Application of Prupe3G307800 gene in determining dormancy breaking time of low-cold-demand peach and method of Prupe3G307800 gene in determining dormancy breaking time of low-cold-demand peach

Abstract

The invention discloses an application of a Prupe3G307800 gene in determining dormancy breaking time of peaches with low cold demand and a method of the Prupe3G307800 gene. According to the invention, the expression quantity of the Prupe3G307800 gene is regularly detected. Before the end of January, the highest expression quantity time of the peach blossom bud Prupe3G307800 gene is the peach dormancy breaking time. The method is easy and convenient to operate, does not depend on human eye observation, is high in accuracy, can be used for peach cultivation management in an auxiliary mode, formulates a scientific management strategy, and is good in application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioinformatics and plant molecular biology, and particularly relates to application of a Prupe_3G307800 gene in determining a low-chilling-requirement peach dormancy release time and a method thereof. BACKGROUND

[0002] Peach trees enter a dormant state after leaf fall in winter, and only when sufficient chilling requirement is accumulated can natural dormancy be successfully completed, which is an important physiological basis for subsequent normal germination, flowering and fruiting. However, in southern regions, insufficient chilling accumulation often leads to incomplete dormancy release due to the influence of warm winter climate, thereby causing uneven germination, prolonged flowering, low fruit setting rate and other problems, which seriously affect yield and fruit quality. Therefore, accurately determining the dormancy release time of peach trees is crucial for formulating scientific cultivation and management strategies and ensuring the economic benefits of orchards. In facility cultivation, the early or late completion of dormancy directly affects the determination of the warming time. If warming is too early, the low-temperature requirement is not met, which can lead to low germination rate and uneven flowering; and if warming is too late, it can delay the fruit maturation period and reduce the economic benefits of facility cultivation. Therefore, precise monitoring of the dormancy release period of peach trees is a key technical link for scientific environmental regulation, early market of fruits and successful cultivation in facility cultivation. At present, the methods for determining the dormancy release period of peach varieties mainly include (1) water culture method: after leaf fall in winter, 10-20 branches of current-year branches are cut and placed in an artificial climate chamber every 3-5 days, the branches are inserted into water about 2-3 cm deep, and the germination rate is counted after 20 days. When the germination rate is more than 50% or the weighted average value is equal to or greater than 2.5, it indicates that dormancy is released. The main defect of this method is that it needs to be determined under artificial climate conditions, and the sampling frequency, sampling amount and observation time are large, and the efficiency is low. (2) Color reaction: the flower bud is longitudinally cut, soaked with a tetrazole solution, and subjected to color reaction in the dark at a set temperature. The flower bud cut surface shows red within a specified time, indicating that dormancy is released. This color reaction judgment relies on the naked eye observation, lacks objective quantitative standard, and may lead to inconsistent results. Moreover, the temperature and experimental conditions of this method need to be strictly controlled, and are easily disturbed by multiple factors. Therefore, it is particularly necessary to seek a more efficient and accurate method for determining the dormancy release time of peach. SUMMARY

[0003] The present application is particularly directed to the problem that the water culture method and color reaction method of the prior art cannot meet the current accurate and efficient method for determining the dormancy release period of peach varieties, and provides application of a Prupe_3G307800 gene in determining a low-chilling-requirement peach dormancy release time and a method thereof.

[0004] The technical scheme of the present application is as follows:

[0005] The application of the Prupe_3G307800 gene in determining the dormancy release time of low chilling-requirement peaches, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The low cooling capacity required is <500h.

[0007] Furthermore, this invention also protects a method for determining the dormancy break time of low-chillation-requirement peaches using the aforementioned Proppe_3G307800 gene. The method uses flower buds from one-year-old branches of peach trees from before dormancy to flowering as material, and periodically detects the relative expression level of the Proppe_3G307800 gene. The sampling date of the flower bud with the highest relative expression level of the Proppe_3G307800 gene before the end of January is the dormancy break time of the low-chillation-requirement peach.

[0008] Specifically, it includes the following steps:

[0009] S1: From the time the peach tree enters dormancy until flowering, regularly take flower buds from one-year-old branches, freeze them in liquid nitrogen, and then store them at -80℃ for later use.

[0010] S2: RNA was extracted from flower buds, and its quality and concentration were detected by agarose gel electrophoresis and NanoDrop 2000.

