A method for identifying low temperature tolerance of Brassica napus during flowering period by silique length

Abstract

The invention provides a method for identifying the low temperature resistance of Brassica napus during flowering by using the length of siliques, and belongs to the technical field of identification of low temperature resistant germplasm resources of Brassica napus. The method comprises the following steps: 1) directly sowing Brassica napus in the field in mid-to-late October of the current year; 2) in March of the second year, when the daily average temperature is lower than 10°C for 3 consecutive days, marking the Brassica napus for low temperature treatment; 3) when the daily average temperature is higher than 16°C for 3 consecutive days, determining the elongated siliques as the normal temperature treatment group of Brassica napus; 4) respectively measuring the lengths of the siliques of the low temperature treatment group and the normal temperature treatment group, and calculating the low temperature resistance index of the siliques, wherein the low temperature resistance index of the siliques=the average length of the siliques of the normal temperature treatment group / the average length of the siliques of the low temperature treatment group. The low temperature resistance of the Brassica napus variety to be detected during flowering is determined according to the low temperature resistance index of the siliques. The method of the invention is simple to operate, and can quickly and effectively identify germplasms resistant to low temperature during flowering under a field environment.

Description

Technical Field

[0001] The invention belongs to the technical field of identification of low-temperature-resistant germplasm resources of rapeseed, and in particular relates to a method for identifying low-temperature resistance of Brassica napus during flowering period. Background Art

[0002] Brassica napus (Brassica napus, AACC, 2n = 38), an allotetraploid crop, is one of China's important oil crops. Brassica napus is a low-temperature tolerant plant, and is therefore the only overwintering oil crop in the Yangtze River Basin. The main overwintering Brassica napus production areas mainly adopt the "oil-rice" or "oil-rice-rice" rotation system for multi-cropping production in one year. In recent years, with the continuous extension of the rice growth period, especially the continuous promotion of the "oil-rice-rice" three-cropping system, in order to adapt to the early maturity of the crop, the demand for rapeseed germplasm has continued to increase. Therefore, in production practice, early-maturing rapeseed germplasm needs to have the advantages of tolerance to delayed sowing, fast emergence at low temperatures, and strong overwintering ability at the seedling stage. At the same time, it needs to have the advantages of early flowering, early flowering, short maturity, strong comprehensive stress resistance, stable and high yield during the flowering period. The Yangtze River Basin is the main producing area of ​​rapeseed. It is very prone to sudden temperature drops due to "late spring cold" from February to March every year. Early-maturing rapeseed is likely to encounter low temperatures during key reproductive growth periods such as bolting, flowering, and silique setting. This can easily cause rapeseed growth stagnation, flower abortion, reduced fruit set rate, slow elongation and development of siliques, or even failure to expand, forming "shaded pods" and segmented fruit setting, leading to large-scale rapeseed production reductions.

[0003] At present, the research on low temperature tolerance of Brassica napus mainly focuses on the germination and seedling stage (before bolting), and the leaves are used for sampling. Through the observation and statistics of agronomic traits and field frost damage index, combined with proline, soluble sugar, malondialdehyde (MDA, M alon d The low temperature tolerance of Brassica napus varieties was identified by measuring physiological and biochemical indicators such as aldehyde. There is a certain correlation between the low temperature tolerance of the seedling stage and the low temperature tolerance of the flowering stage, but there is a lack of relevant research. In actual production practice, there is a relative lack of research on the low temperature that is likely to occur during the flowering stage and cause large-scale yield reduction. The methods and ideas of this study can be applied to rapeseed and other winter crops. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for identifying the low temperature resistance of Brassica napus during flowering by using the length of siliques. This method uses the length of siliques as a statistical indicator to reflect the stress resistance of rapeseed when encountering low temperatures. The operation is simple and can quickly and effectively identify germplasm with low temperature resistance during flowering in a field environment.

