CN115623984B - Apricot plant distant hybridization high-affinity backbone parent selection method and cotyledon abortive hybrid embryo rescue method based on genome heterozygosity - Google Patents

Abstract

The invention discloses a method for selecting a distant hybridization high-affinity backbone parent of apricot plants based on genome heterozygosity and a method for saving cotyledon abortive hybrid embryos; the parent selection method comprises the following steps: selecting a target variety from apricot plants, and calculating the genome heterozygosity of the target variety; and screening apricot plant parents with genome heterozygosity of more than or equal to 1 percent. The method for saving the cotyledon abortive hybrid embryo comprises the following steps: after pollination of the high-affinity backbone parent, collecting hybrid fruits, and taking out kernels of the hybrid fruits; sterilizing the kernels of the hybrid fruits, removing seed coats, taking out hybrid embryos, and putting the hybrid embryos into a primary culture medium for culture; cross-cutting the junction of the epicotyl and the hypocotyl of the hybrid embryo, and then respectively placing the cross-cut epicotyl and the hypocotyl into a secondary culture medium for culture to obtain a propagation plant; separating the propagation plants and placing the plants in a strong seedling culture medium for culture to obtain strong hybrid seedlings; and (3) grafting the robust branches on the robust hybrid seedlings onto the robust branches of the apricot trees. The invention can improve distant hybridization affinity of apricot plants and solve the problem of abortion in the development process of hybrid embryo.

Description

Apricot plant distant hybridization high-affinity backbone parent selection method and cotyledon abortive hybrid embryo rescue method based on genome heterozygosity

Technical Field

The invention relates to the technical field of distant hybridization of apricot plants. In particular to a method for selecting a distant hybridization high-affinity backbone parent of apricot plants based on genome heterozygosity and a method for saving cotyledon abortive hybrid embryos.

Background

Apricot is a traditional fruit tree with long cultivation history in China, and is also a woody grain and oil economic tree species with great development potential. The apricot species and the breed group have unique excellent characters: common apricot fruits are big, and the pulp is sour, sweet and delicious; the kernels of the apricots and the Siberian apricots are full, the content of fatty acid and protein is high, and the quality of the kernels is good; xinjiang apricot fruits are fragrant and sweet, full in kernel, excellent in kernel and not easily affected by cold in the late flowering phase. Hybridization between different varieties and variety groups is favorable for polymerization of excellent fruit and kernel characteristics of apricots, and is an effective way for creating new varieties.

The reproductive isolation and the common hybridization incompatibility phenomenon exist among different apricot seeds and different species groups, seeds are difficult to obtain by hybridization or seeds cannot germinate, and the average setting rate among different apricot seeds and different species groups is found to be 0.71% through many years of hybridization pollination experiments, wherein 52.67% is a shrunken seed for cotyledon abortion, and the setting rate of effective hybridization seeds is only 0.38%; therefore, the selection of the high-affinity backbone parent is of great significance to the efficient breeding of apricots. At present, methods such as repeated pollination, mixed pollen pollination, advanced or delayed pollination, physiologically active substance or ray treatment and the like are often used for overcoming the incompatibility of interspecific hybridization, however, the hybrid seed setting rate obtained by the traditional method is low, the phenomenon of serious embryo abortion exists, and effective seeds are difficult to obtain.

The hybrid embryo between species and species group usually develops about 50 days after flower filling, but the seed leaf stage of endosperm transformation is easy to generate the phenomena of abortive fruit drop, and even if a few abortive hybrid fruits develop and mature, the seeds are shrunken and lose vigor, so that the number of effective hybrid seeds is very small. The cotyledon abortive embryo rescue technology can timely carry out in vitro culture on mature hybrid embryos to obtain propagation-expanding plants, effectively improves the hybridization efficiency among apricot plant species and species groups, and provides technical support for the creation of apricot excellent specific species.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a method for selecting high-affinity backbone parents between apricot plant species and product populations based on genome heterozygosity, which can effectively improve distant hybridization affinity of apricot plants, and a method for saving cotyledon abortive hybrid embryos, so as to solve the abortive problem in the development process of hybrid embryos, greatly improve distant hybridization efficiency between apricot plant species and product populations, and serve the creation of excellent specific new varieties of apricot plants.

