CN114740114A - Method for identifying extremely-low-cold-demand peach germplasm resources - Google Patents
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Abstract
The invention discloses a method for identifying peach germplasm resources with extremely low cold demand, which is mainly characterized in that the peach germplasm resources with extremely low cold demand (<300h) are identified and distinguished by adopting a method for measuring the content of abscisic acid in flower buds 15d before and after a large number of leaf fall periods in winter according to the condition that the content of abscisic acid (ABA) in the flower buds of peaches is kept at a higher level before dormancy breaking and is sharply reduced after dormancy breaking.
Description
[ technical field ] A
The invention belongs to the technical field of plant breeding, and particularly relates to a method for identifying peach germplasm resources with extremely low cold demand.
[ background ] A method for producing a semiconductor device
Peach (Prunus persica L.) is a medium perennial tree of the Rosaceae family (Rosaceae), Prunus (Prunus L.), Prunus (Amygdalus L.). Originally produced in cold and cool areas in western regions of Shaanxi, Gansu, Tibet and the like in China, a certain amount of cold energy is needed to complete dormancy so as to enter a male and female cell differentiation stage. The cold requirement is the effective low-temperature time required for breaking the natural dormancy of deciduous fruit trees, and the temperature is generally less than or equal to 7.2 ℃ as a starting point for calculation. When the cold requirement of the peach trees cannot be met, the buds are irregular, the flowering period is long, flowers are not solid, and the yield cannot be guaranteed. Therefore, the cold requirement is one of the key factors for planting the southern peaches. At present, the peaches are classified according to the cold demand, and can be divided into 5 grades of extremely low cold demand (<300h), low cold demand (300 + 600h), medium cold demand (600 + 900h), high cold demand (900 + 1200h) and extremely high cold demand (>1200 h).
Related researches exist at present, for example, Chinese patent application No. 201410264421.5 discloses a method for rapidly measuring the cold demand of peaches, wherein 3-5 branches of the annual branches of peaches are collected every 4-5 days after the peaches fall off leaves, the branches have the length of 20-30cm, the number of buds and leaves on the branches of each peach variety is not less than 20, the branches are inserted into water for 2-3cm indoors, the lower parts of the peach branches are cut every 3 days to expose new stubbles, water is changed every three days for cleaning treatment, the indoor temperature is 23-25 ℃ in the daytime, 15-17 ℃ in the evening, the time is 14/10 hours, the humidity is 60-70%, the branch illumination intensity is 2500LX, and 50% of the tops of the buds represent that the buds and the buds meet the cold demand of the peaches; and then, a cold demand model is used for counting cold demand, so that the cold demand of the peaches is measured.
For another example, chinese patent application No. 202111261417.X discloses a method for rapidly measuring and calculating the cooling capacity required by peach varieties based on a qRT-PCR detection method, comprising the steps of: detecting the relative expression level of the PpDAM5 gene; grading the relative expression quantity of the PpDAM5 gene and the cold requirement; (III) rapidly measuring and calculating the variety of the peach to be measured; the method takes the determined cold-demand variety as a standard substance, the PpDAM5 gene expression quantity of leaves is measured when the peach variety falls off leaves, a cold-demand grading model is established, the PpDAM5 gene relative expression quantity of the new peach variety to be measured for cold demand is detected, and then matching is carried out according to the established cold-demand grading model, so that the cold demand of the peach variety to be measured can be preliminarily and rapidly measured.
In general, the measuring methods of the cooling capacity required by peaches mainly comprise the following steps:
(1) water culture method: cutting 10-20 branches of the current year every 3-5 days after leaf falling in winter, inserting the branches into water with the depth of 2-3cm, placing the branches in an artificial climate box, counting the germination rate after 21 days, wherein the cold demand is met when the germination rate exceeds 50%, and the hours when the temperature is lower than 7.2 ℃ during counting are the cold demand of the variety;
(2) comparison variety method: the method is characterized in that a series of comparison varieties are used for measuring the cold demand of the variety to be measured, the method is generally applied abroad, the variety to be measured and the comparison peach variety are simultaneously sampled every 7d from the field (the method is the same as a water culture method), and after 21d in a greenhouse, the germination rates of the measured variety and the comparison variety are compared, so that the cold demand of the measured variety can be known;
(3) and (3) molecular marker identification: in 2019, the peach subject group of Zhengzhou fruit tree institute of Chinese academy of agricultural sciences is identified by a molecular marker technology.
