Cultivation method of high-quality cold-resistant large-fruit crisp-pulp storable apples

Abstract

The invention discloses a cultivation method of high-quality cold-resistant large-fruit crisp-pulp storable apples, relates to the technical field of agriculture, and aims to solve the problems that small-fruit varieties represented by Saixihong and the like are high in cold resistance, good in fresh food quality and rapid in development area expansion, but fruits are small and have the problems of easiness in softening and browning, too short shelf life and the like, and many difficulties are brought to post-harvest storage, transportation and sales. And large-area popularization and application are restricted. The invention provides a cultivation method of high-quality cold-resistant large-fruit crisp-pulp storable apples, which comprises the following steps: S1, preparing a cross parent combination: selecting Hanfu (ACS1-2 / 2, storable) apples as male parents and Xiaohong (ACS1-1 / 2, non-storable) apples as female parents, and hybridizing the Hanfu (ACS1-2 / 2, non-storable) apples and the Xiaohong (ACS1-1 / 2, non-storable) apples; s2, detecting and analyzing the storability of the hybrid progeny through PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis experiments; and S3, screening according to an analysis result to obtain four high-quality, cold-resistant, large-fruit, crisp-pulp and storable apple superior lines, namely TL0307, TL0110, TL0115 and TL0279.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technologies, and particularly to a cultivation method for high-quality, cold-resistant, large-fruited, crispy-fleshed and storage-resistant apples. Background Art

[0002] Apples are the largest tree species among deciduous fruit trees in China, with both the area and output accounting for more than half of the world's total, and they are bulk fruits that ensure stable annual supply. In vast high-latitude regions such as Liaoning, Jilin, Heilongjiang, Inner Mongolia, and Xinjiang, which serve as large ecological barriers, there are large areas of forest land and grasslands. Although these areas have harsh climate conditions such as short crop growth seasons and long durations of low winter temperatures and severe cold, conditions such as long sunshine hours, high light intensity, large temperature differences between day and night, and little rainfall during the growth season are conducive to the formation of the fruit quality of cold-region fruit trees. In recent years, cold-region apples have developed rapidly due to good economic benefits and have become an important industry for promoting the income increase of forest farmers in the region. Therefore, reasonably developing and utilizing forest land to develop economic forests such as apples is an important way to achieve the simultaneous realization of ecological, economic, and social benefits.

[0003] The existing distribution area of cold-region apples is located at 40 - 46° north latitude. The main apple varieties planted include Hanfu, Xinping No. 1, Yueyan, Zixiang, Saiwaihong, K9 (July Fresh), Longhong, Longfeng, etc. Among these varieties, large-fruited varieties represented by Hanfu have better cold resistance than Fuji and have pushed the planting area of large apples 200 kilometers northward. However, as a late-maturing variety, Hanfu still has a significant gap in cold resistance compared with small apples such as Saiwaihong. Due to the late fruit maturity and late nutrient return of the tree body, the early frost not only affects fruit tree production, but the superposition of the dual factors of early frost and cold winter weather even endangers the safe overwintering of the tree body. In recent years, there have been frost damages when planting Hanfu in places such as Pingquan, Hebei (41° north latitude), which has affected the industrial development, and the comprehensive quality has been difficult to meet the increasing consumption demands of people. Therefore, the planting area of Hanfu apples has shown a shrinking trend in recent years. Small-fruited varieties represented by Saiwaihong have strong cold resistance and good fresh food quality, and their planting areas have expanded rapidly. However, the fruit size is small (the average single fruit weight is 58.3 g, and the maximum single fruit weight is 80.3 g), and there are problems such as easy softening and browning and too short shelf life, which bring many difficulties to post-harvest storage, transportation, and sales and restrict their large-scale popularization and application. Therefore, although the development space and potential of cold-region apples are huge, the lack of high-quality, large-fruited, cold-resistant, and storage-resistant new varieties is the bottleneck problem restricting the high-quality and efficient development of cold-region apples. Summary of the Invention

[0004] Aiming at the above existing problems, the present invention aims to provide a cultivation method for high-quality, cold-resistant, large-fruited, crispy-fleshed and storage-resistant apples, which can quickly and efficiently screen out candidate excellent lines, without the need to conduct storage experiments on a large number of hybrid offspring, and the traits such as crispy-fleshed and storage-resistant fruits can be determined at the seedling stage, improving the seed selection efficiency and reducing the breeding cost.