In order to solve the technical problems, the invention provides the following technical scheme:

the method for selecting the distant hybridization high-affinity backbone parent of the apricot plant based on the genome heterozygosity comprises the steps of selecting a target variety with excellent characters from the apricot plant; calculating the genome heterozygosity of the target variety; and screening apricot plant parents with genome heterozygosity greater than or equal to 1% from the target variety to obtain the distant hybridization high-affinity backbone parent of the apricot plant. The genome heterozygosity of the general diploid plant is between 0.5 and 1.2 percent, the heterozygosity of the high heterozygosity genome of the apricot is more than 1 percent, and the distant hybridization affinity of the apricot plant can be effectively improved by selecting the high heterozygosity varieties to carry out hybridization pollination among the varieties.

The method for selecting the distant hybridization high-affinity backbone parent of the apricot plant based on the genome heterozygosity comprises the following steps of:

(1) Selecting young leaves of a target variety for DNA extraction; the extraction method of the DNA in the invention is a conventional method, such as CTAB method and the like;

(2) Performing high-throughput sequencing (High Throughput Sequencing, HTS) on the extracted DNA to obtain nucleotide sequence data, and performing quality control to obtain a sequence file; the quality control method mainly comprises removing the adaptor sequence of reads; cutting off inaccurate bases sequenced at two ends of reads; directly intercepting 5 bases at the left end and 5 bases at the right end; removing reads containing N; discarding a pair of reads corresponding to a read when more than 20% of the base mass fraction in the reads is less than 20;

(3) K-mer counting and statistics are carried out on the sequence file by utilizing KMC software, then, matching analysis is carried out on K-mer data by utilizing Genome Scope software through a binomial model (negative binomial model), and Genome heterozygosity is calculated.

The apricot plant distant hybridization high-affinity backbone parent selection method based on genome heterozygosity comprises the following steps of ' trunk on tree ' apricot, ' small white apricot ', ' pearl oil apricot ', ' golden sun ' apricot, ' kernel No. 5' apricot, ' Siberian apricot ' F106', [ Siberian apricot ' F106', see Chen, C., liu, H., gou, N.et al. AprGPD: the apricot genomic and phenotypic database.plant Methods 17,98 (2021) ], excellent ' apricot and Dragon cap ' apricot.

The method for rescuing the cotyledon aborted hybrid embryo comprises the following steps:

step A: collecting hybrid fruits after distant hybridization of apricot plants and pollination of high-affinity backbone parents, and taking out kernels of the hybrid fruits; the apricot plant distant hybridization high-affinity backbone parent is obtained by screening by using the apricot plant distant hybridization high-affinity backbone parent selection method based on genome heterozygosity;

and (B) step (B): sterilizing the kernels of the hybrid fruits, removing seed coats of the kernels by forceps and a surgical knife, peeling off cotyledons, taking out hybrid embryos, and putting the hybrid embryos into a primary culture medium for culture;

step C: respectively culturing two parts obtained after cross cutting of the junction of the epicotyl and the hypocotyl of the hybrid embryo in a secondary culture medium to obtain a propagation plant; the survival rate of the primary culture after the hybrid embryo separation is higher (92.6%), while the survival rate of the whole seed or the decorticated cotyledon culture is low (only 9.1% and 26.1%). After primary culture of hybrid embryo, the young seedling must be transversely cut into two parts along the junction of upper embryo axis and lower embryo axis, and respectively undergo next subculture to make normal differentiation and proliferation, and can not be proliferated and differentiated without transverse cutting, transverse cutting position or other cutting modes, so that the goal of proliferation can not be reached.

Step D: separating the propagation plants and placing the propagation plants in a strong seedling culture medium for culture to obtain strong hybrid seedlings;

step E: and (3) grafting the robust branches on the robust hybrid seedlings onto the robust branches of the apricot trees, thereby completing the rescue of hybrid embryos. Can bloom and fruit after grafting for 2-3 years, and greatly shortens the breeding period.

