitting model was substituted to calculate the contents of thallus photosynthetic pigments phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), and chlorophyll-a (Chla). The spectral preprocessing method and fitting model for PE and Chla are PLSR (partial least squares regression) models combining S-G + standardization preprocessing; the spectral preprocessing method and fitting model for PC and APC are SVR (support vector machine regression) models combining S-G + SNV preprocessing.

[0070] 3. Data Statistical Analysis

[0071] 3.1 Cold resistance coefficient

[0072] In order to eliminate the influence of genetic factors of different strains on each trait, the values ​​of each trait measured at low temperature were compared with those measured at suitable temperature to obtain the cold resistance coefficient (CTC) of each trait. The calculation formula 1 is as follows:

[0073]

[0074] In the formula, X L are the values ​​of various properties measured at low temperature, X N These are the values ​​of various traits measured at optimum temperature.

[0075] 3.2 Membership function value

[0076] The membership function value of the CTC of each trait is calculated to eliminate the difference in the dimensions of different indicators. When the trait used is a positive indicator, formula 4 is used for calculation:

[0077]

[0078] When the trait used is a reverse indicator, Formula 5 is used:

[0079]

[0080] where X′ mn is the membership function value of the CTC value of the nth indicator of the m sample, X max is the maximum value of CTC of a trait in all samples, X min It is the minimum value of CTC of a certain trait in all samples.

[0081] 3.3 Principal component weights

[0082] The indicator weights are calculated using information such as the load coefficient in the variance matrix obtained from the principal component analysis, using Formula 6 to Formula 8:

[0083]

[0084]

[0085]

[0086] Where L represents the linear combination coefficient matrix, L m is the linear combination coefficient of the mth principal component. m is the load coefficient of the kth variable in the mth principal component, which can reflect the information extraction of the principal component for each trait. m is the characteristic root corresponding to the mth principal component. m Refers to the comprehensive score coefficient, S m W refers to the variance explanation rate, and F refers to the cumulative variance explanation rate. m is the weight value of each trait. The comprehensive score coefficient is summed and normalized to obtain the weight value of each indicator.

[0087] 3.4 Comprehensive evaluation indicators

[0088] The comprehensive evaluation index D value is calculated according to the index weight, using formula 9:

[0089]

[0090] Among them, D m is the comprehensive evaluation index value of strain m. The larger the value, the stronger the strain's comprehensive tolerance to low temperature. mn is the membership function value of the CTC value of the nth indicator of the m sample, and Wm is the weight value of each trait.

[0091] 3.5 Construction of low temperature resistance evaluation model

[0092] Multiple regression analysis was performed on the cold tolerance traits to establish a mathematical model that can be used to evaluate the cold tolerance of Porphyra yezoensis thallus. Multiple linear regression analysis was performed with CTC as the independent variable and the D value, a comprehensive evaluation index of low temperature tolerance, as the dependent variable.

[0093] SPSS Statistics 25.0 software (IBM, USA) was used to perform descriptive statistical analysis, principal component analysis, correlation analysis, membership function analysis, cluster analysis, and regression analysis on the above data.

[0094] 4. Results

[0095] 4.1 Phenotypic traits of Porphyra yezoensis at low temperatures

[0096] Under low temperature stress, the numerical distribution of 13 traits of 60 Porphyra yezoensis lines showed normal distribution or skewed normal distribution, which was consistent with the characteristics of quantitative traits ( Figure 1 ). Most of the photosynthetic physiological traits have strong positive correlations, among which Φ PSⅡ The correlation coefficient with qP trait is as high as 0.93, F v / F m The correlation coefficient between NPQ and DGR was 0.74. Among the growth traits, DGR and leaf area were closely correlated, with a correlation coefficient of 0.71. There was also a very significant positive correlation between the pigment content traits, among which the correlation coefficient between PE and chlorophyll a was 0.78, and the correlation coefficient between APC and chlorophyll a was 0.53. In addition, the correlation between the growth traits and the photosynthetic physiology and pigment content traits was weak, and only the leaf area was correlated with qP or Φ PSII There was no correlation between NPQ and PE traits, or between Rfd and DGR.