[0005] The main idea of the technical solution adopted in the present invention: Select high-quality, large-fruited, late-maturing, cold-storage-resistant (ACS1-2 / 2, cold-storage-resistant) apples with complex genetic backgrounds (including excellent genes from germplasms of 3 countries) as male parents, and cold-resistant, mid-maturing "Saiwaihong" (ACS1-1 / 2, not cold-storage-resistant) as female parents to construct a hybrid segregation population. Using the molecular marker patent technology developed in the early stage, rapid detection and analysis of cold-storage resistance were carried out through experiments such as PCR amplification and agarose gel electrophoresis. Four excellent lines of high-quality, cold-resistant, large-fruited, crispy-fleshed, cold-storage-resistant apples, namely TL0307, TL0110, TL0115, and TL0279, were efficiently screened and determined from 12,000 hybrid offspring, and their ACS1 genotypes were all ACS1-2 / 2 (cold-storage-resistant).

[0006] To achieve the above object, the technical solution adopted in the present invention is as follows:

[0007] A method for cultivating high-quality, cold-resistant, large-fruited, crispy-fleshed, cold-storage-resistant apples, comprising the following steps:

[0008] S1. Prepare the hybrid parent combination: Select "Hanfu" (ACS1-2 / 2, cold-storage-resistant) apples as male parents and "Saiwaihong" (ACS1-1 / 2, not cold-storage-resistant) as female parents for hybridization;

[0009] S2. Detect and analyze the cold-storage resistance of the hybrid offspring through PCR amplification and agarose gel electrophoresis experiments;

[0010] S3. Screen according to the analysis results to obtain a total of 4 excellent lines of high-quality, cold-resistant, large-fruited, crispy-fleshed, cold-storage-resistant apples, namely TL0307, TL0110, TL0115, and TL0279.

[0011] Through the above technical solution, further, the hybridization process includes:

[0012] S11. Collect the bell-shaped flowers of "Hanfu" apples, take out the pollen, dry it at room temperature for later use;

[0013] S12. Select the bell-shaped flowers of "Saiwaihong" apples, and use the "continuous petal and stamen removal method" to remove the petals, sepals, filaments, and anthers;

[0014] S13. Dip a cotton swab in the pollen processed in S11 and dot it on the stigma of "Saiwaihong";

[0015] S14. After the fruits mature, pick the fruits without sepals, take out the seeds, mix them with wet sand, and place them in a 4°C refrigerator for stratification treatment;

[0016] S15. Wait for the seeds to germinate, plant them in nutrient pots, and then plant the germinated seeds in the field.

[0017] Through the above technical solutions, further: in the step S3, the ACS1 genotypes of the 4 excellent cold-resistant large-fruit crispy-flesh and storage-resistant apple elite lines are all ACS1-2 / 2, the fruit development period is 120 - 140 days, the single fruit weight is 71.8 - 196 g, and the soluble solids content is 11.9 - 18.9%.

[0018] Through the above technical solutions, further: in the step S3, TL0110, TL0115, and TL0307 are stored at room temperature for 30 days.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. Through scientific selection and mating of hybrid parents, a high-quality large-fruit late-maturing storage-resistant Hanfu (ACS1-2 / 2, storage-resistant) apple with a complex genetic basis (including excellent genes from 3 national germplasms) is used as the male parent, and the cold-resistant mid-maturing Saiwaihong (ACS1-1 / 2, non-storage-resistant) is used as the female parent to construct a hybrid segregation population. By using the molecular marker patent technology developed in the early stage, rapid detection and analysis of storage resistance are carried out through experiments such as PCR amplification and agarose gel electrophoresis. Four excellent cold-resistant large-fruit crispy-flesh and storage-resistant apple elite lines, namely TL0307, TL0110, TL0115, and TL0279, are efficiently screened and determined from 12,000 hybrid offspring. Their ACS1 genotypes are all ACS1-2 / 2 (storage-resistant), the fruit development period is 120 - 140 days, the single fruit weight is 71.8 - 196 g, which are all significantly higher than those of Saiwaihong, and the soluble solids content is 11.9 - 18.9%.

