CN113748787A - Screening method of salt-tolerant or salt-sensitive rice seeds in germination period - Google Patents
Screening method of salt-tolerant or salt-sensitive rice seeds in germination period Download PDFInfo
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Abstract
The invention discloses a screening method of salt-tolerant or salt-sensitive rice seeds in a germination period, which comprises the following steps: measuring each growth index of the rice seeds in the germination period, and calculating a corrected growth index value; calculating to obtain a membership function value by adopting a membership function; classifying according to rice types, and performing correlation analysis to obtain correlation growth indexes; then, carrying out principal component analysis to obtain a weight coefficient; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value; finally, screening out the rice seeds with strong salt tolerance or salt sensitivity in the germination period. According to the method, the rice seeds are classified firstly, and then the salt tolerance sequence of the rice seeds is obtained by using the membership function, the correlation analysis method and the principal component analysis method, so that the rice seeds with good salt tolerance in the germination period are obtained by screening, and a theoretical basis and an application basis are provided for subsequent breeding, cultivation and production.
Description
Technical Field
The invention relates to the technical field of plant seed vitality and treatment, in particular to a screening method of salt-tolerant or salt-sensitive rice seeds in a germination period.
Background
Rice is an important grain crop, is a moderate salt sensitive cereal crop, and the soil affected by salt accounts for about 20% of the total cultivated land area in the world. The Chinese saline-alkali soil is complex in type and wide in distribution, and the total area accounts for about 25% of the total cultivated area. In recent years, under the influence of the problems of extreme weather and imperfect farming and irrigation modes and the like, the phenomena of land salinization and secondary salinization become severe day by day, and the national food safety is directly threatened. In order to fully develop and utilize saline-alkali soil and increase crop cultivated land area so as to improve grain yield, from the 50 th century, scholars at home and abroad begin to carry out research on the identification of the salt tolerance of rice germplasm resources and improve the salt tolerance of rice.
The soil salt damage is one of the environmental factors which have serious influence on the growth and the yield of crops, can inhibit the growth and the development of rice, reduce the plant height, the tillering number, the single-plant green leaf number and the biomass, influence the photosynthesis, and further cause the yield to be reduced. In a word, the salt tolerance of the plant is comprehensive quantitative character controlled by multi-factor combination, and the strength of the salt tolerance of the plant is difficult to be reflected comprehensively and accurately only according to a single index; and the types of rice varieties are various, and the evaluation of the salt tolerance of the rice needs to select a plurality of key indexes for comprehensive evaluation aiming at different types.
The membership function analysis method is a method capable of carrying out systematic analysis on a plurality of index data, has scientific and reliable evaluation results, and is widely applied to screening and identifying stress resistance of crops at present; however, the identification of the salt resistance of the seeds in the rice germination period is not complete, and the application of the membership function analysis method to the identification of the salt resistance of the seeds in the rice germination period is not developed.
Disclosure of Invention
The invention provides a screening method of salt-tolerant or salt-sensitive rice seeds in a germination period, which can effectively and quickly screen rice seeds with better salt tolerance from rice varieties to be screened, and provides theoretical basis and application basis for subsequent breeding, cultivation and production.
The specific technical scheme is as follows:
a screening method of salt-tolerant or salt-sensitive rice seeds in a germination period comprises the following steps:
(1) taking a plurality of rice varieties to be screened, respectively placing seeds of the rice varieties on germination paper which is wet by clear water and is wet by saline water with different salt concentrations, carrying out germination tests, measuring seedling growth indexes of the seeds for germination and germination for 14 days, calculating the ratio of each growth index value of each rice variety under different salt concentrations to each growth index value under zero salt concentration, and marking as a corrected growth index value;
(2) performing membership function analysis on the corrected growth index values of the rice varieties to obtain membership function values;
(3) classifying the rice varieties according to the types of the rice to obtain sample sets of different types; performing correlation analysis on each growth index in the same sample set according to the corrected growth index value in the step (1), and removing the growth index with small correlation to obtain the correlation growth index in each sample set;
(4) performing principal component analysis on each correlation growth index in each sample set according to the correlation growth index in the step (3) to obtain a normalization weight coefficient of each correlation growth index in each sample set;
(5) multiplying the weight coefficient in the step (4) by the membership function value of each correlation growth index of each corresponding rice variety to obtain a correction membership function value of each correlation growth index of each rice variety; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value;
(6) and performing salt tolerance sorting on each rice seed according to the sorting reference value, selecting the rice seeds which are sorted in front or back according to the result of the salt tolerance sorting, and screening the rice seeds with strong salt tolerance or salt sensitivity in the germination period.