The disadvantages of the above method are: firstly, the measurement is carried out under the condition of artificial climate, branches are in an isolated state after being cut off, the natural conditions in the field cannot be completely simulated, and the transportation and the germination of nutrient substances are influenced to a certain extent, so that the error of the measurement result is large; secondly, the workload is large, and the efficiency is low; thirdly, only peach germplasm resources above 500h can be identified and distinguished by adopting a molecular marker technology, and varieties below 500h cannot be distinguished.
[ summary of the invention ]
Aiming at the defects of the prior art, the invention provides a method for identifying peach germplasm resources with extremely low cold demand, and the method is mainly used for identifying and distinguishing the peach germplasm resources with extremely low cold demand (<300h) by measuring the content of abscisic acid in the peach buds in a large leaf fall period according to the fact that the content of abscisic acid in the peach buds keeps a stable level in the large leaf fall period.
The purpose of the invention is realized by the following technical scheme:
a method for identifying peach germplasm resources with extremely low cold demand comprises the following steps:
1) selecting different peach germplasm resources which are healthy in growth and need cooling capacity;
2) sampling: collecting 5.0g of flower buds of robust annual middle fruit branches at the middle upper part of a crown in the early stage of leaf falling, a large amount of leaf falling and 10 days after leaf falling, and quickly freezing in liquid nitrogen and storing in a refrigerator at-80 ℃ for later use;
3) drawing an abscisic acid working curve: weighing 4.0mg of abscisic acid standard substance, respectively using methanol to fix and contain the abscisic acid standard substance in a 100.0mL volumetric flask as mother solution (the concentration is 0.04mg/mL), respectively using 2.0mL, 2.5mL, 3.0mL, 4.0mL, 5.0mL and 6.0mL of mother solution to place in a 10mL volumetric flask, diluting the mother solution into solutions with different concentration gradients by using methanol, filtering the solutions by using a 0.22um organic filtering membrane, measuring the peak areas of abscisic acid with different concentrations by using a high performance liquid chromatograph, and drawing a working curve according to the peak areas and the concentrations of the abscisic acid;
4) extraction of abscisic acid: quickly freezing the fresh flower buds collected in the step 2) by using liquid nitrogen, drying the flower buds to constant weight by using a freeze dryer, fully grinding and crushing the flower buds, accurately weighing 100.0mg of the flower buds, putting the flower buds into a 2.5mL centrifuge tube, adding 1.5mL of extracting solution (methanol: water: formic acid: 80:20:0.2), shaking and extracting the flower buds in a shaking table for 40min, centrifuging the flower buds for 8min (14000r/min, 4 ℃), sucking supernatant after centrifugation, putting the supernatant into a new 5mL centrifuge tube, extracting the supernatant by using the same extraction method for 2 times, then combining the extracting solution after volatilizing the solvent, adding 1mL of 50 percent methanol aqueous solution for redissolving, filtering the mixture by using a 0.22-micron organic filter membrane, and then putting the mixture on a machine for analysis;
5) determination of abscisic acid content: determining the abscisic acid content in flower buds by adopting a high performance liquid chromatograph under the following conditions:
a chromatographic column: YMC-Pack ODS-AM (150X 4.6mm, 3 μm); column temperature: 30.0 ℃; the detection wavelength is 265 nm;
mobile phase A: 0.1% aqueous formic acid; mobile phase B: methanol; flow rate: 0.5 mL/min; the sample injection amount is 10.0 mu L;
time gradient: 0.00-33.00 min: 40% B, 33.01-34.00 min: 100% B, 34.01-46.00 min: 100% B, 46.01-47.00 min: 40% B, 47.01-60.00 min: 40% of B;
6) when the content of abscisic acid in the dried flower buds of the peach germplasm resources with unknown cold demand is less than 14.00 mu g/g, the peach germplasm resources with extremely low cold demand (the cold demand is less than 300h) are identified.