[0005] The present invention provides a method for identifying low temperature tolerance of Brassica napus during flowering period by using silique length, comprising the following steps:

[0006] 1) Sowing different varieties of Brassica napus in mid-to-late October of the same year;

[0007] 2) In March of the second year, when the average daily temperature is below 10°C for three consecutive days, the Brassica napus plants that have entered the flowering and pod-bearing period are marked with a low-temperature treatment mark, and the low-temperature treatment mark is placed below the flowering flowers on the main inflorescence of the rapeseed;

[0008] 3) After 28 to 35 days, 4 to 6 siliques are counted upward from the low temperature treatment mark in step 2), which is the low temperature treatment group; when the average daily temperature is higher than 16°C for 3 consecutive days, 4 to 6 elongated siliques are counted continuously from the top of the main inflorescence from top to bottom, which is the normal temperature treatment group;

[0009] 4) In May of the second year, the length of siliques in the low temperature treatment group and the normal temperature treatment group was measured, and the average length of siliques in the low temperature treatment group and the normal temperature treatment group was calculated, and then used to calculate the silique low temperature tolerance index = the average length of siliques in the normal temperature treatment group / the average length of siliques in the low temperature treatment group;

[0010] Different varieties of Brassica napus were ranked from large to small according to the silique low temperature tolerance index. The varieties with the top 25% of the silique low temperature tolerance index were low temperature intolerant germplasms during flowering, the varieties with the bottom 25% of the silique low temperature tolerance index were low temperature tolerant germplasms during flowering, and the remaining varieties were medium low temperature tolerant germplasms during flowering.

[0011] 5) The Brassica napus variety to be identified is operated according to steps 1) to 4) to obtain the low temperature resistance index of the silique of the Brassica napus variety to be identified;

[0012] When the silique low temperature resistance index is ≥1.380, it is a germplasm that is not tolerant to low temperatures during the flowering period; when the silique low temperature resistance index is between 1.014 and 1.380, it is a germplasm that is moderately tolerant to low temperatures during the flowering period; when the silique low temperature resistance index is ≤1.014, it is a germplasm that is tolerant to low temperatures during the flowering period.

[0013] Preferably, the Brassica napus in step 1) is sown in a field.

[0014] Preferably, the sowing in step 1) is direct sowing.

[0015] Preferably, the number of the Brassica napus variety in step 1) is 30 to 100 seeds.

[0016] Preferably, the low temperature treatment mark is a label tag.

[0017] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses silique length as a statistical index to reflect the stress tolerance of Brassica napus when encountering low temperatures. On the same plant, by setting up a low-temperature treatment group and a normal temperature treatment group in stages, the change in the elongation length of the silique during the process of field temperature recovering from low temperature to normal temperature during the flowering period can be observed more intuitively. By calculating the silique low temperature tolerance index, the low temperature tolerance of different germplasms of Brassica napus during the flowering period, which is a critical period of reproductive growth, can be better reflected. The method described in the present invention improves the evaluation system of low temperature tolerance of Brassica napus during the flowering period; using this method, germplasms that are resistant to low temperatures during the flowering period can be quickly and effectively identified in the field, providing a reference basis for breeding rapeseed lines or varieties that are resistant to low temperatures during the flowering period. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of temperature statistics in Datian, West District, Zijingang Campus, Zhejiang University, Sandun Town, Hangzhou City, Zhejiang Province in March 2024.

[0019] Figure 2 These are the hanging position of the main inflorescence of field Brassica napus and the siliques in the low-temperature and normal temperature treatment groups. The left picture is the hanging position of the main inflorescence of field early-flowering Brassica napus planted in the West District of Zijingang, Zhejiang University on March 1, 2024, as shown by the white line; the right picture is the siliques of the low-temperature treatment group near the hanging position on April 3, 2024, and the siliques of the normal temperature treatment group near the top of the main inflorescence. The white vertical line is a scale with a length of 1 cm.

[0020] Figure 3 To identify early-flowering rapeseed germplasms with different low-temperature tolerance during flowering period by using silique low-temperature tolerance index.