Further, in the step (1), the number of the rice varieties to be screened is more than or equal to 15.
Further, in the step (1), the different salt concentrations are at least two or more salt concentrations, namely a low salt concentration and a high salt concentration.
Furthermore, the low salt concentration is 50-160 mM, and the high salt concentration is 165-300 mM.
Preferably, the low salt concentration is 150mM and the high salt concentration is 200 mM.
Further, in step (1), the growth indicator includes: germination vigor, germination rate, seedling rate, germination index, vigor index, average germination time, fresh weight and dry weight.
Further, in step (2), the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
Further, the classification in step (3) is as follows: (A) classifying indica rice and japonica rice into the following categories: indica, japonica and indica-japonica rice; or, (B) classified by breeding method into: hybrid rice and conventional rice; or (C) classifying the rice into glutinous rice and non-glutinous rice according to the quality of the rice.
Further, in step (4), the SPSS software is used for principal component analysis and the normalization weight coefficient is calculated.
Further, in the step (3), the growth index with small correlation is as follows: the ratio of the number of other growth indexes having correlation with the growth index to the total number of the growth indexes is less than or equal to 1/3.
Further, in the step (6), the rice seeds with salt tolerance or salt sensitivity in the germination period screened by adopting different classification modes are counted, and the rice variety with the highest occurrence frequency is selected to obtain the rice variety with salt tolerance or salt sensitivity in the germination period.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the rice seeds are classified firstly, and then the salt tolerance sequence of the rice seeds is obtained by utilizing a correlation analysis method and a principal component analysis method and combining membership functions, so that the rice seeds with good salt tolerance in a germination period are obtained by screening, and a theoretical basis and an application basis are provided for subsequent breeding, cultivation and production.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
Reagents required for the following examples: sodium chloride for salt treatment was purchased from aladin company.
18 rice varieties: qian you No. 1, Yuan you Huazhan, Nei 5 you 8015, Jing you 534, Ji you 06, Y you 689, Meng you Huazhan, Zhe you 274, Chun you 84, Chun you 927, Jiafengyou No. 2, Jiayou No. 5, Yongyou No. 10, Chun Jiannuo No. 6, Xiushui 121, Jia 58, Jiazaofeng 18, and Yongnong 15. According to different classification modes of rice, the rice can be classified into indica rice, indica rice and japonica rice, hybrid rice and conventional rice, glutinous rice and non-glutinous rice (see table 1 in particular).
All experimental procedures used in the following examples are conventional unless otherwise specified.
TABLE 1 classification of rice varieties used in the examples
Example 1 growth index of Rice varieties germinating at different salt concentrations
(1) The rice seeds were placed on three layers of 0, 150 or 200mM saline-wetted germination paper for germination testing, which was repeated 4 times for 100 seeds. The seeds were placed in an incubator at 30 ℃ (16 hours dark)/20 ℃ (8 hours light) at an intensity of 250. mu. mol. -%m-2·s-1. And taking the seed radicle breaking through the seed coat as the germination standard, and recording the germination number every day. And counting the germination vigor on the 5 th day of germination, and counting the germination rate and the seedling rate on the 14 th day. When the germination rate is counted, 40 seedlings are randomly selected to measure the fresh weight, then the sample is placed in an oven at 80 ℃ for 24 hours, and is taken out and placed in a dryer for cooling, and then the dry weight of the sample is measured. And finally calculating the germination index, the vitality index and the average germination time.
Wherein, the germination potential (GE,%) is multiplied by 100 percent (the germination number of seeds/the number of tested seeds in 5 days);
germination rate (GP,%) (number of seeds germinated/number of seeds examined within 14 days) × 100%;
normal seedling rate (SP,%) (number of robust seedlings/number of seeds tested on day 14) × 100%;
germination Index (GI) ═ sigma (Gt/Dt), wherein Gt is the number of germination seeds per day and Dt is the number of corresponding germination days;
the Vitality Index (VI) is GI multiplied by DW, wherein GI is the germination index, and DW is the dry weight;
the average germination time (MGT, day) ═ Σ (Gt × Tt)/∑ Gt, where Gt is the number of germinating seeds on a daily basis and Dt is the number of corresponding germinating days.
TABLE 2 Rice seed germination and seedling growth indices under salt stress of different concentrations
As can be seen from Table 2, the growth indexes such as germination percentage and the like of 10 rice varieties at 0mM salt concentration are different in level, and the salt tolerance of the 10 rice varieties is different and can not be judged directly according to the difference of the growth indexes at 150mM salt concentration and 200mM salt concentration to draw a conclusion.