[0097] 4.2 Cold tolerance coefficient of Porphyra yezoensis and its membership function

[0098] The coefficient of variation of CTC of each trait is arranged from large to small as Rfd>area>NPQ>Φ PSⅡ >DGR>PE>APC>Chla>qP>qL>Aspect Ratio>PC>F v / F m The larger the coefficient of variation, the greater the discreteness and the more unstable the cold resistance coefficient. v / F m The coefficient of variation of , qL and length-width ratio traits is lower than 30%, which is weak variation, indicating that the cold resistance coefficient is relatively stable; while the coefficient of variation of Rfd and area traits is greater than 50%, which is strong variation; the coefficient of variation of other traits ranges from 30% to 50%, which is moderate variation, and the cold resistance coefficient is relatively unstable.

[0099] The membership function values ​​of each trait CTC range from 0.24 to 0.70, among which F v / F m , qP, qL, leaf area, DGR, and PE were the first six traits exceeding 0.50 ( Table 1 ).

[0100] Table 1 CTC and membership functions of each trait

[0101]

[0102] Generally speaking, a single indicator can only reflect one aspect, but not the whole. If each trait is used to analyze the cold tolerance of Porphyra yezoensis separately, the relationship between multiple traits on cold tolerance will be ignored, which is not enough to accurately describe the situation at the overall level. Therefore, it is necessary to further conduct a comprehensive evaluation of multiple traits.

[0103] 4.3 Principal component analysis of low temperature tolerance traits of Porphyra yezoensis

[0104] Through principal component analysis of 13 traits of 60 Porphyra yezoensis strains, 13 individual traits were transformed into 4 principal components (represented by P1, P2, P3, and P4), with a cumulative contribution rate of 72.46%, among which the first principal component had the largest contribution rate of 36.04% (Table 2). v / F m , Φ PSⅡ The eigenvectors of NPQ, qL, qP, area, aspect ratio, and DGR traits were relatively large. In the second principal component, the eigenvectors of PE and PC traits were relatively large, with a contribution rate of 14.33%. In the third principal component, the eigenvectors of Rfd and Chla traits were relatively large, with a contribution rate of 11.24%. In the fourth principal component, the eigenvector of APC traits was relatively large, with a contribution rate of 10.09%.

[0105] The weights of each indicator in each principal component are obtained by using formulas VI to VIII: area, DGR, Chl a, PE, APC, Φ PSII ,qL,qP,PC,F v / F m, NPQ, Rfd and L / W. The weight reflects the contribution of each indicator to the comprehensive indicator, that is, the weight is used to calculate the D value. The indicator with a large weight value contributes more to the D value and is an important factor in selecting trait indicators. From the above results, it can be seen that the indicators with high weight rankings include area, DGR, Chl a, PE, APC, and DGR is a trait calculated based on area, and the two have a high correlation and belong to the same similar indicators. The area trait contributes the most to the low temperature resistance comprehensive index. Therefore, the area trait indicator is selected to abandon DGR for the construction of a comprehensive evaluation D value. At the same time, the four principal components (area, chla, APC and F v / F m ) were quite different from the characteristic values ​​of their corresponding traits, indicating that the measured traits play a role in the cold-tolerance phenotypic diversity of Porphyra yezoensis.

[0106] Table 2 Principal component analysis of low temperature resistance traits

[0107]

[0108]

[0109] 4.4 Evaluation model of low temperature tolerance of Porphyra yezoensis

[0110] A total of 13 equation models were fitted, and 13 variables were included in the model (Table 3). When there was only one variable, area, the regression equation determination coefficient R 2 The F test was 0.714, and the F test reached an extremely significant level, indicating that the regression equation has a certain explanatory power. As the independent variables selected into the equation increase successively, the determination coefficient also gradually increases. In order to avoid the collinearity effect and simplify the evaluation while maintaining satisfactory accuracy, 0.387×area+0.122×Chl a+0.242×Fv / Fm+0.066×APC-0.086 was selected as the cold tolerance evaluation model, and its determination coefficient was 0.955. The F test reached an extremely significant level, so it can be used to evaluate the cold tolerance of Porphyra yezoensis. It also shows that area, Chl a, and F under low temperature stress v / F m Both PCR and APC traits can be used as evaluation indicators.