[0021] Figure 4 Statistical analysis of the silique length of Brassica napus germplasm that is intolerant to low temperatures and that is tolerant to low temperatures during flowering; the left figure shows the silique length of Brassica napus germplasm that is intolerant to low temperatures during flowering in the normal temperature treatment group (dark green) and the low temperature tolerant group (light green); the right figure shows the silique length of Brassica napus germplasm that is tolerant to low temperatures during flowering in the normal temperature treatment group (dark blue) and the low temperature intolerant group (light blue); Through T test analysis, ** indicates that the p value is less than 0.01, and there is a significant difference; ns indicates that the p value is greater than 0.05, and there is no significant difference.

[0022] Figure 5 These are pictures of the siliques of the low-temperature-intolerant and low-temperature-tolerant germplasms of Brassica napus treated with low temperatures during flowering. The left picture shows the siliques of 3 Brassica napus germplasms that are intolerant to low temperatures during flowering in the first 25%; the right picture shows the siliques of 3 Brassica napus germplasms that are tolerant to low temperatures during flowering in the last 25%; the white vertical line is the scale bar with a length of 1 cm.

[0023] Figure 6It is a bar graph of malondialdehyde (MDA) content in leaves of Brassica napus that are intolerant to low temperature and those that are tolerant to low temperature during flowering after low temperature treatment before bolting; the left graph is the MDA content of 3 Brassica napus germplasms that are intolerant to low temperature during flowering in the first 25%; the right graph is the MDA content of 3 Brassica napus germplasms that are tolerant to low temperature during flowering in the last 25%. DETAILED DESCRIPTION

[0024] The present invention provides a method for identifying low temperature tolerance of Brassica napus during flowering period, comprising the following steps:

[0025] First, different varieties of Brassica napus are sown in mid-to-late October of the current year. In the present invention, the number of varieties of Brassica napus is preferably 30 to 100 varieties that flower early in previous years, and further preferably 40 to 70 varieties that bolt and flower early in the current year; the present invention determines the elongation length of siliques of different varieties under low temperature treatment by sowing different varieties of Brassica napus, and determines the standard of low temperature resistance during flowering period based on statistical data and silique length as an indicator. Siliques of rapeseed are its yield organs, and their elongation length will be significantly affected because they do not produce seeds and the development of siliques themselves is affected by low temperature. Because it is easy to observe, the growth and yield of siliques are usually judged in the field by the elongation length of siliques.

[0026] In the present invention, in March of the second year, when the average daily temperature is lower than 10°C for 3 consecutive days, the Brassica napus that has entered the flowering and silique fruiting period is marked for low-temperature treatment, and the low-temperature treatment mark is placed below the flowering flowers on the main inflorescence of the rapeseed; the low-temperature treatment mark is preferably a label tag, which is convenient for marking and statistics.

[0027] According to the present invention, 28 to 35 days after the low-temperature treatment mark is set, 4 to 6 siliques are counted upward from the low-temperature treatment mark, which is a low-temperature treatment group; when the average daily temperature is higher than 16°C for 3 consecutive days, 4 to 6 elongated siliques are counted continuously from the top to the bottom from the top of the main inflorescence, which is a normal temperature treatment group; the flowers and siliques of the low-temperature treatment group are close to the label tag position, and the flowers and siliques of the normal temperature treatment group are far from the label tag position and close to the top position of the main inflorescence.

[0028] The invention counts and photographs the growth length of siliques in the main inflorescence 28 to 35 days after low-temperature treatment marking, which is used as a technical index of low-temperature resistance during flowering; the length of siliques in the low-temperature treatment group and the normal temperature treatment group are measured respectively, and the average length of siliques in the low-temperature treatment group and the normal temperature treatment group is calculated, and the low-temperature resistance index of siliques = the average length of siliques in the normal temperature treatment group / the average length of siliques in the low-temperature treatment group;

[0029] Different varieties of Brassica napus were ranked from large to small according to the silique low temperature tolerance index. The varieties with the top 25% of the silique low temperature tolerance index were low temperature intolerant germplasms during flowering, the varieties with the bottom 25% of the silique low temperature tolerance index were low temperature tolerant germplasms during flowering, and the remaining varieties were medium low temperature tolerant germplasms during flowering.