(2) And (3) calculating the ratio of each growth index value of the rice seeds under different salt concentrations to each growth index value under zero salt concentration according to the growth index values in the step (1) to obtain a corrected growth index value.
TABLE 3 Rice seed germination and seedling correction indices under salt stress of different concentrations
GE: relative germination vigour; GP: relative germination rate; SP: relative seedling rate; GI: relative germination index; VI: a relative viability index; MGT: relative average germination time; FW: relatively fresh weight; DW: relative dry weight.
(3) Calculating a membership function value of each growth index of each rice seed in the sample set by adopting a membership function according to the corrected growth index value in the step (2);
the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
And obtaining a sorting reference value according to the membership function value, sorting the salt tolerance of each rice seed according to the sorting reference value, and selecting the rice seed with the top sorting according to the result of the salt tolerance sorting, namely the rice seed with good salt tolerance in the germination period. According to the comprehensive ranking result of index membership function values under 150mM salt concentration, the ranks of Jingliangyou 534, Yongxian 15 and Chuanyou 927 are 1 st, 2 nd and 3 rd, the ranks of Zhejiang liangyou 274, Xiushui 121 and Chunjiannuo 6 are 16 th, 17 th and 18 th, and the comprehensive ranking result under 200mM salt concentration shows that the ranks of Yongxian 15, Jingliangyou 534 and Y Shuangyou 689 varieties are 1 st, 2 nd and 3 th, and the ranks of Zhejiang liangyou 274, Chunjiannuo 6 and Xishui 121 are 16 th, 17 th and 18 th. The varieties of Yongliang 15, Jingliangyou 534, Chuanyou 927 and Y liangyou 689 can be basically determined as salt-resistant varieties, and Zhejiang Youyou 274, Chunjiang 6 and Xiushui 121 as salt-sensitive varieties. And under different salt concentrations, the salt tolerance of the rice is slightly different.
TABLE 4 Rice seed germination and seedling growth indices under salt stress of different concentrations
RGE: relative germination vigour; RGP: relative germination rate; RSP: relative seedling rate; RGI: relative germination index; RVI: a relative viability index; RMGT: relative average germination time; RFW: relatively fresh weight; RDW: relative dry weight.
Example 2 salt tolerance screening of indica and japonica rice varieties
The specific screening method is as follows:
(1) taking 18 rice seeds to be screened (namely Qian you No. 1, Jing you Huazhan, Nei 5 you 8015, Jing you 534, Zhangyou 06, Y you 689, Meng you Huazhan, Zhe you 274, Jia Zaofeng 18, Yonglong rice 15, Chun you 84, Chun you 927, Jiafengyou No. 2, Jiayou No. 5, Yongyou No. 10, Chun Jiangguo No. 6, Xiushui 121 and Jia 58), respectively placing the rice seeds on three layers of germinating paper wetted by 0 (clear water) and 150 or 200mM saline for germination test, and repeating for 4 times, wherein each time is 100 seeds. The seeds were placed in an incubator at 30 ℃ (16 hours dark)/20 ℃ (8 hours light) at an intensity of 250 μmol · m-2·s-1. Calculating the difference value between each growth index value of the rice seeds under different salt concentrations and each growth index value under zero salt concentration to obtain a corrected growth index value;
and taking the seed radicle breaking through the seed coat as the germination standard, and recording the germination number every day. And counting the germination vigor on the 5 th day of germination, and counting the germination rate and the seedling rate on the 14 th day. When the germination rate is counted, 40 seedlings are randomly selected to measure the fresh weight, then the sample is placed in an oven at 80 ℃ for 24 hours, and is taken out and placed in a dryer for cooling, and then the dry weight of the sample is measured. And finally calculating the germination index, the vitality index and the average germination time.
Wherein, the germination potential (GE,%) is multiplied by 100 percent (the germination number of seeds/the number of tested seeds in 5 days);
germination rate (GP,%) (number of seeds germinated/number of seeds examined within 14 days) × 100%;
normal seedling rate (SP,%) (number of robust seedlings/number of seeds tested on day 14) × 100%;
germination Index (GI) ═ sigma (Gt/Dt), wherein Gt is the number of germination seeds per day and Dt is the number of corresponding germination days;
the Vitality Index (VI) is GI multiplied by DW, wherein GI is the germination index, and DW is the dry weight;
the average germination time (MGT, day) ═ Σ (Gt × Tt)/∑ Gt, where Gt is the number of germinating seeds on a daily basis and Dt is the number of corresponding germinating days.
Calculating to obtain a membership function value of each growth index of each rice seed in the sample set by adopting a membership function according to the corrected growth index value;
the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
The results are shown in example 1.