[0021] Among them, when TL0110 and TL0279 are stored at room temperature for 30 days, the fruits still remain hard and crispy. When Saiwaihong is stored at room temperature for 15 days, the fruit hardness decreases by 29%, and by 39% after 30 days of storage. While when TL0279 is stored at room temperature for 15 days, the fruit hardness hardly changes, and after 25 days of storage, the fruit hardness only decreases by 19.6%. When TL0110, TL0115, and TL0307 are stored at room temperature for 30 days, the hardness only decreases by 18.5%, 14%, and 5.2% respectively, and they still remain hard, crispy, and juicy.

[0022] 2. Compared with the traditional evaluation method, this method has strong predictability, and there is no need to conduct storage experiments on a large number of hybrid offspring. The traits such as crispy-flesh and storage resistance of the fruits can be determined at the seedling stage, and candidate elite lines can be quickly and efficiently screened out, locking the target for subsequent variety cultivation and approval, greatly improving the seed selection efficiency, and significantly reducing the breeding cost. Description of the Drawings

[0023] Figure 1 It is the pedigree diagram of the elite lines of the hybrid offspring of Hanfu and Saiwaihong;

[0024] Figure 2 It is the fruit mature phenotype diagram of the elite lines of the hybrid offspring of Hanfu and Saiwaihong;

[0025] Figure 3 Electrophoretogram for genotype identification of the key ethylene synthesis gene ACS1 of excellent lines in the hybrid offspring of Hanfu and Sawaihong

[0026] Figure 4 Variation diagram of fruit firmness during ambient temperature storage of excellent lines in the hybrid offspring of Hanfu and Sawaihong Specific implementation manners

[0027] To make the objectives, technical solutions and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are part rather than all of the embodiments of this application. Usually, the components of the embodiments of this application described and illustrated in the accompanying drawings here can be arranged and designed in various different configurations.

[0028] The inventors' research found that small-fruit varieties represented by Sawaihong have strong cold resistance and good fresh food quality, and the development area has expanded rapidly. However, the fruit size is on the small side (average single fruit weight is 58.3 g, and the maximum single fruit weight is 80.3 g), and there are problems such as easy softening and browning and too short shelf life, which bring many difficulties to post-harvest storage, transportation and sales, restricting its large-scale popularization and application. Therefore, although the development space and potential of cold-region apples are huge, the lack of new high-quality large-fruit cold-resistant and storage-resistant varieties is the bottleneck problem restricting the high-quality and efficient development of cold-region apples.

[0029] Based on the above findings, this application proposes a method for cultivating high-quality cold-resistant large-fruit crispy-flesh storage-resistant apples. The high-quality large-fruit late-maturing storage-resistant Hanfu (ACS1-2 / 2, storage-resistant) apples with a complex genetic basis (including excellent genes of 3 national germplasms) are selected as the male parent, and the cold-resistant mid-maturing Sawaihong (ACS1-1 / 2, not storage-resistant) is used as the female parent to construct a hybrid segregation population. Using the molecular marker patent technology developed in the early stage, rapid detection and analysis of storage resistance are carried out through experiments such as PCR amplification and agarose gel electrophoresis. Four excellent lines of high-quality cold-resistant large-fruit crispy-flesh storage-resistant apples, namely TL0307, TL0110, TL0115, and TL0279, are efficiently screened and determined from 12,000 hybrid offspring, and the ACS1 genotype is ACS1-2 / 2 (storage-resistant) for all of them.

