A high-yield molecular marker and breeding method combining common oil-tea camellia fruit traits

Abstract

The present invention provides a high-yield molecular marker and breeding method combined with common tea oil fruit traits. It belongs to the field of gene breeding technology. The present invention has developed three high-yield molecular markers, ISSR815, ISSR835 and ISSR858, which are closely linked to the common tea oil hybrid fruit traits, and obtained four gene loci, 815-11, 835-12, 858-2 and 858-3. Combined with the two traits of fruit height and seed fresh weight of hybrid fruits, high-yield and excellent families and plants of common tea oil were selected from 19 hybrid families in natural populations. The selection efficiency reached 75% and the accuracy rate was 100%. According to the method, high-yield families of common tea oil were obtained, with high yield and an average unit crown width yield of 2.86kg / m 2 , much higher than the average unit crown width yield of 1.2kg / m 2 In each hybrid generation, high-yield varieties can be successfully screened out within 2 years, which greatly shortens the breeding cycle compared to the existing technology of more than ten to twenty years, and solves the problems of long cycle, poor stability and low efficiency of existing oil tea hybrid breeding.

Description

Technical Field

[0001] The invention relates to the technical field of gene breeding, and more specifically to a high-yield molecular marker and breeding method combining common oil-tea camellia fruit traits. Background Art

[0002] Camellia oleifera Camellia oleifera Abel.) is a woody oil-bearing tree species unique to my country and an important woody edible oil-bearing species in southern my country. Camellia oleifera is mainly planted in the southern subtropical region and widely distributed in the southern hilly areas. The cultivated area is large, among which the common camellia oleifera has the largest planting area. At present, the yield of camellia oleifera forest is relatively low, which is in great contradiction with the rapidly growing market demand for camellia oil, becoming one of the main difficulties faced by the development of the camellia oleifera industry. Therefore, cultivating new varieties of high-yield camellia oleifera is the key to the steady development of the camellia oleifera industry.

[0003] Quantitative traits show continuous variation, are controlled by micro-effect polygenes, and are easily affected by the environment. Traditional breeding technology is mainly aimed at phenotypic selection, and the results of selection and identification are easily affected by the environment and produce deviations. Yield is a quantitative trait. In order to improve the accuracy of selection, it is necessary to plant and select for many years. For woody plants, it takes more than ten to twenty years. With the development of molecular marker technology, molecular marker-assisted selection (MAS) provides an effective means for the efficient selection of quantitative traits. After discovering the beneficial alleles of candidate genes with significant phenotypic effects through association analysis, allele-specific PCR primers are designed, markers are developed, and they are verified in germplasm resources with a broad genetic basis for use in MAS under a broad genetic background, thereby improving the efficiency of selection. Using functional markers for MAS, on the one hand, the gene itself can be selected to ensure the accuracy of selection and improve the efficiency of selection. On the other hand, using association analysis, specific functional domains that affect trait variation can be selected to ensure the effect of selection.

[0004] In genetic breeding, fruit-related phenotypic evaluation needs to be carried out after hybrid seedlings are planted and bloom and bear fruit. There are problems such as long identification cycle, occupation of land resources and high labor costs, which cannot meet the needs of rapid and accurate selection according to breeding goals. Molecular marker-assisted selection (MAS) technology is an emerging technology that uses molecular markers closely linked to target genes to screen dominant genotypes and obtain target individuals. It can improve the accuracy of selection, shorten the breeding period, and improve breeding efficiency. ISSR (Inter-Simple Sequence Repeats) is a dominant marker with simple operation, simple primer design, good polymorphism, high repeatability, low experimental cost, and high experimental stability. It is a good DNA fingerprint marker.

[0005] However, at present, molecular marker technology is mainly used in the fields of genetic diversity analysis, variety identification, kinship analysis and hybrid identification of tea oil plants. Some molecular markers related to tea oil quality have been developed, but molecular markers closely linked to yield have not yet been developed, and there are no research reports on the application of developed molecular markers in hybrid breeding.