In the method for rescuing the cotyledon aborted hybrid embryo, in the step A, the time for collecting the hybrid fruits is 50-60 days after pollination.

In the step B, the disinfection treatment method comprises the following steps: sterilizing with 70% alcohol for 30 seconds, and flushing with sterile water for 3-5 times; then sterilizing for 30 minutes by using sodium hypochlorite solution with the mass concentration of 1wt%, and then flushing for 3-5 times by using sterile water. The alcohol sterilizing capability is strong, but the penetrating power is extremely strong, and the embryo can be killed when the sterilizing time is too long, so that the sterilizing time is not longer than 30 seconds; the sodium hypochlorite disinfectant is efficient, high-speed, wide-area, nontoxic, harmless and free of residue pollution, can be rapidly decomposed after disinfection, and has no harm to the environment. The single use of a disinfectant can not thoroughly sterilize, and the combination of the disinfectant and the disinfectant can effectively disinfect seeds and ensure the activity of embryos.

In step B, the primary culture medium is used for improving MS (1/2 NH) ₄ NO ₃ ) The culture medium is a basic culture medium, the mass concentration of 6-benzylaminopurine in the primary culture medium is 0.5mg/L, the mass concentration of naphthylacetic acid is 0.5mg/L, the mass concentration of agar is 6g/L, and the mass concentration of sucrose is 30g/L; the pH of the primary culture medium was 5.8; the culture time of the primary culture is 7 days; in the primary culture, the illumination condition is set to 2500LX, and the illumination period is 16h/d.

In step C, the medium is replaced with a modified MS (1/2 NH) ₄ NO ₃ ) The culture medium is a basic culture medium, the mass concentration of 6-benzylaminopurine in the secondary culture medium is 0.5mg/L, the mass concentration of dichlorophenoxyacetic acid is 0.5mg/L, the mass concentration of agar is 6g/L, and the mass concentration of sucrose is 30g/L; the pH of the secondary culture medium is 5.8; the culture time of the subculture is 20 days; in the secondary culture, the illumination condition is set to 1500LX, and the illumination period is 8h/d.

Modified MS (1/2 NH) used in the present invention ₄ NO ₃ ) Trade name of the MediumCalled MS medium salts (1/2 NH) ₄ NO ₃ Vitamin-containing), model: PM1271, specification 2X 250g, manufacturer: produced by Beijing cool Lai Bo technology Co., ltd.

In the method for rescuing the cotyledon aborted hybrid embryo, in the step D, the seedling strengthening culture medium is used for improving MS (1/2 NH) ₄ NO ₃ ) The culture medium is a basic culture medium, the mass concentration of the zeatin in the strong seedling culture medium is 1.0mg/L, the mass concentration of the indoleacetic acid is 0.1mg/L, the mass concentration of the agar is 6g/L, and the mass concentration of the sucrose is 30g/L; the pH of the seedling strengthening culture medium is 5.8; the culture time of strong seedling culture is 30 days; when strong seedlings are cultivated, the illumination condition is set to 1500LX, and the illumination period is 8h/d.

In the step E, the robust branches with the diameter larger than or equal to 0.5cm on the robust hybrid seedlings are grafted to the robust branches with the diameter of 0.8-1.0 cm on the healthy apricot trees.

The technical scheme of the invention has the following beneficial technical effects:

the invention uses the variety with high heterozygosity genome to carry out parent configuration, thereby increasing the fusion of genes and improving the hybridization affinity between varieties. The distant hybridization high-affinity backbone parent of the apricot plant obtained by screening by the method can effectively improve the hybridization affinity, and the hybrid seedling is obtained by embryo rescue technology, so that the apricot plant is grafted in a high position on a big tree in the current year, blooms and fruit in advance, and the creation process of a new excellent specific variety of the apricot plant is greatly promoted.

The invention selects the high heterozygosity parent combination to carry out the hybridization between apricot seeds and breed clusters, the average fruit setting rate reaches 7.03 percent (3.07-11.07 percent), and compared with the average fruit setting rate of 0.13 percent (0.00-0.25 percent) of two low heterozygosity parents, the invention improves 53 times, and remarkably improves the hybridization fruit setting rate between breed clusters.