[0030] Example 1

[0031] Refer to Figures 1 - 4, This application discloses a cultivation method for high-quality cold-resistant large-fruited crispy-fleshed and storage-resistant apples. The existing distribution area of cold-region apples is located at 40-46° north latitude. The main apple varieties planted are Hanfu, Xinping No. 1, Yueyan, Zixiang, Sairenghong, K9 (July Fresh), Longhong, Longfeng, etc. Among these varieties, the large-fruited varieties represented by Hanfu have better cold resistance than Fuji and have pushed the planting area of large apples 200 kilometers northward. However, as a late-maturing variety, Hanfu still has a significant gap in cold resistance compared with small apples such as Sairenghong. Due to the late ripening of the fruit and the late return of tree nutrients, the early frost not only affects fruit tree production, but the superposition of the dual factors of cold winter weather even endangers the safe overwintering of the tree body.

[0032] In recent years, there have been frost damages when planting Hanfu in places such as Pingquan, Hebei (41° north latitude), which has affected the industrial development, and the comprehensive quality has been difficult to meet the increasing consumption demands of people. Therefore, the planting area of Hanfu apples has shown a shrinking trend in recent years.

[0033] Small-fruited varieties represented by Sairenghong have strong cold resistance and good fresh food quality, and their planting areas have expanded rapidly. However, the fruit size is small (the average single fruit weight is 58.3 g, and the maximum single fruit weight is 80.3 g), and there are problems such as easy softening and browning and too short shelf life, which bring many difficulties to post-harvest storage, transportation and sales, restricting their large-scale popularization and application.

[0034] Therefore, although there is great development space and potential for cold-region apples, the lack of high-quality large-fruited cold-resistant and storage-resistant new varieties is the bottleneck problem restricting the high-quality and efficient development of cold-region apples.

[0035] Therefore, breeding new varieties of high-quality large-fruited cold-resistant crispy-fleshed and storage-resistant apples is the key to breaking through the high-quality development of cold-region apples. The selection of hybridization parents not only affects the size of breeding achievements, but even determines the success or failure of breeding. The selection and matching of traditional hybridization breeding parents basically rely on experience, with a large number of hybridization populations constructed and high breeding costs. With the continuous development of molecular biology, the role played by molecular markers in fruit tree breeding has become increasingly important. Developing and utilizing reliable molecular markers, through genetic background analysis and molecular marker identification, reasonably selecting hybridization parents, and achieving the goal of quickly identifying target traits by identifying the target genes of hybrid offspring can significantly improve breeding efficiency and reduce breeding costs.

[0036] Therefore, based on the previous evaluation and research of apple cold-resistant germplasm resources, through scientific selection and matching of hybridization parents, a high-quality large-fruited late-maturing storage-resistant Hanfu (ACS1-2 / 2, storage-resistant) apple with a complex genetic basis (including excellent genes from 3 national germplasms) was selected as the male parent, and the cold-resistant mid-maturing Sairenghong (ACS1-1 / 2, not storage-resistant) was used as the female parent (as Figure 1 ), and a hybridization segregation population was constructed.

[0037] The hybridization process includes: collecting the bell-shaped flowers of Hanfu apples, taking out the pollen and drying it at room temperature for later use; selecting the bell-shaped flowers of Saimanhong apples, and removing the petals, sepals, filaments and anthers together using the "continuous petal emasculation method" (pinch the bottom of the sepal with your fingernail and turn it around and lift it up); then using a cotton swab to dip the pollen of Hanfu and dot it on the stigma of Saimanhong; after the fruits mature, pick the fruits without sepals, take out the seeds, mix them with wet sand and place them in a 4°C refrigerator for stratification treatment. Wait for the seeds to germinate and plant them in nutrient pots. After there is no late spring cold snap in spring, plant them in the field for normal management.