(2) 18 rice varieties were classified according to indica, indica and japonica, wherein indica: qian you No. 1, Ling you Huazhan, Nei 5 you 8015, Jing you 534, Zhao you 06, Y you 689, Meng you Huazhan, Zhe you 274, Jia Zao Feng 18, Yonglong rice 15; indica rice: 84 percent of spring you, 927 percent of spring you and 2 percent of Jiafengyou; japonica rice: jiayou No. 5, Yongyou No. 10, Chunjiannuo No. 6, Xiushui 121 and Jia 58; obtaining sample sets of different categories (indica rice, indica rice and japonica rice);
(3) performing correlation analysis according to the corrected growth index value in the step (1), and eliminating growth indexes with small correlation to obtain correlation growth indexes in each sample set; performing principal component analysis on each correlation growth index in each sample set according to the corrected growth index value in the step (1) to obtain a weight coefficient of each correlation growth index in each sample set; and (4) carrying out principal component analysis by adopting SPSS software, and calculating a normalization weight coefficient.
The results of the correlation analysis are shown in table 5.
TABLE 5 correlation coefficient between salt tolerance indexes
And indicates significance at P <0.05 and P <0.01 levels, respectively. RGE: relative germination vigour; RGP: relative germination rate; RSP: relative seedling rate; RGI: relative germination index; RVI: a relative viability index; RMGT: relative average germination time; RFW: relatively fresh weight; RDW: relative dry weight.
The results in table 5 show that under the salt concentration of 150mM, GI and MGT are not highly correlated with other indexes, and these 2 data are rejected; under the salt concentration of 200mM, the correlation between MGT and other indexes is not high, and the 1 data is rejected. The indica rice has correlation between each index under the salt concentration of 150mM and 200mM, and all indexes are reserved. Under the condition of 150mM salt concentration, all indexes of the japonica rice have correlation and are completely reserved; under the salt concentration of 200mM, the correlation between MGT and other indexes is not high, and the 1 data is rejected.
The weight of each index having a correlation is determined using principal component analysis. And (4) carrying out principal component analysis by adopting SPSS software, and calculating a normalization weight coefficient. The main component analysis result shows that under the salt concentration of 150mM and 200mM, the index only has 1 main component, and the cumulative contribution rate respectively reaches 77.713% and 81.168%. Under the salt concentration of 150mM and 200mM, the indexes of indica-japonica rice have 2 main components, and the cumulative contribution rate reaches 100.000%. Under the salt concentration of 150mM and 200mM, the indexes of japonica rice have 2 main components, and the cumulative contribution rates reach 90.643% and 91.246%.
Each index weight coefficient is the index score coefficient/the sum of each index score coefficient, wherein the index score coefficient is the square root of the score/the sum of each index score formed by the orthogonal rotation method with kaiser normalization. Indica rice has a higher weight of VI at 150 and 200mM salt concentration, 0.180 and 0.219 respectively. The weight of GP is higher at 150mM and 200mM salt concentration, and is 0.202 and 0.163 respectively. Japonica rice was weighted higher for FW at 150 and 200mM salt concentrations, 0.195 and 0.412 respectively (table 6).
TABLE 6 weight and contribution ratio of Each index of salt stress
(4) Multiplying the weight coefficient in the step (3) by the corresponding membership function value of each correlative growth index of each rice seed to obtain a correction membership function value of each correlative growth index of each rice seed; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value;
and combining the result of the membership function with the weight of the corresponding index to obtain a new membership function value sorting table (see table 7).
Weight sorting of salt tolerance coefficients of table 718 rice varieties
RGE: relative germination vigour; RGP: relative germination rate; RGI: relative germination index; RVI: a relative viability index; RDW: relative dry weight.
(5) And performing salt tolerance sorting on each rice seed according to the sorting reference value, and selecting the rice seed with the top sorting according to the result of the salt tolerance sorting, namely the rice seed with good salt tolerance in the germination period.
According to the comprehensive ranking result of index membership function values under 150mM salt concentration, the ranking of the Chongxian 15 and Jingliangyou 534 is No. 1 and No. 2, the ranking of the Zhejiang Shuangyou 274 and the Chunjiannuo 6 is No. 17 and No. 18, while the comprehensive ranking result under 200mM salt concentration shows that the ranking of the Chongxian 15 and the Jingliangyou 534 is No. 1 and No. 2, the ranking of the Zhejiang Shuangyou 274 and the Xiushui 121 is No. 17 and No. 18, and the ranking of the Chunjiano 6 is No. 14. It can be basically determined that the Yongliang 15 and Jingliangyou 534 are salt-resistant varieties, and the Zhejiang Youyou 274, Chunjiannuo No. 6 and Xiushui 121 are salt-sensitive varieties. And under different salt concentrations, the salt tolerance of the rice is slightly different.
