Method for identifying low temperature resistance of brassica napus in flowering period through silique length

Abstract

The invention provides a method for identifying the low temperature resistance of brassica napus in the flowering period through the silique length, and belongs to the technical field of rape low temperature resistance germplasm resource identification, the method comprises the following steps: 1) directly sowing brassica napus in a field in mid-to-late October in the current year; (2) in March of the second year, when the daily average temperature is lower than 10 DEG C for 3 days continuously, performing low-temperature treatment marking on the brassica napus; (3) when the daily average temperature is higher than 16 DEG C for 3 days, determining the extending siliques as a normal-temperature treatment group of the brassica napus; (4) the silique lengths of the low-temperature treatment group and the normal-temperature treatment group are measured respectively, the silique low-temperature-resistant index is calculated, and the silique low-temperature-resistant index is equal to the average silique length of the normal-temperature treatment group / the average silique length of the low-temperature treatment group. And determining the flowering phase low temperature resistance of the to-be-detected brassica napus variety according to the silique low temperature resistance index. The method disclosed by the invention is simple to operate, and can be used for quickly and effectively identifying the germplasm with low temperature resistance in the flowering phase in a field environment.

Description

Technical Field

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

[0002] Brassica napus (Brassica napus, AACC, 2n=38), an allotetraploid crop, is one of China's important oilseed crops. Due to its low-temperature tolerance, it is the only overwintering oilseed crop in the Yangtze River Basin. Major overwintering rapeseed production areas primarily utilize a "oilseed-rice" or "oilseed-rice-rice" rotation system, achieving multiple cropping per year. In recent years, with the continuous extension of the rice growing season, particularly the increasing adoption of the "oilseed-rice-rice" triple-cropping system, the demand for rapeseed germplasm has increased to accommodate early maturation. Therefore, in production practice, early-maturing rapeseed germplasm needs to exhibit tolerance to delayed sowing, rapid emergence at low temperatures, and strong overwintering resistance. Furthermore, it needs to exhibit early flowering, early flowering, a short maturity period, strong overall stress resistance, and stable and high yields. 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 to stagnate, flowers to abort, fruit set rate to decrease, silique elongation to develop slowly or even fail to expand, forming "shaded pods" and segmented fruit setting, leading to large-scale rapeseed yield 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. M alon d The cold tolerance of Brassica napus varieties is assessed by measuring physiological and biochemical indicators such as aldehyde (IDA). While there is a correlation between cold tolerance during the seedling stage and cold tolerance during the flowering stage, relevant research is lacking. Furthermore, research on the high likelihood of cold weather during the flowering stage, which can lead to large-scale yield losses, is relatively lacking. The methods and approaches 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 tolerance 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 tolerance of rapeseed when encountering low temperatures. The method is simple to operate and can quickly and effectively identify germplasm that is resistant to low temperatures during flowering in a field environment.

[0005] The present invention provides a method for identifying low-temperature tolerance of Brassica napus during flowering 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 stages are marked with a low-temperature treatment mark, with the low-temperature treatment mark placed below the flowering flowers on the main inflorescence of the rapeseed;

[0008] 3) After 28 to 35 days, count 4 to 6 siliques from the low-temperature treatment mark in step 2) upwards, indicating the low-temperature treatment group; when the average daily temperature is above 16°C for 3 consecutive days, count 4 to 6 elongating siliques from the top of the main inflorescence downwards, indicating the normal-temperature treatment group;

[0009] 4) In May of the second year, the silique lengths of the low-temperature treatment group and the normal-temperature treatment group were measured, and the average silique lengths of the low-temperature treatment group and the normal-temperature treatment group were calculated. This was then used to calculate the silique cold tolerance index (average silique length of the normal-temperature treatment group / average silique length of the low-temperature treatment group).

[0010] Different varieties of Brassica napus were ranked from highest to lowest according to their silique cold tolerance index. The top 25% of varieties were classified as cold-intolerant during flowering, the bottom 25% were classified as cold-tolerant during flowering, and the remaining varieties were classified as moderately cold-tolerant during flowering.

[0011] 5) performing steps 1) to 4) on the Brassica napus variety to be identified to obtain a low temperature tolerance index of the Brassica napus var.

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

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

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

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

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

[0017] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses silique length as a statistical indicator 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 through staged listing, 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 Brassica napus germplasms 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 Brassica napus low temperature tolerance during flowering; using this method, germplasms that are resistant to low temperatures during flowering 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 flowering. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 These are the hanging positions of the main inflorescences of field-grown 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-grown early-flowering Brassica napus planted in the Zijingang West District of Zhejiang University on March 1, 2024, as indicated 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 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 rapeseed germplasms treated with low temperatures during flowering. The left picture shows the siliques of three rapeseed germplasms in the first 25% that are intolerant to low temperatures during flowering; the right picture shows the siliques of three rapeseed germplasms in the last 25% that are tolerant to low temperatures during flowering. The white vertical line is a scale bar with a length of 1 cm.