[0030] According to this classification standard, the classification value of the silique low temperature resistance index is determined. When the silique low temperature resistance index is ≥1.380, it is a germplasm that is not resistant to low temperatures during the flowering period; when the silique low temperature resistance index is between 1.014 and 1.380, it is a germplasm that is moderately resistant to low temperatures during the flowering period; when the silique low temperature resistance index is ≤1.014, it is a germplasm that is resistant to low temperatures during the flowering period.

[0031] For a single Brassica napus variety to be identified, the Brassica napus variety to be identified is operated according to the above steps to obtain the silique low temperature resistance index of the Brassica napus variety to be identified; and the flowering period low temperature resistance classification of the Brassica napus variety to be identified is determined according to the silique low temperature resistance index and the above classification standards.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] On October 26, 2023, the R series of Brassica napus germplasm resources were planted by direct seeding in the large field of the West Agricultural Experiment Station of Zhejiang University Zijingang Campus in Sandun Town, Xihu District, Hangzhou City, Zhejiang Province (120.1 degrees east longitude, 30.3 degrees north latitude). Through the detection of field temperature during its growth period, it is scheduled to be listed on March 1, 2024 for the 60 Brassica napus germplasms that have already bloomed ( Figure 2 ) and defined it as an early flowering broccoli-type rapeseed germplasm. On April 3, 2024, the weather station (Top Yunnong, https: / / smart.topyn.cn ) detection data, and obtained the temperature record in March ( Figure 1 ), and from March 1st, the growth status and low temperature stress of the main inflorescence siliques of different germplasms were counted and photographed as collected data.

[0035] Based on the date of low temperature weather and the flowering and silique growth, the length of the main inflorescence siliques of each germplasm was divided into two groups for statistics and analysis. The first group was the low temperature treatment group, which experienced low temperature weather conditions with an average daily temperature below 10°C for three consecutive days during flowering and silique elongation. The average silique length counted was the average silique length of the low temperature treatment group of this germplasm, and this part of the siliques was close to the hanging place on the main inflorescence.

[0036] The second group is the normal temperature treatment group. The average daily temperature during flowering and silique elongation in this group is basically higher than the normal temperature weather conditions of 16℃ and above. The average silique length counted is the average silique length of the normal temperature treatment group of this germplasm. These siliques are close to the top of the main inflorescence.

[0037] Among the 60 early-flowering Brassica napus germplasms, the elongation length of the siliques of a small number of germplasms became significantly shortened after low temperature stress during the flowering period, and were greatly affected by low temperature. The elongation length of the siliques of another small number of germplasms did not shorten significantly, that is, the flowering period was not significantly affected by low temperature. However, the elongation length of the siliques of most germplasms shortened to varying degrees.

[0038] A statistical comparison of 30 materials with obvious phenotypes revealed that during the reproductive growth and development process, a small number of flowers and siliques were subjected to low temperature stress. On their main inflorescence, the average length of siliques in the low temperature treatment group did not change much, and they were identified as low temperature resistant germplasm during the flowering period of Brassica napus. However, the average length of siliques in a small number of low temperature treatment groups shortened significantly, and they were identified as Brassica napus germplasm that was not low temperature resistant during the flowering period. For most germplasms, after experiencing low temperature stress, the average length of siliques in the low temperature treatment group shortened, and they were identified as moderately low temperature resistant germplasm during the flowering period of Brassica napus. Through this experiment, the low temperature resistance of early-flowering Brassica napus in the field can be identified during the flowering period of reproductive growth, providing a method for breeding rapeseed germplasm, lines or varieties with low temperature resistance during the flowering period.

[0039] The specific experimental steps are as follows:

[0040] 1 Experimental design

[0041] 1.1 Test materials

[0042] The 30 early-flowering Brassica napus germplasms for phenotypic statistics were obtained from the Institute of Crop Sciences, Zhejiang University. The R series numbers are shown in Table 1 below.