[0111] Table 3 Multiple regression analysis of cold tolerance evaluation of Porphyra yezoensis

[0112]

[0113] Example 2

[0114] A comprehensive evaluation method for cold tolerance of Porphyra yezoensis

[0115] 1) Calculation of cold resistance coefficient

[0116] The phenotypic indexes of the sample to be tested under low temperature and suitable temperature conditions screened in Example 1 are measured respectively, and then substituted into Formula 1 to calculate the cold resistance coefficient (CTC, Coefficient of cold resistance);

[0117]

[0118] In the formula, X L is the phenotypic index data measured under 2℃ low temperature stress, X N It is the phenotypic index data measured under the optimum temperature of 10℃.

[0119] 2) Calculation of cold resistance comprehensive index D value

[0120] Substitute the cold resistance coefficient of the above screening index into formula 2 to obtain the cold resistance comprehensive index D value.

[0121] D = 0.387 × CTC Area +0.122×CTC Chl a +0.242×CTC Fv / Fm +0.066×CTC APC -0.086 Formula 2

[0122] Among them, R 2 =0.955.

[0123] 3) Cold resistance evaluation

[0124] The cold resistance level of Porphyra yezoensis is evaluated according to its five levels of low temperature resistance:

[0125] When D≥0.69, level I is strong resistance type;

[0126] When 0.59≤D<0.69, level II is the tolerance type;

[0127] When 0.45≤D<0.59, Grade III is moderately tolerant;

[0128] When 0.25≤D<0.45, level IV is moderately sensitive;

[0129] When D<0.25, level V is sensitive.

[0130] The results of the determination of four phenotypic indicators of 60 Porphyra yezoensis strains under low temperature and moderate temperature conditions are as follows Figure 1 As shown. The mean values ​​of leaf area and chlorophyll a at low temperature were lower than those at optimum temperature, Fv / Fm was similar at low temperature and optimum temperature, and the mean value at low temperature was slightly higher; the mean value of APC content at low temperature was higher than that at optimum temperature, and the differences were not significant ( Figure 3 ).

[0131] Table 4 CTC, comprehensive evaluation index D value and cold tolerance classification of 60 lines for four evaluation index traits

[0132]

[0133]

[0134] Comprehensive indicators for low temperature resistance evaluation and classification of cold resistance levels are shown in Figure 2 and Table 3. The D values ​​of 60 Porphyra yezoensis strains ranged from 0.18 to 0.75, with an average of 0.50 ( Figure 2 ). The cold resistance of the test materials was evaluated based on the D value. The larger the D value, the stronger the cold resistance. The cold resistance of 60 Porphyra yezoensis strains was divided into 5 levels. Level I was strong cold resistance (D≥0.69), with 7 strains, accounting for 11.7%; Level II was cold resistance (0.59≤D<0.69), with 13 strains, accounting for 21.7%; Level III was medium cold resistance (0.45≤D<0.59), with 19 strains, accounting for 31.6%; Level IV was medium sensitive (0.25≤D<0.45), with 13 strains, accounting for 21.7%; Level V was sensitive (D<0.25), with 8 strains, accounting for 13.3%. The cold resistance decreased from level I to level V.