[0011] S3: Reverse transcription of RNA into cDNA;

[0012] S4: Quantitative fluorescence detection of the relative expression level of the Prope_3G307800 gene.

[0013] Furthermore, in S1, the term "regularly" refers to every 1 to 15 days.

[0014] Furthermore, in S4, peach actin primers were used as internal controls, and analysis was performed using a real-time PCR instrument. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, for 40 cycles.

[0015] Furthermore, in S4, the nucleotide sequence of the upstream primer of the peach actin primer set is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0016] Furthermore, in S4, the nucleotide sequence of the upstream primer of the primer set used for quantification of the Prope_3G307800 gene is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention determines the dormancy break date of peaches by periodically detecting the expression level of the Proppe_3G307800 gene. Since peaches requiring less than 500 hours of chilling generally break dormancy by the end of January in southern regions, the dormancy break date is determined by the time when the expression level of the Proppe_3G307800 gene on peach flower buds is highest before the end of January in southern regions. The method of this invention is simple to operate, does not rely on human observation, has high accuracy, and can be used to assist in peach cultivation management, helping to formulate scientific management strategies, showing good application prospects. Attached Figure Description

[0019] Figure 1 The relative expression levels of the Prope_3G307800 gene in flower buds of different chilling-required peach varieties at different times. Detailed Implementation

[0020] To illustrate the technical effects of this invention, the applicant provides the following experimental process and data:

[0021] The present invention will be further described below with reference to embodiments, but this does not limit the scope of the implementation of the present invention.

[0022] Example 1

[0023] The method for determining the dormancy break time of low chilling-requirement peaches using the aforementioned Proppe_3G307800 gene includes the following steps:

[0024] S1: From the time the peach tree enters dormancy until flowering, take flower buds from one-year-old branches every 1-15 days, freeze them in liquid nitrogen, and then store them at -80℃ for later use.

[0025] S2: RNA was extracted from flower buds, and its quality and concentration were detected by agarose gel electrophoresis and NanoDrop 2000.

[0026] S3: Reverse transcription of RNA into cDNA;

[0027] S4: The relative expression level of the Prope_3G307800 gene was detected by real-time PCR using peach actin primers as an internal control. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, for 40 cycles.

[0028] The nucleotide sequence of the Prope_3G307800 gene is shown in SEQ ID NO.1.

[0029] In S4, the nucleotide sequence of the upstream primer of the peach actin primer set is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0030] In S4, the nucleotide sequence of the upstream primer used for quantification of the Prope_3G307800 gene is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0031] experiment:

[0032] The peach varieties involved in the experiment were: Tropical Prince, Nan Gui Peach No. 1, Nanshan Sweet Peach, Florida King, Florida Crown, Early Red No. 2, Zhong Tao No. 8, Zhongyou 20, and Beijing 40.

[0033] method:

[0034] From before peach tree enters dormancy until flowering, flower buds from one-year-old branches were collected every 15 days, flash-frozen in liquid nitrogen, and then stored at -80℃ for later use. RNA was extracted from the flower buds using a plant RNA extraction kit manufactured by Beijing Huayueyang Biotechnology Co., Ltd., and its quality and concentration were detected by agarose gel electrophoresis and NanoDrop 2000 (Thermo). Reverse transcription was performed using the FonSuper™ RTⅢ All-in-One Mix (with dsDNase) kit manufactured by Wuhan Fengsheng Bell Technology Co., Ltd. to convert RNA into cDNA. Quantitative PCR was performed using the MonAmp™ SYBR Green qPCR Mix kit. Peach actin primers were used as internal controls, and analysis was performed using a BIO CFX Opus 96 quantitative PCR instrument (USA). The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, for 40 cycles. Two... -ΔΔCT The relative expression level of the Prope_3G307800 gene was calculated using this method. The primers used in this study included a peach actin primer set and a primer set for Prope_3G307800 gene quantification. The nucleotide sequence of the upstream primer in the peach actin primer set is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 3. The nucleotide sequence of the upstream primer in the primer set used for Prope_3G307800 gene quantification is shown in SEQ ID NO. 4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 5.

[0035] The relative expression levels of the Prope_3G307800 gene at different time stages for each peach variety are shown in the figure. Figure 1 .