Example 3 salt tolerance screening of hybrid and conventional Rice varieties
The specific screening method is as follows:
(1) taking 18 rice seeds to be screened (namely Qian you No. 1, Jing you Huazhan, Nei 5 you 8015, Jing you 534, Zhangyou 06, Y you 689, Meng you Huazhan, Zhe you 274, Jia Zaofeng 18, Yonglong rice 15, Chun you 84, Chun you 927, Jiafengyou No. 2, Jiayou No. 5, Yongyou No. 10, Chun Jiangguo No. 6, Xiushui 121 and Jia 58), respectively placing the rice seeds on three layers of germinating paper wetted by 0 (clear water) and 150 or 200mM saline for germination test, and repeating for 4 times, wherein each time is 100 seeds. The seeds were placed in an incubator at 30 ℃ (16 hours dark)/20 ℃ (8 hours light) at an intensity of 250 μmol · m-2·s-1. Calculating the difference value between each growth index value of the rice seeds under different salt concentrations and each growth index value under zero salt concentration to obtain a corrected growth index value;
and taking the seed radicle breaking through the seed coat as the germination standard, and recording the germination number every day. And counting the germination vigor on the 5 th day of germination, and counting the germination rate and the seedling rate on the 14 th day. When the germination rate is counted, 40 seedlings are randomly selected to measure the fresh weight, then the sample is placed in an oven at 80 ℃ for 24 hours, and is taken out and placed in a dryer for cooling, and then the dry weight of the sample is measured. And finally calculating the germination index, the vitality index and the average germination time.
Wherein, the germination potential (GE,%) is multiplied by 100 percent (the germination number of seeds/the number of tested seeds in 5 days);
germination rate (GP,%) (number of seeds germinated/number of seeds examined within 14 days) × 100%;
normal seedling rate (SP,%) (number of robust seedlings/number of seeds tested on day 14) × 100%;
germination Index (GI) ═ sigma (Gt/Dt), wherein Gt is the number of germination seeds per day and Dt is the number of corresponding germination days;
the Vitality Index (VI) is GI multiplied by DW, wherein GI is the germination index, and DW is the dry weight;
the average germination time (MGT, day) ═ Σ (Gt × Tt)/∑ Gt, where Gt is the number of germinating seeds on a daily basis and Dt is the number of corresponding germinating days.
Calculating a membership function value of each growth index of each rice seed in the sample set by adopting a membership function according to the corrected growth index value;
the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
The results are shown in example 1.
(2) 18 rice varieties were classified according to hybrid rice and conventional rice, in which hybrid rice: qian you No. 1, Lin you Huazhan, Nei 5 you 8015, Jing you 534, Zhangliangyou 06, Y you 689, Meng you Huazhan, Zhe you 274, Chun you 84, Chun you 927, Jiafengyou No. 2, Jiayou No. 5 and Yongyou No. 10; conventional rice: jiazaofeng 18, Yongzi 15, Chunjiannuo No. 6, Xiushui 121 and Jia 58. Obtaining different types of sample sets (hybrid rice and conventional rice);
(3) performing correlation analysis on each growth index in each sample set according to the corrected growth index value in the step (1), and removing growth indexes with small correlation to obtain correlation growth indexes in each sample set; and (2) performing principal component analysis on each correlation growth index in each sample set according to the corrected growth index value in the step (1) to obtain a weight coefficient of each correlation growth index in each sample set, performing principal component analysis by adopting SPSS software, and calculating a normalized weight coefficient. (ii) a
The results of the correlation analysis are shown in table 8.
The results show that there is a correlation between the respective indices at 150mM salt concentration and at 200mM salt concentration for both hybrid and conventional rice, all remaining. Wherein the correlation among various indexes of the conventional rice is obviously higher than that of the hybrid rice.
TABLE 8 correlation coefficient between salt tolerance indexes
And indicates significance at P <0.05 and P <0.01 levels, respectively. RGE: relative germination vigour; RGP: relative germination rate; RSP: relative seedling rate; RGI: relative germination index; RVI: a relative viability index; RMGT: relative average germination time; RFW: relatively fresh weight; RDW: relative dry weight.
The weight of each index having a correlation is determined using principal component analysis. And (4) carrying out principal component analysis by adopting SPSS software, and calculating a normalization weight coefficient. The analysis result of the main components shows that the indexes of the hybrid rice have 3 and 2 main components respectively under the salt concentrations of 150 and 200mM, and the cumulative contribution rates reach 91.161 percent and 81.391 percent respectively. Under the salt concentration of 150mM and 200mM of the conventional rice, the index has 1 main component, and the cumulative contribution rate reaches 81.470% and 86.420% respectively.