[0038] Using the molecular marker patent technology developed in the early stage, rapid detection and analysis of storage tolerance were carried out through experiments such as PCR amplification and agarose gel electrophoresis. Four excellent apple lines with high quality, cold resistance, large fruits, crispy flesh and storage tolerance, namely TL0307, TL0110, TL0115 and TL0279, were efficiently screened and determined from 12,000 hybrid offspring. Their ACS1 genotypes are all ACS1-2 / 2 (storage tolerant) (as Figure 3 ), the fruit development period is 120-140 days, the single fruit weight is 71.8-196 g, which are all significantly higher than those of Saimanhong, and the soluble solids are 11.9-18.9%.

[0039] PCR amplification conditions: Extract the leaf DNA of the candidate lines, take 1 μL of DNA as the template, 1 μL of each upstream and downstream primer, 10 μL of PCR mix, and 7 μL of ddH2O, add them to the PCR tube and mix well.

[0040] Table 1 PCR reaction conditions

[0041]

[0042] Gel electrophoresis experiment: Prepare a 2% agarose gel. After PCR, take 10 μL of the PCR product for sample loading and electrophoresis. Use 2000 DNA marker as a reference marker, electrophoresis at 154 v for 30 min, place it in a gel imager for observation, photography and analysis, and judge the genotype according to the band position and type.

[0043] Table 2 Traits of excellent lines of the hybrid offspring of Hanfu and Saimanhong apples

[0044]

[0045] Among them, when TL0110 and TL0279 are stored at room temperature for 30 days, the fruits still remain hard and crispy (as shown in Table 2 and Figure 2)。The hardness of Saiwaihong decreased by 29% after 15 days of storage at room temperature and by 39% after 30 days. While for TL0279 stored at room temperature for 15 days, the fruit hardness hardly changed, and after 25 days of storage, the fruit hardness only decreased by 19.6%. For TL0110, TL0115, and TL0307 stored at room temperature for 30 days, the hardness only decreased by 18.5%, 14%, and 5.2% respectively, and still remained hard, brittle, and juicy (such as Figure 4 )。

[0046] Compared with the traditional evaluation method, this method has strong predictability, and there is no need to conduct storage experiments on a large number of hybrid offspring. The traits such as fruit crispness and storability can be determined at the seedling stage, and candidate excellent lines can be quickly and efficiently screened out, locking in the target for subsequent variety cultivation and approval, greatly improving the seed selection efficiency and significantly reducing the breeding cost.

[0047] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for cultivating high-quality cold-resistant big-fruited and crispy-fleshed long-storage apples, characterized in that, It includes the following steps: S1. Prepare the hybrid parent combination: Select Hanfu apples as the male parent and Saiwaihong apples as the female parent for hybridization; S2. Detect and analyze the storage tolerance of the hybrid offspring through PCR amplification and agarose gel electrophoresis experiments; S3. Screen out a total of 4 high-quality cold-resistant, large-fruited, crispy-fleshed and storage-tolerant apple elite lines, namely TL0307, TL0110, TL0115 and TL0279, according to the analysis results.

2. The cultivation method of high-quality cold-resistant large-fruited crispy-fleshed and storage-resistant apples according to claim 1, characterized in that, In the above S1, the hybridization process includes: S11. Collect the bell-shaped flowers of Hanfu apples, take out the pollen and dry it at room temperature for later use; S12. Select the bell-shaped flowers of Saiwaihong apples, and remove the petals, sepals, filaments and anthers by the "petal-removing emasculation method"; S13. Dip a cotton swab in the pollen treated in S11 and dot it on the stigma of Saiwaihong; S14. After the fruits are mature, pick the fruits without sepals, take out the seeds, mix them with wet sand, and place them in a 4°C refrigerator for stratification treatment; S15. Wait for the seeds to germinate and plant them in nutrient pots, and then plant the germinated seeds in the field.

3. The cultivation method of high-quality cold-resistant large-fruited crispy-fleshed and storage-resistant apples according to claim 2, wherein: In the above S3, the ACS1 genotypes of the 4 high-quality cold-resistant, large-fruited, crispy-fleshed and storage-tolerant apple elite lines are all ACS1-2 / 2, the fruit development period is 120 - 140 days, the single fruit weight is 71.8 - 196 g, and the soluble solids are 11.9 - 18.9%.

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