[0135] Example 4

[0136] 1) Acquisition and preliminary screening of cold-tolerant mutants

[0137] The monospores released from the pure line of Porphyra yezoensis RZ in the laboratory algae germplasm bank were mutagenized with ethyl methyl carbonate (EMS) to obtain the mutant population of Porphyra yezoensis, and 57 thallus samples were obtained. The thallus length was about 2-3 cm, and they were divided into multiple parallel groups for low-temperature (2°C) water circulation culture. The growth and photosynthetic physiology, pigment content and other indicators related to cold tolerance were measured. Finally, two EMS-induced mutants with cold tolerance, fast growth rate and strong photosynthetic ability were obtained through preliminary screening. The strains were named D37 and D52, and their progeny populations were obtained through asexual reproduction. At the same time, the laboratory pure line RZ strain was included in the treatment group to comprehensively evaluate the cold tolerance characteristics.

[0138] 2) Comprehensive evaluation of cold tolerance of mutant progeny

[0139] The EMS-induced mutant strains obtained in the initial screening were reproduced asexually and sexually to obtain offspring, expanded and cultured, and reproduced for multiple generations to obtain sample groups of different offspring of each strain. The sample groups were cultured at 2℃ and 10℃ for 15 days, and the leaf area, maximum photochemical quantum yield Fv / Fm, chlorophyll a content (Chla, mg / g), and allophycocyanin content (APC, mg / g) of the samples were measured. The cold resistance coefficient (CTC) was calculated step by step according to the comprehensive evaluation method of cold resistance of Porphyra yezoensis.

[0140]

[0141] In the formula, X L is the phenotypic index data measured under 2℃ low temperature stress, X N It is the phenotypic index data measured under the suitable temperature condition of 10℃. Substitute the cold resistance coefficient of the above screening index into formula II to obtain the cold resistance comprehensive index D value.

[0142] D = 0.387 × CTC Area +0.122×CTC Chl a +0.242×CTC Fv / Fm +0.066×CTC APC -0.086 Formula 9

[0143] Among them, R 2 =0.955.

[0144] The cold resistance level is evaluated based on the calculated cold resistance comprehensive index D value:

[0145] When D≥0.69, level I is strong resistance type;

[0146] When 0.59≤D<0.69, level II is the tolerance type;

[0147] When 0.45≤D<0.59, Grade III is moderately tolerant;

[0148] When 0.25≤D<0.45, level IV is moderately sensitive;

[0149] When D<0.25, level V is sensitive.

[0150] A comprehensive cold resistance evaluation was conducted on the EMS-induced mutant strains and their progeny, the cold resistance of the cold-tolerant mutants and their progeny was analyzed, and the stability of the heritability of the cold-tolerant trait was verified.

[0151] Results:

[0152] The first generation D37, the second generation D37, the first generation D52, the second generation D52, the third generation D52 obtained by the asexual reproduction method and the sexual offspring of D52 obtained by the sexual reproduction method were cultured and the CTCs of the four evaluation index traits were determined to obtain the cold tolerance grading results.

[0153] Table 5 CTC of four evaluation index traits, comprehensive evaluation index D value and cold tolerance classification of D37, D52 mutant progeny and laboratory RZ strain

[0154]

[0155] The comprehensive cold tolerance evaluation index and cold tolerance level are shown in Table 5. The laboratory RZ strain belongs to the Class III moderate tolerance type, the D37 strain monospore generation 1 shows the Class I strong tolerance type, but the monospore generation 2 shows the Class IV moderate sensitivity type. Its cold tolerance trait is unstable and cannot be used as a cold tolerance mutant for further research. The asexual offspring of the D52 strain, D52 generation 1, D52 generation 2, D52 generation 3, and the D52 sexual offspring obtained by sexual reproduction are all Class I strong tolerance types. It can be determined that the EMS-induced Porphyra yezoensis D52 strain is a strong cold tolerance mutant, which can be used to explore key genes for low temperature tolerance and verify their functions.