[0036] Determining the date of dormancy end in peaches using hydroponics:

[0037] From December 1st to late February of the following year, one-year-old branches with a diameter of 0.4–0.7 cm and a length of 20–40 cm were cut every 7 days from five directions (east, south, west, north, and center) in the upper part of the tree canopy. This was repeated three times, resulting in a total of 15 branches. Five branches per group were placed in a 25℃ artificial climate chamber for hydroponics. The light intensity was 1200 lx, the light / dark cycle was 14h / 10h, the relative humidity was 70%, and the water was tap water at a depth of 2–3 cm, changed every 3 days. A small portion of the base of each branch was trimmed to expose the new shoot. After 3 weeks, the budding status was assessed. The bud grading criteria were: Grade 1, no budding; Grade 2, budding; Grade 3, green tip showing; Grade 4, red tip showing; Grade 5, flower open. The sampling date was considered the end of dormancy when the weighted average of each grade was equal to or greater than 2.5. Calculation formula: In the formula: i represents the flower bud germination level; Xi represents the number of buds at each level.

[0038] The dormancy end dates for each peach variety are shown in Table 1 below.

[0039] Table 1. Dormancy end dates for various peach varieties determined by hydroponics.

[0040]

[0041] As shown in Table 1, Tropical Prince, Nangui Peach No. 1, and Nanshan Sweet Peach broke dormancy on December 20; Florida King and Florida Crown broke dormancy on January 3; Zaohong No. 2 broke dormancy on January 31; and Zhongtao No. 8, Zhongyou 20, and Beijing 40 broke dormancy on February 14.

[0042] right Figure 1 Analysis of the expression of the Proppe_3G307800 gene in flower buds of various peach varieties under different chilling requirements during dormancy revealed a positive correlation between gene expression and low-temperature accumulation. In varieties requiring ≤200h chilling (Tropical Prince, Nan Gui Peach No. 1, and Nanshan Sweet Peach), gene expression in flower buds began to increase in November, peaking on December 20th. In varieties requiring approximately 400h chilling (Florida King and Florida Crown), gene expression began to increase in December, peaking on January 3rd. In varieties requiring 500h or more chilling (Tropical Prince and Nan Gui Peach No. 1), gene expression began to increase in December, peaking on January 17th. The peak expression time in flower buds of peach varieties requiring less than 500h chilling coincided with the dormancy completion time, allowing the gene expression level to be used to determine the dormancy end date for low-chilling-requirement (<500h) peach varieties.

[0043] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. The application of the Prope_3G307800 gene in determining the dormancy release time of low chilling-requirement peaches, characterized by: The nucleotide sequence of the Prope_3G307800 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The required cooling capacity is <500h.

3. A method for determining the dormancy release time of low-chillation peaches using the Proppe_3G307800 gene as described in claim 1 or 2, characterized in that: The method uses flower buds from one-year-old branches of peach trees before they enter dormancy and during the flowering period as material, and regularly detects the relative expression level of the Proppe_3G307800 gene. The sampling date of the flower bud with the highest relative expression level of the Proppe_3G307800 gene before the end of January is the dormancy release time of low chilling requirement peach.

4. The method according to claim 3, characterized in that: It includes the following steps: S1: From the time the peach tree enters dormancy until flowering, regularly take flower buds from one-year-old branches, freeze them in liquid nitrogen, and then store them at -80℃ for later use. S2: RNA was extracted from flower buds, and its quality and concentration were detected by agarose gel electrophoresis and NanoDrop 2000. S3: Reverse transcription of RNA into cDNA; S4: Quantitative fluorescence detection of the relative expression level of the Prope_3G307800 gene.

5. The method according to claim 4, characterized in that: In S1, "regularly" refers to every 1 to 15 days.

6. The method according to claim 4, characterized in that: In S4, peach actin primers were used as internal controls, and analysis was performed using a real-time PCR instrument. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 59℃ annealing for 30 s, 72℃ extension for 20 s, for 40 cycles.

7. The method according to claim 6, characterized in that: In S4, the nucleotide sequence of the upstream primer of the peach actin primer set is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

8. The method according to claim 6, characterized in that: In S4, the nucleotide sequence of the upstream primer used for quantification of the Prope_3G307800 gene is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

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