[0006] Therefore, how to develop a molecular marker and breeding method that is closely linked to the yield of common tea oil is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0007] In view of this, the present invention provides a high-yield molecular marker and breeding method combined with the fruit traits of common oil-tea camellia. Three high-yield molecular markers, ISSR815, ISSR835 and ISSR858, which are closely linked to the hybrid seed traits of common oil-tea camellia, were developed, and four gene loci, 815-11, 835-12, 858-2 and 858-3, were obtained. Combined with the two traits of fruit height and seed fresh weight of the F1 generation and backcross progeny, high-yield plants of common oil-tea camellia were screened in the backcross progeny. This technology was used to select high-yield excellent families and high-yield individual plants, with a selection efficiency of 75% and a selection accuracy of 100%. High-yield individual plants can be cultivated into high-yield new varieties, and can also be directly used as hybrid and backcross parents, solving the problems of long cycle, poor stability and low efficiency of existing oil-tea camellia hybrid breeding.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A high-yield molecular marker combined with common oil-tea camellia fruit traits, comprising tightly linked molecular markers ISSR815, ISSR835 and / or ISSR858; the molecular marker ISSR815 comprises a 815-11 gene locus; the molecular marker ISSR835 comprises a 835-12 gene locus; the molecular marker comprises two gene loci, 858-2 and 858-3; the primer sequence for amplifying the molecular marker ISSR815 is 5'-CTCTCTCTCCTCCTCTG-3', SEQ ID NO: 1; the primer sequence for amplifying the molecular marker ISSR835 is 5'- AgA gAg AgA gAg AgA gYC-3', SEQ ID NO: 2; the primer sequence for amplifying the molecular marker ISSR858 is 5'- TGT GTG TGT GTG TGT GRT-3', SEQ ID NO: 3.

[0010] Preferably, the gene locus 815-11 of the molecular marker ISSR815 is extremely significantly correlated with the dry weight of the kernel; the gene locus 835-12 of the molecular marker ISSR835 is extremely significantly correlated with the seed fresh weight and the single plant yield; the gene locus 858-2 of the molecular marker ISSR858 is extremely significantly correlated with the fruit height, and the gene locus 858-3 is extremely significantly correlated with the seed fresh weight.

[0011] Preferably, the yield per plant of common tea oil trees is extremely significantly positively correlated with fruit height and seed fresh weight.

[0012] Preferably, the gene loci 835-12, 858-2 and 858-3 are high-yield dominant genes; the gene loci 815-11 is a high-yield recessive gene; the gene loci 835-12 is paternally inherited, and the gene loci 858-2 and 858-3 are maternally inherited.

[0013] Another object of the present invention is to provide the application of the above-mentioned high-yield molecular marker combined with common oil-tea camellia fruit traits in the high-yield breeding of common oil-tea camellia.

[0014] Another object of the present invention is to provide a method for high-yield breeding of common camellia oleifera, comprising the following steps:

[0015] (1) Parent selection: Select parents with a peak production period of more than 10 years and a unit crown area yield of more than 1.2 kg / m 2 The oil-tea tree was used as the candidate parent, and the molecular markers ISSR815, ISSR835 and ISSR858 were used to assist in screening the parents, and the high-yield and superior strains showing one or more gene loci of 835-12, 858-2 or 858-3 were selected as the parents;

[0016] (2) Parent configuration and hybridization: select the high-yield superior strain with 858-2 and / or 858-3 gene loci in step (1) as the female parent, and the high-yield superior strain with 835-12 gene loci as the male parent, and hybridize them to obtain the F1 generation;

[0017] (3) Backcrossing: Select the high-yield superior plant of the F1 generation as the female parent, select the male parent with the 835-12 gene locus in step (2) as the backcross male parent for backcrossing, select the high-yield superior plant in the backcross progeny as the recurrent female parent, and backcross it with the male parent with the 835-12 gene locus in step (2) for multiple times to obtain backcross progeny;

[0018] (4) Screening of high-yield and superior plants in backcross progeny: Based on the fruit traits and genotypes of ordinary Camellia oleifera backcross progeny, high-yield and superior plants in hybrid progeny were screened from the backcross progeny population, as follows:

[0019] ① Screening excellent families based on phenotype: In the fruit population of backcross progeny, families with fruit height higher than the population average, or seed fresh weight higher than the population average, are selected as excellent families;

[0020] ② Select superior individual plants in superior families by combining phenotypes: Based on superior families and combining the fruit traits of backcross progeny, select superior seeds with high fruit height and high seed fresh weight, and cultivate them into seedlings;

[0021] ③ Using molecular markers, select the excellent individual plants expressing the 815-11 gene locus and at least one of 835-12, 858-2 or 858-3 from the seedlings cultivated in step ② using molecular markers ISSR815, ISSR835 and ISSR858, which are common high-yield varieties of tea oil.

[0022] Preferably, in the high-yield superior plant screening process of steps (1) and (4), when a single band of 1600 bp is amplified, it means that the tested plant has the 815-11 gene locus; when a single band of 850 bp is amplified, it means that the tested plant has the 835-12 gene locus; when a single band of 300 bp is amplified, it means that the tested plant has the 858-2 gene locus; when a single band of 350 bp is amplified, it means that the tested plant has the 858-3 gene locus.