Each index weight coefficient is the index score coefficient/the sum of each index score coefficient, wherein the index score coefficient is the square root of the score/the sum of each index score formed by the orthogonal rotation method with kaiser normalization. The weights of GP and FW were higher at 150 and 200mM salt concentration, 0.180 and 0.173, respectively, for hybrid rice. Conventional rice has higher weight of VI at 150 and 200mM salt concentration, 0.168 and 0.170 respectively (Table 9).
TABLE 9 weight and contribution ratio of Each index of salt stress
(4) Multiplying the weight coefficient in the step (3) by the corresponding membership function value of each correlative growth index of each rice seed to obtain a correction membership function value of each correlative growth index of each rice seed; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value;
and combining the result of the membership function with the weight of the corresponding index to obtain a new membership function value sorting table (see table 10).
Table 1018 rice variety salt tolerance coefficient weight sorting
RGE: relative germination vigour; RGP: relative germination rate; RGI: relative germination index; RVI: a relative viability index; RDW: relative dry weight.
(5) And performing salt tolerance sorting on each rice seed according to the sorting reference value, and selecting the rice seed with the top sorting according to the result of the salt tolerance sorting, namely the rice seed with good salt tolerance in the germination period.
According to the comprehensive ranking results of growth index membership function values under 150mM salt concentration, the ranks of Jingliangyou 534 and Jiayou 5 are 1 st and 2 nd, the ranks of Xiushui 121 and Chunfuyou 6 are 17 th and 18 th, and the ranks of Jingliangyou 534 and Jiafengyou 2 are 1 st and 2 nd, and the ranks of Chunfuyou 6 and Jiashui 121 are 17 th and 18 th. Basically, the Jingliangyou 534, Jiayou No. 5 and Jiafengyou No. 2 are determined as salt-resistant varieties, and Xiushui 121 and Chunjiannuo No. 6 are determined as salt-sensitive varieties. Under different salt concentrations, the salt-tolerant and salt-sensitive varieties of rice are relatively consistent.
Example 4 salt tolerance screening of waxy and non-waxy rice varieties
(1) Taking 18 rice seeds to be screened (namely, Qian you No. 1, Jing you Huazhan, Nei 5 you 8015, Jing you 534, Shuang you 06, Y two)You 689, Mengliangyouzhan, Zhejiang 274, Jiazaofeng 18, Yonglong 15, Chunyou 84, Chunyou 927, Jiafengyou No. 2, Jiayou No. 5, Yongyou No. 10, Chunyuan glutinous No. 6, Xiushui 121 and Jia 58), placing the rice seeds on three layers of germination paper wetted by 0 (clear water) and 150 or 200mM saline respectively to carry out germination test, repeating for 4 times, and repeating for 100 seeds each time. The seeds were placed in an incubator at 30 ℃ (16 hours dark)/20 ℃ (8 hours light) at an intensity of 250 μmol · m-2·s-1. Calculating the difference value between each growth index value of the rice seeds under different salt concentrations and each growth index value under zero salt concentration to obtain a corrected growth index value;
and taking the seed radicle breaking through the seed coat as the germination standard, and recording the germination number every day. And counting the germination vigor on the 5 th day of germination, and counting the germination rate and the seedling rate on the 14 th day. When the germination rate is counted, 40 seedlings are randomly selected to measure the fresh weight, then the sample is placed in an oven at 80 ℃ for 24 hours, and is taken out and placed in a dryer for cooling, and then the dry weight of the sample is measured. And finally calculating the germination index, the vitality index and the average germination time.
Wherein, the germination potential (GE,%) is multiplied by 100 percent (the germination number of seeds/the number of tested seeds in 5 days);
germination rate (GP,%) (number of seeds germinated/number of seeds examined within 14 days) × 100%;
normal seedling rate (SP,%) (number of robust seedlings/number of seeds tested on day 14) × 100%;
germination Index (GI) ═ sigma (Gt/Dt), wherein Gt is the number of germination seeds per day and Dt is the number of corresponding germination days;
the Vitality Index (VI) is GI multiplied by DW, wherein GI is the germination index, and DW is the dry weight;
the average germination time (MGT, day) ═ Σ (Gt × Tt)/∑ Gt, where Gt is the number of germinating seeds on a daily basis and Dt is the number of corresponding germinating days.
Calculating a membership function value of each growth index of each rice seed in the sample set by adopting a membership function according to the corrected growth index value;
the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
The results are shown in example 1.