After pollination, about 50d, the hybrid kernels are easy to generate abortion phenomenon in the cotyledon filling stage, so that the hybrid fruits fall off in advance, and even if few of the abortion hybrid fruits develop and mature, the seeds are shrunken and lose vigor, and the acquisition of healthy hybrid seeds is seriously affected. The invention uses cotyledon abortive embryo rescue method to culture in vitro mature hybrid embryo of about 50d, the survival rate of primary culture reaches over 90%, the increment coefficient of secondary culture is 3-6, the propagation expansion seedling reaches semi-lignification degree after strong seedling culture, the seedling grows robustly after grafting, a small amount of flower buds can differentiate in the same year, the result can be obtained in 2-3 years, and the crossbreeding process is greatly shortened.

Drawings

FIG. 1 is a schematic diagram of the calculation result of the genome heterozygosity of the excellent apricot (heterozygosity is 1.32%);

FIG. 2 is a graph showing the result of calculation of the genome heterozygosity of kernel No. 5' apricot (heterozygosity of 0.93%);

FIG. 3 is a graph showing the result of calculation of the genome heterozygosity of pearl oil apricot (heterozygosity: 1.20%);

FIG. 4 is a schematic diagram showing the result of calculation of the genome heterozygosity of small white apricot (heterozygosity: 1.30%);

FIG. 5 is a schematic diagram showing the result of calculation of genome heterozygosity of the trunk apricot on the tree (heterozygosity is 1.24%);

FIG. 6 is a graph showing the result of calculation of the genome heterozygosity of golden sun apricot (heterozygosity: 1.06%);

FIG. 7 is a schematic representation of the calculation of the genomic heterozygosity of Siberian apricot ('F106') (heterozygosity 0.92%);

FIG. 8 is a schematic diagram of the calculation result of the genome heterozygosity of the Dragon cap apricot (heterozygosity: 1.36%);

FIG. 9 is a graph comparing normal fruit (left) with aborted fruit (right) at 50d after pollination;

FIG. 10 is a graph showing a comparison of normal fruit (left) and abortive fruit (right) cut at 50d after pollination;

FIG. 11 is a graph showing the kernel comparison of normal fruit (left) and aborted fruit (right) at 50d after pollination;

FIG. 12 is a graph comparing normal fruit (left) with aborted fruit (right) at maturity;

FIG. 13 is a graph showing a comparison of normal fruit (left) and abortive fruit (right) after seed kernels are dissected at maturity;

FIG. 14 is a graph showing the kernel comparison of normal fruit (left) and abortive fruit (right) when ripened;

FIG. 15 is a flowchart of hybrid embryo rescue and post-emergence grafting.

Detailed Description

(one) high-affinity backbone parent selection method

In this example, the selection method of the distant hybridization high-affinity backbone parent of the apricot plant is as follows: selecting a target variety with excellent characters from apricot plants; calculating the genome heterozygosity of the target variety; and screening apricot plant parents with genome heterozygosity greater than or equal to 1% from the target variety to obtain the distant hybridization high-affinity backbone parent of the apricot plant.

In this example, for selection of high affinity backbone parents, 8 varieties of Xinjiang cultivated apricots ('trunk on tree', 'small white apricots', 'pearl oil apricots'), ordinary apricots ('golden sun'), mountain apricots ('kernel No. 5'), siberian apricots ('F106'), see Chen, c, liu, h, gou, n.et al aprgpd: the apricot genomic and phenotypic database.plant Methods 17,98 (2021), kernel apricots ('you' and 'dragon cap') were heterozygous detected and high heterozygous parents were selected for hybridization.