In the invention:
the peach germplasm resources with different cold requirements in the step 1) are selected from tropical prince (150h), Nanshan sweet peach (200h), Flordiaglo (200h), Malyvia (250h), Sunrayter (300h), Zaohong No. 2 (500h), Zhongyou No. 13 (550h), Shuangfo (550h), Meiting (550h), Wuyuehuo (550h), Amken (600h), Chunmei (600h), Jinchunpun (600h), brocade (800h) and Meijin (800 h).
The high performance liquid chromatograph in the step 3) is selected from Shimadzu LC-20 AT.
The solution diluted by methanol to different concentration gradients in the step 3) is a solution with the concentration of 0.008mg/mL, 0.001mg/mL, 0.0012mg/mL, 0.0016mg/mL, 0.0020mg/mL and 0.0024 mg/mL.
Compared with the prior art, the invention has the following advantages:
1. according to the method for identifying the peach germplasm resources with extremely low cold demand, the abscisic acid content of peach bud is measured, and the abscisic acid content of the peach germplasm resources with extremely low cold demand (the cold demand is less than 300h) is lower than 13.50 mu g/g, and the abscisic acid content of the remaining peach germplasm resources with the cold demand of more than 300h is higher than 13.50 mu g/g. Therefore, the unknown cold-demand peach germplasm resources are 15 days before and after a large amount of leaves fall in winter, the extremely-low cold-demand peach germplasm resources can be accurately identified by measuring the abscisic acid content in the dry flower buds, and technical support can be provided for peach variety hybridization breeding, variety division, facility cultivation and development of the peach industry in subtropical regions.
2. According to the method for identifying the peach germplasm resources with extremely low cold demand, the ABA content in the flower buds of a plurality of peach varieties can be continuously measured in multiple batches by an HPLC method, the time consumption is short, the efficiency is high, the repeated measurement and verification can be performed for multiple times, and the result is accurate and reliable.
3. The method for identifying the peach germplasm resources with extremely low cold demand is not influenced by external conditions such as regions, climates and the like by measuring the content of abscisic acid in a large number of flower buds in the defoliation period.
[ description of the drawings ]
FIG. 1 is an abscisic acid HPLC chromatogram of an example of the invention.
FIG. 2 is an HPLC chromatogram of mallotus officinalis buds according to an embodiment of the present invention.
[ detailed description ] embodiments
The following examples are provided to further illustrate the embodiments of the present invention.
Example (b):
a method for identifying peach germplasm resources with extremely low cold demand comprises the following steps:
1 materials and methods
1.1 test materials
Selecting 15 healthy peach germplasm resources with different cold requirements as test materials, namely tropical prince (150h), Nanshan sweet peach (200h), Flordiaglo (200h), Maryvira (250h), Sunrayter (300h), Zaohong No. 2 (500h), Zhongzhou No. 13 (550h), Shuangfo (550h), Meiting (550h), Wuyuelio (550h), Amken (600h), Chunmei (600h), Jinchun (600h), brocade (800h) and Meijin (800 h).
1.2 reagents
Abscisic acid (ABA) was purchased from shanghai-source phylloto biotechnology limited; methanol (GR) and formic Acid (AR) were purchased from Xilongsu chemical Co., Ltd.