(2) The 18 rice varieties were classified according to non-glutinous and glutinous, where non-glutinous: qian you No. 1, Yuan you Huazhan, Nei 5 you 8015, Jing you 534, Ji you 06, Y you 689, Meng you Huazhan, Zhe you 274, Chun you 84, Chun you 927, Jiafeng you No. 2, Jiayou No. 5, Jiazao Feng 18, Yonglong rice 15, Xiushui 121, Jia 58; glutinous rice: chunjiannuo No. 6 and Yongyou No. 10. Obtaining different classes of sample sets (non-waxy rice and waxy rice);
(3) performing correlation analysis on each growth index in each sample set according to the corrected growth index value in the step (1), and removing growth indexes with small correlation to obtain correlation growth indexes in each sample set; performing principal component analysis on each correlation growth index in each sample set according to the corrected growth index value in the step (1) to obtain a weight coefficient of each correlation growth index in each sample set; and (4) carrying out principal component analysis by adopting SPSS software, and calculating a normalization weight coefficient.
The results of the correlation analysis are shown in table 11.
TABLE 11 correlation coefficient between salt tolerance indexes
And indicates significance at P <0.05 and P <0.01 levels, respectively. RGE: relative germination vigour; RGP: relative germination rate; RSP: relative seedling rate; RGI: relative germination index; RVI: a relative viability index; RMGT: relative average germination time; RFW: relatively fresh weight; RDW: relative dry weight.
The results show that the correlation between each index of the non-glutinous rice under the salt concentration of 150mM is not high, wherein the correlation between FW and other indexes is not high, and the 1 datum is rejected; at 200mM salt concentration, there was a correlation between the indices, all of which remained. The data of each index of the non-glutinous rice are all kept.
The weight of each index having a correlation is determined using principal component analysis. The analysis result of the main components shows that under the salt concentration of 150mM and 200mM, the indexes of the non-glutinous rice have 2 main components, and the cumulative contribution rate respectively reaches 83.893% and 83.908%. Under the salt concentration of 150mM and 200mM, the growth index of the glutinous rice has 1 main component, and the cumulative contribution rate reaches 100.000 percent. Each index weight coefficient is the index score coefficient/the sum of each index score coefficient, wherein the index score coefficient is the square root of the score/the sum of each index score formed by the orthogonal rotation method with kaiser normalization. Non-waxy rice was weighted higher for GP and DW at 150 and 200mM salt concentrations, 0.200 and 0.177 respectively (table 12).
TABLE 12 weight and contribution ratio of Each index of salt stress
(4) Multiplying the weight coefficient in the step (3) by the corresponding membership function value of each correlative growth index of each rice seed to obtain a correction membership function value of each correlative growth index of each rice seed; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value;
and combining the result of the membership function with the weight of the corresponding index to obtain a new membership function value sorting table (see table 13).
Table 1318 weight sorting of salt tolerance coefficients of rice varieties
RGE: relative germination vigour; RGP: relative germination rate; RGI: relative germination index; RVI: a relative viability index; RDW: relative dry weight.
According to the comprehensive ranking result of growth index membership function values under the 150mM salt concentration, the ranking of the Yonglong 15 and Jiazaofeng 18 varieties is 1 st and 2 nd, the ranking of the Yongyou No. 10 and Chunzhinuo No. 6 is 17 th and 18 th, the ranking of the Yonglong 15 and Jingliang 534 varieties is 1 st and 2 nd, and the ranking of the Yongyou No. 10 and Chunzhinuo No. 6 is 17 th and 18 th. It can be basically determined that Yongyou 15, Jingliangyou 534 and Jiazaofeng 18 are salt-resistant varieties, Yongyou No. 10 and Chunjiannuo No. 6 are salt-sensitive varieties. At different salt concentrations, there were subtle differences in the salt tolerance of rice.
Example 5 Integrated analysis of salt tolerance analysis of different types of Rice varieties
The salt tolerance analysis of the rice is comprehensively compared according to different types, and the results show that the screening results of the salt-tolerant and salt-sensitive varieties after different classification analysis are slightly different. Under the analysis of different classification modes, the frequency of occurrence of salt-tolerant varieties of the indica rice 15 and the Jingliangyou 534 is the highest under the condition of 150mM salt concentration, and the frequency of occurrence of salt-sensitive varieties of the glutinous rice 6 of the spring river is the highest; under the condition of 200mM salt concentration, the frequency of occurrence of the Yongzi 15 and Jingliangyou 534 is highest, and the frequency of occurrence of the salt-sensitive varieties of Chunjiannuo No. 6 and Xishui 121 is highest. Comprehensively, the Yonglong 15 and Jingliangyou 534 can be determined as salt-tolerant varieties, and the Chunjiannuo No. 6 is a salt-sensitive variety. In addition, the prediction indexes under different classification analysis also have differences. The correlation between the prediction indexes obtained by the analysis methods of indica rice, indica rice and japonica rice and the comprehensive ranking is the highest, and the correlation exceeds 90%, so that the reliability is higher.