[0023] Figure 6This is a bar chart showing the malondialdehyde (MDA) content in the leaves of Brassica napus germplasms that are intolerant to low temperatures during flowering and those that are tolerant to low temperatures before bolting after low temperatures. The left figure shows the MDA content of three Brassica napus germplasms that are intolerant to low temperatures during flowering in the first 25%; the right figure shows the MDA content of three Brassica napus germplasms that are tolerant to low temperatures 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, 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 Brassica napus varieties 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 the siliques of different varieties under low-temperature treatment by sowing different varieties of Brassica napus. Based on statistical data, the silique length is used as an indicator to determine the standard for low-temperature resistance during the flowering period. The silique of rape is its yield organ, and its elongation length will be significantly affected by the lack of seed production and the influence of low temperature on the development of the silique itself. Because it is easy to observe, the growth and yield of rapeseed are usually judged by the elongation length of the silique in the field.

[0026] In the present invention, in March of the second year, when the average daily temperature is lower than 10°C for three consecutive days, the Brassica napus that has entered the flowering and pod-bearing period is marked for low-temperature treatment. 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 elongating 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 hangtag position, while the flowers and siliques of the normal-temperature treatment group are away from the label hangtag position and close to the top position of the main inflorescence.

[0028] The present invention counts and photographs the length of siliques in the main inflorescence 28 to 35 days after low-temperature treatment and marking, and uses this as a technical indicator of low-temperature resistance during flowering; measures the length of siliques in the low-temperature treatment group and the normal-temperature treatment group, and calculates the average length of the siliques in the low-temperature treatment group and the normal-temperature treatment group; and calculates the low-temperature resistance index of the siliques = the average length of the siliques in the normal-temperature treatment group / the average length of the siliques in the low-temperature treatment group.

[0029] Different varieties of Brassica napus were ranked from highest to lowest according to their silique cold tolerance index. The top 25% of varieties were classified as cold-intolerant during flowering, the bottom 25% were classified as cold-tolerant during flowering, and the remaining varieties were classified as moderately cold-tolerant during flowering.

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

[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 low temperature resistance classification of the Brassica napus variety to be identified is determined based on 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] On October 26, 2023, the R series Brassica napus germplasm resources were planted by direct seeding in a large field at the West Agricultural Experiment Station of the Zijingang Campus of Zhejiang University 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 the growth period, the 60 Brassica napus germplasms that have already bloomed will be listed on March 1, 2024. Figure 2 ) and defined it as an early flowering 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, statistics and photos of the growth status and low temperature stress of the main inflorescence siliques of different germplasms were taken as collected data.

[0035] Based on the dates of low temperatures, flowering, and silique growth, the length of the main inflorescence siliques of each accession was divided into two groups for statistical analysis. The first group was the low-temperature treatment group. This group experienced three consecutive days of low temperatures with an average daily temperature below 10°C during flowering and silique elongation. The average silique length calculated was the average length of the low-temperature treatment group for that accession. These siliques were located near the hanging sign on the main inflorescence.

[0036] The second group is the normal temperature treatment group. The average daily temperature in this group during the flowering and silique elongation process 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 60 early-flowering Brassica napus accessions, a small number of accessions showed a significant shortening of silique length after low temperature stress during flowering, indicating that they were greatly affected by low temperature. A small number of accessions showed no significant shortening of silique length, indicating that the flowering period was not significantly affected by low temperature. However, the silique length of most accessions shortened to varying degrees.

[0038] 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 inflorescences, the average length of siliques in the low-temperature treatment group did not change much, and they were identified as Brassica napus germplasm with low temperature tolerance during flowering. However, a small number of low-temperature treatment groups had a significant shortening of the average length of siliques, and they were identified as Brassica napus germplasm with intolerant to low temperature during flowering. 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 Brassica napus germplasm with moderate tolerance to low temperature during flowering. Through this experiment, the low-temperature tolerance 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 tolerance to low temperature during flowering.

[0039] The specific experimental steps are as follows:

[0040] 1 Experimental design

[0041] 1.1 Test materials

[0042] The 30 early-flowering Brassica napus accessions 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 rapeseed germplasm numbers

[0044]

[0045] 1.2 Test methods

[0046] 1.2.1 Seed sowing and low temperature stress

[0047] Brassica napus seeds were sown directly in the fields of Zhejiang University's Zijingang site (120.1°E, 30.3°N) on October 26, 2023. Flowering seeds were labeled on March 1, 2024, with the labels placed directly below the opening flowers of the main inflorescence. Field temperatures were recorded daily for one month, along with temperature fluctuations and the growth of flowers and siliques on the main inflorescence. Phenotypes of stress, such as dehydration, necrosis, and whitish tissues, were observed in both low-temperature-stressed and normal-temperature-growing plants, as well as in inflorescences, flowers, and siliques exposed to low temperatures.