1.3 instruments and devices
High performance liquid chromatograph (Shimadzu LC-20AT)
1.4 methods
1.4.1 sampling
Collecting 5.0g of flower buds of robust annual middle branches at the middle upper part of a crown respectively at 11-month and 15-day (early stage of defoliation) in 2021, 11-month and 30-day (large amount of defoliation) in 2021 and 12-month and 15-day (10 d after defoliation) in 2022, placing in liquid nitrogen for quick freezing, and storing at-80 deg.C for later use;
1.4.2 abscisic acid working curve
Weighing standard abscisic acid 4.0mg, respectively using methanol to fix in a 100.0mL volumetric flask as mother solution (the concentration is 0.04mg/mL), respectively using 2.0mL, 2.5mL, 3.0mL, 4.0mL, 5.0mL and 6.0mL mother solution to place in a 10mL volumetric flask, diluting with methanol to obtain solutions with different concentration gradients (0.008mg/mL, 0.001mg/mL, 0.0012mg/mL, 0.0016mg/mL, 0.0020mg/mL and 0.0024mg/mL), filtering with a 0.22um organic filtering membrane, measuring abscisic acid by using a high performance liquid chromatograph, and determining abscisic acid peak area and peak area according to abscisic acid peak area and peak areaDrawing a working curve by concentration, and drawing an abscisic acid standard sample tR＝31.811min；
1.4.3 extraction of Abscisic acid
Respectively collecting fresh flower buds, quickly freezing by using liquid nitrogen, drying by using a freeze dryer to constant weight, fully grinding and crushing, accurately weighing 100.0mg, placing in a 2.5mL centrifuge tube, adding 1.5mL of extracting solution (methanol: water: formic acid is 80:20:0.2), shaking and extracting for 40min in a shaking table, centrifuging for 8min at 14000r/min, absorbing supernatant, placing in a new 5mL centrifuge tube, extracting for 2 times by using the same extraction method, combining the extracting solutions, volatilizing a solvent by using a nitrogen blower, adding 1mL of 50% methanol aqueous solution for redissolving, filtering by using a 0.22-micron organic filter membrane, then performing on-machine analysis, repeating the determination for 3 times on each peach variety flower bud, and taking an average value;
1.4.4 determination of Abscisic acid content
Determining the content of abscisic acid in flower buds by using a high performance liquid chromatograph; a chromatographic column: YMC-Pack ODS-AM (150X 4.6mm, 3 μm); mobile phase A: 0.1% aqueous formic acid, mobile phase B: methanol; column temperature: 30.0 ℃; flow rate: 0.5 mL/min; the sample injection amount is 10.0 mu L; the detection wavelength is 265 nm; time gradient: (0-33.00 min: 40% B), (33.01-34.00 min: 100% B), (34.01-46.00 min: 100% B), (46.01-47.00 min: 40% B), (47.01-60.00 min: 40% B), abscisic acid t in flower budR＝31.919min。
2 results and analysis
2.1 abscisic acid working curve
Drawing a standard working curve by taking different concentrations of abscisic acid as abscissa and peak area as ordinate, and obtaining a regression equation of y 55948 x-0.9761, R2＝0.999。
2.2 different cooling capacity requirements of the content of abscisic acid in flower buds of peach germplasm resources in the dormant period
As can be seen from Table 1, the abscisic acid content in the flower buds of different refrigeration-required peach germplasm resources is stable and has small fluctuation from 15 days at 11 months to 15 days at 12 months. The content of abscisic acid in 5 peach varieties with the refrigeration requirement of less than 300h is less than 13.50 mu g/g, and the content of abscisic acid in the other 10 peach germplasm resources with the refrigeration requirement of more than 300h is more than 13.50 mu g/g, and detailed data are shown in a table 1.
TABLE 1 content of abscisic acid in flower bud of different cold-requiring peach germplasm resource dormant periods
3 conclusion
The abscisic acid content in the peach germplasm resource dry flower buds with extremely low cold requirement (the cold requirement is less than 300h) is lower than 13.50 mu g/g. Therefore, the extremely low cold demand peach germplasm resources can be identified and distinguished by measuring the abscisic acid content in the dried flower buds by using a high performance liquid chromatograph 15 days before and after a large number of leaf fall periods in winter.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the inventive concept of the present invention, which falls into the protection scope of the present invention.