[0043] Table 1 Early flowering Brassica napus germplasm number

[0044]

[0045] 1.2 Test methods

[0046] 1.2.1 Seed sowing and low temperature stress

[0047] Seeds of Brassica napus germplasm were sown on October 26, 2023 in the fields of Zijingang, Zhejiang University (120.1°E, 30.3°N). On March 1, 2024, the germplasm that had already bloomed was labeled, and the label was located directly below the flowers that were opening on the main inflorescence. The field temperature was counted for one month, and the statistics were kept daily. At the same time, combined with the temperature changes and the growth of flowers and siliques on the main inflorescence, the overall and normal growth of plants under low temperature stress and normal temperature stress, as well as the stress phenotypes of inflorescences, flowers, and siliques after low temperature stress, such as dehydration and necrosis of tissues, were observed and recorded.

[0048] 1.2.2 Statistics of silique length after low temperature stress

[0049] The continuous low temperature during the reproductive growth period, especially during the flowering period, of Brassica napus will affect the elongation length of the siliques. According to the field temperature change data, the siliques on the main inflorescence were divided into two groups. The flowers and siliques experienced a low temperature with an average daily temperature below 10°C for three consecutive days. Five siliques near the hanging place on the main inflorescence were selected, and the elongation length of the siliques in the low temperature treatment group was measured after taking photos. As the temperature rose, the flowers and siliques experienced normal temperature weather with an average daily temperature of 16°C or above. Generally, five consecutive siliques near the top of the main inflorescence that could represent the elongation length of the siliques of the material were selected, and the length of the siliques in the normal temperature treatment group was measured after taking photos. For 30 early-flowering Brassica napus, the statistical data of the elongation length of the siliques in the normal temperature treatment group and the low temperature treatment group are shown in Table 2.

[0050] Table 2 Statistics of silique length of 30 early-flowering Brassica napus germplasms

[0051]

[0052]

[0053]

[0054] 1.2.3 Statistics of low temperature tolerance index of siliques

[0055] In order to quantitatively evaluate the cold tolerance phenotype of siliques, the cold tolerance index of siliques was calculated, which was the value of the average silique length of the normal temperature treatment group divided by the average silique length of the low temperature treatment group. The cold tolerance index of siliques of 30 early flowering Brassica napus accessions is shown in Table 3.

[0056] Table 3 Statistics of low temperature tolerance index of siliques of 30 early flowering Brassica napus germplasms

[0057]

[0058] 1.2.4 Identification of early flowering rapeseed germplasm with tolerance to low temperature during flowering period

[0059] By statistically analyzing the low temperature resistance index of 30 early flowering Brassica napus varieties, the low temperature resistance level of early flowering Brassica napus varieties during flowering was divided into three levels, namely, intolerant to low temperature during flowering, moderately tolerant to low temperature during flowering, and tolerant to low temperature during flowering. According to the distribution of the low temperature resistance index of the silique, the top 25% of the 7 germplasms, whose silique low temperature index was greater than or equal to ≥1.380, had their silique elongation length seriously affected, and were identified as germplasms intolerant to low temperature during flowering. Most of the germplasms, including 16, had a silique low temperature index between 1.014 and 1.380, and their silique elongation length was slightly affected, and were identified as germplasms with moderate low temperature resistance during flowering. In addition, the 7 germplasms in the bottom 25%, whose silique low temperature index was less than or equal to ≤1.014, had their silique elongation length not much affected, and were identified as germplasms with low temperature resistance during flowering. The specific level division is shown in Table 4.