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

[0049] Sustained low temperatures during the reproductive growth period, especially during the flowering phase, can affect silique elongation in Brassica napus. Based on field temperature data, siliques on the main inflorescence were divided into two groups. For plants whose flowers and siliques experienced three consecutive days of low temperatures with average daily temperatures below 10°C, five siliques near the main inflorescence where the material was placed were photographed and measured for silique elongation in the low-temperature treatment group. As temperatures rose, the flowers and siliques experienced normal temperatures with average daily temperatures exceeding 16°C. Five consecutive siliques near the top of the main inflorescence, representing the elongation of the material, were photographed and measured for silique length in the normal-temperature treatment group. Table 2 shows the statistical data for silique elongation in 30 accessions of early-flowering Brassica napus for the normal-temperature and low-temperature treatment groups.

[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] To quantitatively assess the cold tolerance phenotype of siliques, we calculated the cold tolerance index (CTI) by dividing the average silique length of the normal temperature treatment group by the average silique length of the low temperature treatment group. The CTIs for 30 early-flowering Brassica napus accessions are shown in Table 3.

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

[0057]

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

[0059] By analyzing the cold tolerance index of siliques of 30 early-flowering Brassica napus accessions, the cold tolerance of early-flowering Brassica napus accessions was divided into three levels: intolerant to cold during flowering, moderately tolerant to cold during flowering, and tolerant to cold during flowering. Based on the distribution of the cold tolerance index, the top 25% of seven accessions were identified as cold-intolerant during flowering, with a silique cold tolerance index greater than or equal to 1.380. Their silique elongation length was severely affected and they were identified as cold-intolerant during flowering. The majority of accessions, including 16, had a silique cold tolerance index between 1.014 and 1.380, with minimal impact on silique elongation, and they were identified as moderately tolerant during flowering. The remaining seven accessions, in the bottom 25%, had a silique cold tolerance index less than or equal to 1.014, with minimal impact on silique elongation, and they were identified as cold-tolerant during flowering. The specific grading is shown in Table 4.

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

[0061]

[0062] 1.2.5 Determination of malondialdehyde (MDA) content in leaves before bolting of early flowering rapeseed germplasms with low temperature tolerance and low temperature intolerant

[0063] Using the silique cold tolerance index, early-flowering Brassica napus accessions were classified for cold tolerance during flowering. A total of seven accessions were identified for cold tolerance during flowering, 16 accessions for moderate cold tolerance during flowering, and seven accessions for cold intolerant during flowering. Three accessions each were selected from the selected accessions with cold tolerance and cold intolerant during flowering. Based on previous literature, Brassica napus accessions experience a period of low temperatures in the field before bolting, in late December of the year, making it convenient to sample leaves for malondialdehyde (MDA) content. This method was used to validate the selected accessions for cold tolerance and cold intolerant during flowering. Specific MDA contents are shown in Table 5. The MDA content in leaves of the cold intolerant accessions before bolting ranged from 32.86 to 61.29 nmol / g. The MDA content in leaves of the cold tolerant accessions before bolting was 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

[0065]

[0066] As can be seen from the above examples, the present invention sets up a low-temperature treatment group and a normal-temperature treatment group on the same plant, which can more intuitively observe the change in the length of the silique elongation during the process of field temperature recovery from low temperature to normal temperature during the flowering period. By calculating the silique low-temperature tolerance index, the low-temperature tolerance of different Brassica napus germplasms during the flowering period, a critical period of reproductive growth, can be better reflected. By measuring the MDA content of the leaves before bolting, the flowering period low-temperature intolerant and low-temperature tolerant germplasms screened by the silique low-temperature tolerance index are verified, further illustrating the feasibility of this method of identifying the low-temperature tolerance of early-flowering Brassica napus in the field by the silique low-temperature tolerance 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 principles of the present invention. These improvements and modifications should also be regarded as within 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, 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; 4) In May of the second year, the lengths of the siliques of the low temperature treatment group and the normal temperature treatment group were measured, and the average lengths of the siliques of the low temperature treatment group and the normal temperature treatment group were calculated. The silique low temperature tolerance index was calculated as follows: the average length of the siliques of the normal temperature treatment group / the average length of the siliques of 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) 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; 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: The sowing in step 1) is direct seeding.

4. The method according to claim 1, characterized in that In step 1), the number of seeds of the Brassica napus variety 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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