Claims (4)
1. A method for identifying peach germplasm resources with extremely low cold demand is characterized by comprising the following steps: the method comprises the following steps:
1) selecting different peach germplasm resources which are healthy in growth and need cooling capacity;
2) sampling: collecting 5.0g of flower buds of robust annual middle fruit branches at the middle upper part of a crown 15 days before and after a large number of leaf fall periods in winter, and quickly freezing in liquid nitrogen, and storing in a refrigerator at-80 ℃ for later use;
3) drawing an abscisic acid working curve: weighing 4.0mg of abscisic acid standard substance, respectively using methanol to be fixedly contained in a 100.0mL volumetric flask as mother liquor with the concentration of 0.04mg/mL, respectively using 2.0mL, 2.5mL, 3.0mL, 4.0mL, 5.0mL and 6.0mL of mother liquor to be placed in a 10mL volumetric flask, diluting the mother liquor into solutions with different concentration gradients by using methanol, filtering the solutions by using a 0.22um organic filter membrane, measuring the peak areas of abscisic acid with different concentrations by using a high performance liquid chromatograph, and drawing a working curve according to the peak areas and the concentrations of the abscisic acid;
4) extraction of abscisic acid: quickly freezing the fresh flower buds collected in the step 2) by using liquid nitrogen, drying the flower buds to constant weight by using a freeze dryer, fully grinding and crushing, accurately weighing 100.0mg of the flower buds, placing the flower buds into a 2.5mL centrifuge tube, adding an extracting solution, adding methanol, namely 80:20:0.2 and 1.5mL of formic acid, shaking and extracting for 40min in a shaking table, centrifuging for 8min, 14000r/min at 4 ℃, absorbing supernatant after centrifuging, placing the supernatant into a new 5mL centrifuge tube, extracting for 2 times by using the same extraction method, then combining the extracting solution, volatilizing a solvent by using a nitrogen blower, adding 1mL of 50% methanol aqueous solution for redissolving, filtering by using a 0.22-micrometer organic filter membrane, and then placing on a machine for analysis;
5) determination of abscisic acid content: determining the abscisic acid content in flower buds by adopting a high performance liquid chromatograph under the following conditions:
a chromatographic column: YMC-Pack ODS-AM, 150X 4.6mm, 3 μm; column temperature: 30.0 ℃; the detection wavelength is 265 nm;
mobile phase A: 0.1% aqueous formic acid; mobile phase B: methanol; flow rate: 0.5 mL/min; the sample injection amount is 10.0 mu L;
time gradient: 0.00-33.00 min: 40% B, 33.01-34.00 min: 100% B, 34.01-46.00 min: 100% B, 46.01-47.00 min: 40% B, 47.01-60.00 min: 40% of B;
6) when the abscisic acid content in the dried flower buds of the peach germplasm resources with unknown cold demand is less than 13.50 micrograms per gram, the peach germplasm resources with extremely low cold demand are identified, and the cold demand is less than 300 hours.
2. The method for identifying peach germplasm resources with extremely low cold demand according to claim 1, wherein the method comprises the following steps: the different refrigeration capacity-requiring peach germplasm resources in the step 1) are selected from tropical prince, Nanshan sweet peach, Flordiaglo, Maryvira, Sunrayter, Zaohong No. 2, Zhongyou No. 13, Shuangfo, Meiting, Mayuelian, Amiken, Chunmei, Jinchun, brocade and Meijin.
3. The method for identifying peach germplasm resources with extremely low cold demand according to claim 1, wherein the method comprises the following steps: the high performance liquid chromatograph in the step 3) is selected from Shimadzu LC-20 AT.
4. The method for identifying peach germplasm resources with extremely low cold demand according to claim 1, wherein the method comprises the following steps: the solution diluted by methanol to different concentration gradients in the step 3) is a solution with the concentration of 0.008mg/mL, 0.001mg/mL, 0.0012mg/mL, 0.0016mg/mL, 0.0020mg/mL and 0.0024 mg/mL.
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| CN114231602A (en) * | 2021-10-28 | 2022-03-25 | 广西壮族自治区农业科学院 | Method for rapidly measuring and calculating cooling capacity required by peach variety based on qRT-PCR detection method |
| CN114231602B (en) * | 2021-10-28 | 2024-04-26 | 广西壮族自治区农业科学院 | Method for rapidly measuring and calculating cold demand of peach variety based on qRT-PCR detection method |
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