The Xinjiang cultivated apricots, the common apricots and the mountain apricots in the implementation belong to different geographical ecological groups (variety groups) of the common apricots (Prunus armeniaca L.), large character differences exist among the variety groups, wherein 'trunk on trees' belongs to the sub-group of the quaigher-illite ecological groups, and the fruits are fragrant and sweet, the nuts are excellent, and the cold resistance is strong; 'small white apricot', 'pearl oil apricot' belongs to a sub-population of the medium sub-fine ecological group kukuku vehicle, the pulp has high sugar content, and the kernel is sweet and the kernel is excellent; 'Jinsun' belongs to European ecological group, the fruits are extremely early ripe, and the pulp is sweet and sour; "middle kernel No. 5" is a common variety (Prunus armeniaca var. Ansu maxim.) belonging to the North China sub-population of the North China ecological group, high yield, full kernel and high fatty acid and protein content. Siberian apricot (Prunus sibirica L.) has the characteristics of full kernel and thin shell, and is an important bitter almond resource. The kernel apricot variety 'you Yi' and 'Longwang cap' are natural interspecific varieties (Prunus armeniaca×prunus sibirica) of Siberian apricot and common apricot, and the kernel is big and sweet, and is an important sweet almond resource. Hybridization between different species and variety groups is beneficial to the polymerization of excellent fruit and kernel characteristics of apricots.

The method for calculating the genome heterozygosity of the target variety comprises the following steps:

(1) Respectively selecting young leaves of apricots of different varieties to extract DNA; the extraction method of the DNA in the embodiment is a CTAB method;

(2) High-throughput sequencing is carried out on the extracted DNA, and a sequence file is obtained after the quality control of the obtained nucleotide sequence data; the quality control method mainly comprises removing the adaptor sequence of reads; cutting off inaccurate bases sequenced at two ends of reads; directly intercepting 5 bases at the left end and 5 bases at the right end; removing reads containing N; discarding a pair of reads corresponding to a read when more than 20% of the base mass fraction in the reads is less than 20;

(3) And (3) counting and counting K-mers by utilizing KMC software, and then carrying out fitting analysis on the K-mer data by utilizing Genome Scope software through a negative binomial model, so as to obtain Genome heterozygosity by calculation. The results of the calculation of the heterozygosity of the genome in this example are shown in FIGS. 1 to 8.

According to the result of calculating genome heterozygosity, three types of hybridization combinations of high heterozygosity and high heterozygosity, high heterozygosity and low heterozygosity, low heterozygosity and low heterozygosity are set for distant hybridization among product populations, and 16 hybridization combinations are set (see table 1). And counting the fruit setting rate according to the number of the finally obtained hybrid fruits, sowing the hybrid fruits, counting the seedling emergence rate, and counting the healthy seed setting rate according to the number of the seedling emergence seeds.

Method for saving hybrid embryo of cotyledon abortive

As shown in fig. 9-14, the hybrid fruits between apricot populations are usually in cotyledon filling stage (around 50), embryo has developed and matured, however, cotyledon filling is problematic, resulting in massive hybrid fruit abortion and abscission, very few fruits develop to maturity, and seeds are also in a shrunken state. Thus, 50d after flowers are filled is a key period for embryo rescue.

The method for rescuing the sub-leaf abortive hybrid embryo in the embodiment comprises the following steps:

step A: after distant hybridization of apricot plants and pollination of high-affinity backbone parents for 50-60 d, collecting hybridized fruits, and taking out kernels of the hybridized fruits; the apricot plant distant hybridization high-affinity backbone parent is obtained by screening by using the apricot plant distant hybridization high-affinity backbone parent selection method based on genome heterozygosity;

and (B) step (B): collecting hybrid fruits after pollination, taking out kernels, sterilizing for 30s by using 70% alcohol, and flushing for 3-5 times by using sterile water; then sterilizing for 30min by using sodium hypochlorite solution with the mass concentration of 1wt% and washing for 3-5 times by using sterile water. Screening an inoculation mode: the three modes of peeling the whole seeds, peeling the cotyledon seeds and separating embryo are respectively put into a primary culture medium, and the results show that the survival rates of the three modes are respectively 9.1 percent, 26.1 percent and 92.6 percent, so that the embryo separation is the optimal inoculation culture mode;