TABLE 14 salt tolerance analysis of different types of rice
Claims (10)
1. A screening method of salt-tolerant or salt-sensitive rice seeds in a germination period is characterized by comprising the following steps:
(1) taking a plurality of rice varieties to be screened, respectively placing seeds of the rice varieties on germination paper which is wet by clear water and is wet by saline water with different salt concentrations, performing a germination test, measuring seedling growth index growth indexes of the seeds for germination and germination for 14 days, calculating the ratio of each growth index value of each rice variety under different salt concentrations to each growth index value under zero salt concentration, and marking as a corrected growth index value;
(2) performing membership function analysis on the corrected growth index values of the rice varieties to obtain membership function values;
(3) classifying the rice varieties according to the types of the rice to obtain sample sets of different types; performing correlation analysis on each growth index in the same sample set according to the corrected growth index value in the step (1), and removing the growth index with small correlation to obtain the correlation growth index in each sample set;
(4) performing principal component analysis on each correlation growth index in each sample set according to the correlation growth indexes in the step (3), and calculating to obtain a normalized weight coefficient of each correlation growth index in each sample set;
(5) multiplying the weight coefficient in the step (4) by the membership function value of each correlation growth index of each corresponding rice variety to obtain a correction membership function value of each correlation growth index of each rice variety; then calculating the average value of the correction membership function values of all the correlation growth indexes of each rice seed to obtain a sequencing reference value;
(6) and performing salt tolerance sorting on each rice seed according to the sorting reference value, selecting the rice seeds which are sorted in front or back according to the result of the salt tolerance sorting, and screening the rice seeds with strong salt tolerance or salt sensitivity in the germination period.
2. The screening method of salt-tolerant rice seeds in germination period as claimed in claim 1, wherein in step (1), the number of rice varieties to be screened is not less than 15.
3. The method for screening salt-tolerant or salt-sensitive rice seeds during germination of claim 1, wherein in step (1), the different salt concentrations are at least two or more salt concentrations, i.e., a low salt concentration and a high salt concentration.
4. The method of screening salt-tolerant or salt-sensitive rice seeds with a germination period as claimed in claim 3, wherein the low salt concentration is 50-160 mM and the high salt concentration is 165-300 mM.
5. The method for screening salt-tolerant or salt-sensitive rice seeds in germination stage according to claim 1, wherein in step (1), the growth index comprises: germination vigor, germination rate, seedling rate, germination index, vigor index, average germination time, fresh weight and dry weight.
6. The method for screening salt-tolerant or salt-sensitive rice seeds with a germination period as claimed in claim 1, wherein in the step (2), the formula of the membership function is: r (xj.i) ═ x j.min)/(x j.max-x j.min);
wherein Xj.i represents the corrected growth index value of the jth growth index of the ith variety; xj.min represents the minimum value of the corrected growth index value of the jth growth index; max represents the maximum value of the corrected growth index value for the jth growth index; r (Xj.i) represents the membership function value of j growth index of the ith variety.
7. The method of screening salt-tolerant or salt-sensitive rice seeds at germination stage according to claim 1,
in the step (3), the classification mode is at least one of the following modes:
(A) classifying indica rice and japonica rice into indica rice, japonica rice and indica japonica rice;
or (B) classifying the rice into hybrid rice and conventional rice in a breeding mode;
or (C) classifying the rice into glutinous rice and non-glutinous rice according to the quality of the rice.
8. The method for screening salt-tolerant or salt-sensitive rice seeds during germination of claim 1, wherein in step (4), SPSS software is used for principal component analysis and the normalized weight coefficient is calculated.
9. The method of screening salt-tolerant or salt-sensitive rice seeds in germination period as claimed in claim 1, wherein the growth index with small correlation is: the ratio of the number of other growth indexes having correlation with the growth index to the total number of the growth indexes is less than or equal to 1/3.
10. The method for screening salt-tolerant or salt-sensitive rice seeds in the germination period as claimed in claim 1, wherein in the step (6), the salt-tolerant or salt-sensitive rice seeds in the germination period screened by different classification methods are counted to select the rice variety with the highest frequency of occurrence, and the salt-tolerant or salt-sensitive rice variety in the germination period is obtained.
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