[0060] Table 4 Classification of low temperature tolerance during flowering period of early flowering rapeseed

[0061]

[0062] 1.2.5 Determination of malondialdehyde (MDA) content in leaves before bolting of cold-tolerant and cold-intolerant early flowering rapeseed germplasms

[0063] The early flowering Brassica napus rapeseed was divided into low temperature resistance grades during flowering by the silique low temperature resistance index, and a total of 7 low temperature resistant germplasms during flowering, 16 medium low temperature resistant germplasms during flowering, and 7 low temperature intolerant germplasms during flowering were screened. Three materials were selected from each of the selected low temperature resistant and low temperature intolerant germplasms during flowering. According to previous literature experience, in the early stage of bolting of Brassica napus, at the end of December of the year, rapeseed will experience a period of low temperature in the field, which is convenient for sampling its leaves, and then the MDA (malondialdehyde) content was determined. In this way, the selected low temperature resistant and low temperature intolerant germplasms during flowering were verified. The specific MDA content is shown in Table 5, where the content of low temperature intolerant germplasms in the leaves before bolting ranges from 32.86 to 61.29 nmol / g. The content of low temperature resistant germplasms in the leaves before bolting is significantly lower, ranging from 18.43 to 25.12 nmol / g. After bolting, the leaves at the base of Brassica napus senesce and fall off.

[0064] Table 5 Determination of MDA content in leaves before bolting of low temperature tolerant and low temperature intolerant germplasms during flowering period

[0065]

[0066] It can be seen from the above embodiments that the present invention sets up a low temperature treatment group and a normal temperature treatment group on the same plant, and can more intuitively observe the change in the elongation length of the silique during the process of field temperature recovering from low temperature to normal temperature during the flowering period. By calculating the silique low temperature resistance index, the low temperature resistance of different germplasms of Brassica napus during the flowering period, which is a critical period of reproductive growth, can be better reflected. By measuring the MDA content of the leaves before bolting, the flowering period intolerant and low temperature resistant germplasms screened by the silique low temperature resistance index are verified, further illustrating the feasibility of the method of identifying the low temperature resistance of early flowering Brassica napus in the field by the silique low temperature resistance index.

[0067] 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 method for identifying low temperature tolerance of Brassica napus during flowering period by silique length, characterized in that: The following steps are involved: 1) Sowing different varieties of Brassica napus in mid-to-late October of the same year; 2) In March of the second year, when the average daily temperature is below 10°C for three consecutive days, the Brassica napus plants that have entered the flowering and pod-bearing period are marked with a low-temperature treatment mark, and the low-temperature treatment mark is placed below the flowering flowers on the main inflorescence of the rapeseed; 3) After 28 to 35 days, count 4 to 6 siliques from the low temperature treatment mark in step 2), which is the low temperature treatment group; when the average daily temperature is higher than 16°C for 3 consecutive days, count 4 to 6 elongated siliques from the top of the main inflorescence from top to bottom, which is the normal temperature treatment group; 4) In May of the second year, the length of siliques in the low temperature treatment group and the normal temperature treatment group were measured, and the average length of siliques in the low temperature treatment group and the average length of siliques in the normal temperature treatment group were calculated. The silique low temperature tolerance index was calculated as the average length of siliques in the normal temperature treatment group / the average length of siliques in the low temperature treatment group; Different varieties of Brassica napus were ranked from large to small according to the silique low temperature tolerance index. The varieties with the top 25% of the silique low temperature tolerance index were low temperature intolerant germplasms during flowering, the varieties with the bottom 25% of the silique low temperature tolerance index were low temperature tolerant germplasms during flowering, and the remaining varieties were medium low temperature tolerant germplasms during flowering. 5) Carry out the operations of the Brassica napus variety to be identified according to steps 1) to 4) to obtain the low temperature tolerance index of the Brassica napus variety to be identified; When the silique low temperature resistance index is ≥1.380, it is a germplasm that is not tolerant to low temperatures during the flowering period; when the silique low temperature resistance index is between 1.014 and 1.380, it is a germplasm that is moderately tolerant to low temperatures during the flowering period; when the silique low temperature resistance index is ≤1.014, it is a germplasm that is tolerant to low temperatures during the flowering period.

2. The method according to claim 1, characterized in that Step 1) The Brassica napus L. is sown in a field.

3. The method according to claim 1 or 2, characterized in that: Step 1) The sowing is direct seeding.

4. The method according to claim 1, characterized in that: Step 1) The number of Brassica napus varieties is 30 to 100.

5. The method according to claim 1, characterized in that The low temperature treatment mark is a label tag.

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