three basic culture mediums are tested in the embodiment, gradients are set according to plant hormones, pH and illumination intensity, and the optimal culture medium and culture condition for apricot hybrid embryo growth are screened. The settings were as follows: minimal medium was set to WPM, MS, modified MS (1/2 NH) ₄ NO ₃ ) The method comprises the steps of carrying out a first treatment on the surface of the 6-benzylaminopurine (6-BA) set concentration 0.25mg/L, 0.5mg/L, 1mg/L; naphthalene Acetic Acid (NAA) set concentration 0.2mg/L, 0.5mg/L, 0.8mg/L; setting the concentration of the dichlorophenoxyacetic acid (2, 4-D) to be 0.2mg/L, 0.5mg/L and 0.8mg/L; the concentration of zeatin is set to 0.5mg/L, 1.0mg/L and 2.0mg/L; the concentration of the indoleacetic acid (IAA) is set to 0.05mg/L, 0.1mg/L and 0.15mg/L; illumination combinations 1000LX, 1500LX, 2000LX; the pH value is set to be 5.5, 5.8 and 6.0; finally, 30g/L of sucrose and 6g/L of agar are added into each formula for test. The final optimal medium was determined as follows, primary medium: comprising an improved MS (1/2 NH ₄ NO ₃ ) 6-BA 0.5mg/L, NAA 0.5mg/L,6g/L agar, 30g/L sucrose, pH 5.8, 2500LX, light cycle 16h/d. Subculture medium: including modified MS (1/2 NH) ₄ NO ₃ ) 6-BA 0.5mg/L,2, 4-D0.5 mg/L,6g/L agar, 30g/L sucrose, pH 5.8, 1500LX, 12h/D light cycle. Seedling strengthening culture medium: comprising 1/2MS (1/2 NH ₄ NO ₃ ) 1.0mg/L zeatin, 0.1mg/L IAA,6g/L agar, 30g/L sucrose, pH 58, 1500LX, 12h/d of illumination period. Modified MS (1/2 NH) used in this example ₄ NO ₃ ) The trade name of the medium is MS medium salt (1/2 NH) ₄ NO ₃ Vitamin-containing), model: PM1271, specification 2X 250g, manufacturer: produced by Beijing cool Lai Bo technology Co., ltd.

Step C: respectively culturing two parts obtained after cross cutting of the junction of the epicotyl and the hypocotyl of the hybrid embryo in a secondary culture medium to obtain a propagation plant;

step D: separating the propagation plants and placing the propagation plants in a strong seedling culture medium for culture to obtain strong hybrid seedlings;

step E: and (3) grafting the robust branches with the diameter larger than or equal to 0.5cm on the robust hybrid seedlings to the robust branches with the diameter of 0.8-1.0 cm on the apricot trees for high grafting, thus completing the rescue of hybrid embryos. Can bloom and fruit after grafting for 2-3 years, and greatly shortens the breeding period.

When the concentration of 6-BA is too low, the growth promoting effect on the tissue culture seedlings is small, when the concentration is too high, branches are too large in propagation, and the vitrification rate of the branches is high; when the NAA concentration is too low, the tissue culture seedlings grow slowly, and when the NAA concentration is too high, the branches are easy to brown; when the concentration of 2,4-D is too low, the growth of the secondary seedlings is weak, and when the concentration is too high, the proliferation coefficient is reduced; the concentration of the zeatin is too low, which is not beneficial to lignification, the seedling strengthening effect can not be achieved, and the deformity rate of branches with too high concentration is increased; too low IAA concentration does not have a strong seedling effect, and too high IAA concentration inhibits growth; the solidification process of the culture medium is too low when the pH value is 5.5, and the solidification degree of the culture medium is too high when the pH value is 6.0, which is not beneficial to the growth of tissue culture seedlings; too strong illumination is unfavorable for radial growth of seedlings.