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A comprehensive evaluation method for cold tolerance of Porphyra yezoensis, characterized in that: The following steps are involved: The phenotypic indexes of Porphyra yezoensis cultured under low temperature stress of 1.5-2.5°C and cultured under suitable temperature of 9.5-10.5°C were measured to obtain phenotypic index data; the phenotypic indexes included leaf area of ​​the thallus of Porphyra yezoensis, maximum light quantum efficiency Fv / Fm, photosynthetic pigment chlorophyll a and allophycocyanin; According to the phenotypic index data of Porphyra yezoensis, the cold tolerance coefficient CTC was calculated according to Formula 1; In formula I, X L It is the phenotypic index data measured under low temperature stress of 1.5-2.5℃. N It is the phenotypic index data measured under the optimum temperature of 9.5-10.5℃; Substitute the cold tolerance coefficients of different phenotypic indicators of Porphyra yezoensis into the cold tolerance comprehensive evaluation formula 2 to calculate and obtain the D value of the tested Porphyra yezoensis; D = 0.387 × CTC Area +0.122×CTC Chla +0.242×CTC Fv / Fm +0.066×CTC APC -0.086 Formula 2 In Formula II, CTC Area represents the cold tolerance coefficient of the leaf area of ​​the Porphyra yezoensis thallus to be tested; CTC Chla It represents the cold tolerance coefficient of the photosynthetic pigment chlorophyll a in the thallus of Porphyra yezoensis; CTC Fv / Fm The cold tolerance coefficient representing the maximum light quantum efficiency Fv / Fm of the thallus of Porphyra yezoensis; CTC APC It represents the cold tolerance coefficient of allophycocyanin in the thallus of Porphyra yezoensis; The cold tolerance is judged according to the D value of the Porphyra yezoensis: the larger the D value is, the stronger the cold tolerance of the Porphyra yezoensis is.

2. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 1, characterized in that: The light intensity of the low temperature stress culture or the suitable temperature culture of Porphyra yezoensis is 47 to 53 μmol·photons·m -2 ·s -1 The light-dark cycle of the low temperature stress culture or the suitable temperature culture of the Porphyra yezoensis is 12h:12h.

3. The comprehensive evaluation method for cold resistance of Porphyra yezoensis according to claim 1, characterized in that: The culture solution for low temperature stress culture or suitable temperature culture of Porphyra yezoensis is a PES culture solution with a mass percentage of 2%; The time for low temperature stress culture or suitable temperature culture of Porphyra yezoensis is 10 to 20 days.

4. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 1, characterized in that: The length of the thallus of Porphyra yezoensis is 2 to 3 cm.

5. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 1, characterized in that: The method for measuring the leaf area of ​​the thallus of Porphyra yezoensis comprises obtaining an RGB image of the thallus of Porphyra yezoensis, and processing and analyzing the image using Image J software to obtain the thallus area.

6. The comprehensive evaluation method for cold tolerance of Porphyra yezoensis according to claim 1, characterized in that: The method for determining the maximum light quantum efficiency Fv / Fm includes determining it using a chlorophyll fluorescence imager.

7. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 1, characterized in that: The method for determining the photosynthetic pigment chlorophyll a or allophycocyanin comprises obtaining a hyperspectral image of a thallus using a hyperspectral imager; extracting hyperspectral data from the hyperspectral image, preprocessing using The Unscrambler X 10.4 software, and then substituting the preprocessed data into a fitting model in MATLAB R2019b software to calculate the content of the photosynthetic pigment chlorophyll a or allophycocyanin.

8. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 7, characterized in that: The fitting model of the allophycocyanin includes a support vector regression model; the preprocessing method is completed by standardized grouping combined with standard normal vector.

9. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to claim 7, characterized in that: The fitting model of the photosynthetic pigment chlorophyll a includes a partial least squares regression model; the preprocessing method includes standardized grouping and combined standardization.

10. The method for comprehensive evaluation of cold tolerance of Porphyra yezoensis according to any one of claims 1 to 9, characterized in that: The cold tolerance of Porphyra yezoensis is divided into five levels according to the D value: When the D value is ≥0.69, level I is strong resistance type; When 0.59≤D value<0.69, level II is tolerant; When 0.45≤D value<0.59, Grade III is moderately tolerant; When 0.25≤D value<0.45, level IV is moderately sensitive; When the D value is <0.25, level V is sensitive.

Images