(III) hybrid seedling grafting and management

Selecting branches with the length of more than 0.5cm of the hybrid tissue culture seedling as scions, grafting in the middle and late ten days of 6 months at the temperature of about 20-25 ℃, and grafting in a square bud grafting mode in sunny windless weather. Cutting square bud pieces with the length of 2-3 cm and the width of 0.5-1 cm on the scion, cutting a epidermis with the size similar to that of the bud pieces on a branch with the thickness of 0.8-1.0 cm of the stock, and cutting a diversion trench with the size of about 1cm on the lower part of the incision; putting the bud slice into the incision, keeping one side and the lower part of the slice tightly attached to the cambium of the stock, and wrapping and sealing with a film to expose the bud. The water of the stock is thoroughly poured once a week before grafting, so that the sufficient moisture of the stock is ensured, the bark is easy to separate, and the healing of the grafting opening is facilitated. The grafting mouth heals well (about 7 days), after the grafting buds sprout, the grafting is cut at a position 1cm away from the top end of the grafting mouth, and the binding film is removed. After the grafting buds grow robustly (about 20 days), the redundant buds on the stock are erased, and trace element foliar fertilizer is sprayed according to the situation so as to ensure the growth of the grafted buds.

(IV) implementation effects

Based on the parent heterozygosity, 16 hybrid combinations among the species groups of the types of high heterozygosity and high heterozygosity, high heterozygosity and low heterozygosity, low heterozygosity and low heterozygosity are respectively set, and the test is carried out in the institute of forestry science and forestry research Meng Zhouji. The hybridization results are shown in Table 1, the fruit setting rate of hybridization of two high heterozygosity parents is 3.07-11.07%, and the average fruit setting rate is 7.03%; the hybridization fruit setting rate of the high heterozygosity and low heterozygosity parents is 1.18-2.50 percent, and the average 1.93 percent; the fruit setting rate of hybridization between two parents with low heterozygosity is 0.00-0.25%, and the average fruit setting rate is 0.13%. It can be seen that the high heterozygosity helps to increase the cross-compatibility and fruit setting rate between clusters. However, the hybrid fruits fall off in advance or the seeds are shrunken due to the cotyledon abortion, the healthy seeds are few, the healthy seed setting rate of the hybridization of two high heterozygosity parents is 0.00-2.79%, and the average fruit setting rate is 0.87%; the fruit setting rate of the high heterozygosity and low heterozygosity parent healthy seeds is 0.00-0.50 percent, and the average fruit setting rate is 0.15 percent; healthy seed setting rates for crosses between two low heterozygosity parents were 0.00%. It can be seen that the premature abscission of hybrid fruits or the shrunken kernel caused by the cotyledon abortion seriously affects the acquisition of healthy hybrid seeds.

TABLE 1 hybridization parent configuration and fruit setting rate between apricot variety groups based on heterozygosity

In the key period of cotyledon abortion (50 d after flower filling), the seeds of the hybridization of Yi-Jinsun are collected in time for embryo rescue. Sterilizing the hybrid kernels for 30s by using 70% alcohol with the volume fraction, flushing for 3-5 times by using sterile water, sterilizing for 30min by using sodium hypochlorite solution with the mass concentration of 1wt%, flushing for 3-5 times by using sterile water, removing seed coats by using forceps and a surgical knife, taking out embryos, placing the embryos into a primary culture medium for culturing for 7d, transecting the junction of an epicotyl and a hypocotyl for respectively carrying out secondary culture for 20d, separating the amplified plants, and placing the plants into a strong seedling culture medium for culturing for 30d to obtain robust hybrid seedlings. At the end of 6 months, selecting branches with the length of more than Miao Zhijing 0.5.5 cm as scions, grafting the scions onto branches with the length of 0.8-1 cm of the strong mountain apricot stock by adopting a square bud grafting mode, cutting off the stock, wiping buds, and irrigating in time to obtain strong plants, wherein the technical process is shown in figure 15. The cotyledon abortive embryo rescue and grafting technology is applied to rescue hybrid seeds in time, and the hybrid seeds are rapidly grafted into seedlings, so that the seedlings can bloom and bear fruits in two years, and the breeding period is effectively shortened.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While the obvious variations or modifications which are extended therefrom remain within the scope of the claims of this patent application.

Claims (5)

1. The method for selecting the distant hybridization high-affinity backbone parent of the apricot plant based on the genome heterozygosity is characterized in that a target variety with excellent properties is selected from the apricot plant; calculating the genome heterozygosity of the target variety; screening apricot plant parents with genome heterozygosity of more than or equal to 1% from target varieties, namely, distant hybridization high-affinity backbone parents of the apricot plants;

the method for calculating the genome heterozygosity of the target variety comprises the following steps:

(1) Selecting young leaves of a target variety for DNA extraction; the target varieties include 'tree trunk' apricots, 'small white apricots', 'pearl oil apricots', 'golden sun' apricots, 'excellent one' apricots and 'dragon king cap' apricots;

(2) High-throughput sequencing is carried out on the extracted DNA to obtain nucleotide sequence data, and a sequence file is obtained after quality control;

(3) And (3) counting and counting K-mers by utilizing KMC software, and then carrying out fitting analysis on the K-mer data by utilizing Genome Scope software through a negative binomial model, so as to obtain Genome heterozygosity by calculation.

2. The method for rescuing the cotyledon aborted hybrid embryo is characterized by comprising the following steps:

step A: collecting hybrid fruits after distant hybridization of apricot plants and pollination of high-affinity backbone parents, and taking out kernels of the hybrid fruits; the apricot plant distant hybridization high-affinity backbone parent is obtained by screening by the method for selecting the apricot plant distant hybridization high-affinity backbone parent based on genome heterozygosity according to the claim 1;

and (B) step (B): sterilizing the kernels of the hybrid fruits, removing seed coats of the kernels, peeling cotyledons, taking out hybrid embryos, and putting the hybrid embryos into a primary culture medium for culture; the primary culture medium takes an MS culture medium with halved ammonium nitrate as a basic culture medium, wherein the mass concentration of 6-benzyl aminopurine in the primary culture medium is 0.5mg/L, the mass concentration of naphthylacetic acid is 0.5mg/L, the mass concentration of agar is 6g/L, and the mass concentration of sucrose is 30g/L; the pH of the primary culture medium was 5.8; the culture time of the primary culture is 7 days; in primary culture, the illumination condition is set to 2500LX, and the illumination period is 16h/d;

step C: respectively placing two parts obtained after cross cutting of the junction of the epicotyl and the hypocotyl of the hybrid embryo into a secondary culture medium for culture to obtain a propagation plant; the secondary culture medium takes an MS culture medium with halved ammonium nitrate as a basic culture medium, wherein the mass concentration of 6-benzylaminopurine in the secondary culture medium is 0.5mg/L, the mass concentration of dichlorphenoxyacetic acid is 0.5mg/L, the mass concentration of agar is 6g/L, and the mass concentration of sucrose is 30g/L; the pH of the secondary culture medium is 5.8; the culture time of the subculture is 20 days; during subculture, the illumination condition is set to 1500LX, and the illumination period is 12h/d;

step D: separating the propagation plants and placing the plants in a strong seedling culture medium for culture to obtain strong hybrid seedlings; the seedling strengthening culture medium takes an MS culture medium with halved ammonium nitrate as a basic culture medium, wherein the mass concentration of the zeatin in the seedling strengthening culture medium is 1.0mg/L, the mass concentration of the indoleacetic acid is 0.1mg/L, the mass concentration of agar is 6g/L, and the mass concentration of sucrose is 30g/L; the pH of the seedling strengthening culture medium is 5.8; the culture time of strong seedling culture is 30 days; when strong seedlings are cultivated, the illumination condition is set to 1500LX, and the illumination period is 12h/d;

step E: and (3) grafting the robust branches on the robust hybrid seedlings onto the robust branches of the apricot trees, thereby completing the rescue of hybrid embryos.

3. The method of claim 2, wherein the time for collecting hybrid fruits is 50 to 60 days after pollination.

4. The method for rescuing the cotyledon aborted hybrid embryo according to claim 2, wherein in step B, the sterilization treatment method is as follows: sterilizing with 70% alcohol for 30 seconds, and flushing with sterile water for 3-5 times; then sterilizing for 30 minutes by using sodium hypochlorite solution with the mass concentration of 1wt%, and then flushing for 3-5 times by using sterile water.

5. The method for rescuing the cotyledon aborted hybrid embryo according to claim 2, wherein in step E, the robust shoots with the diameter of 0.5cm or more on the robust hybrid seedlings are grafted onto the robust shoots with the diameter of 0.8-1.0 cm on the robust apricot trees.

Images