[0023] Preferably, the total number of backcrossings in step (3) is ≥ 2 times.

[0024] Preferably, the screening process of the F1 generation high-yield superior plants in step (3) is the same as the screening process of the backcross generation high-yield superior plants in step (4).

[0025] Another object of the present invention is to provide an amplification primer set combined with a high-yield molecular marker of common oil-tea tea fruit traits, including amplification primers for molecular markers ISSR815, ISSR835 and ISSR858, the primer sequence for amplifying the molecular marker ISSR815 is shown in SEQ ID NO: 1; the primer sequence for amplifying the molecular marker ISSR835 is shown in SEQ ID NO: 2; the primer sequence for amplifying the molecular marker ISSR858 is shown in SEQ ID NO: 3.

[0026] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention constructed a hybrid population to complete the accumulation and homozygosity of the high-yield gene of common tea oil. At the same time, combined with ISSR-PCR and the hybrid fruit traits of common tea oil, the molecular markers ISSR815, ISSR835 and ISSR858 that are closely linked to the high yield of common tea oil were developed and screened, and the four gene loci 815-11, 835-12, 858-2 and 858-3 were obtained. The above molecular markers were used to select high-yield and excellent families and plants of common tea oil from 19 families in the natural population. The selection efficiency was 75% and the accuracy rate was 100%. According to the method, high-yield families of common tea oil were obtained, with high yield and an average unit crown width yield of 2.86 kg / m 2 , much higher than the average unit crown width yield of 1.2 kg / m 2 national industry standards.

[0028] (2) The breeding method of the present invention can obtain superior offspring with stable traits and high yield for variety testing. In each hybrid generation, high-yield and excellent varieties can be successfully obtained within two years. Compared with the existing technology that requires more than ten to twenty years, the breeding cycle is greatly shortened, and the problems of long cycle, poor stability and low efficiency of existing oil tea hybrid breeding are solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 : Amplification spectrum of 30 materials using primers of molecular marker ISSR835. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example 1

[0032] (I) A high-yield molecular marker combined with common oil-tea tea fruit traits, including tightly linked molecular markers ISSR815, ISSR835 and / or ISSR858; molecular marker ISSR815 includes 815-11 gene locus; molecular marker ISSR835 includes 835-12 gene locus; molecular markers include two gene loci, 858-2 and 858-3.

[0033] The primer sequence for amplifying the molecular marker ISSR815 is 5'-CTCTCTCTCCTCCTCTG-3', SEQ ID NO: 1; the primer sequence for amplifying the molecular marker ISSR835 is 5'- AgA gAg AgA gAg AgA gYC-3', SEQ ID NO: 2; the primer sequence for amplifying the molecular marker ISSR858 is 5'- TGT GTG TGT GTG TGT GRT-3', SEQ ID NO: 3.

[0034] (II) Through research, it was found that there are 8 gene loci closely linked to the high-yield traits of common oil-tea camellia, including gene loci 815-5, 815-11, 815-12 of molecular marker ISSR815, gene loci 835-3, 835-12 of molecular marker ISSR835 and gene loci 858-2, 858-3, 858-10 of molecular marker ISSR858. The association between the above gene loci and fruit traits and single-plant yield was analyzed respectively. The experimental results are shown in Tables 1 and 2:

[0035] Traits Associated site Significance test value Phenotypic variation Fruit High 858-2 0.0005 35.02 Fruit width 858-2 0.0005 24.73 Number of seeds 815-12 0.0076 27.4 Fresh fruit weight 858-2 0.0017 23.97 Fresh weight of peel 858-2 0.0008 24.36 Seed fresh weight 815-5 0.0066 29.55 835-12 0.0002 45.59 858-3 0.0091 27.72 Seed kernel fresh weight 835-12 0.001 45.57 Dried fruit weight 835-12 0.0003 41.87 Seed dry weight 835-12 0.0001 54.42 858-10 0.0055 35.49 858-3 0.0036 38.05 Kernel dry weight 815-11 0.0029 39.64 835-12 0.0002 52.70 858-10 0.006 35.23 Fruit moisture content 815-11 0.0043 37.34 835-12 0.0066 34.62 Fresh seed rate 858-3 0.0097 27.41 Yield per plant 835-3 0.0082 29.82 835-12 0.0078 30.08 Results: Among the yield traits of individual plants, 835-12 and 835-3 were significantly correlated, so 835-12 and 835-3 were determined to be high-yield closely associated gene loci of common oil-tea camellia. The variation explanation rate of 835-12 was greater than that of 835-3; at the same time, there were significantly correlated with the other six fruit shape traits, namely, seed fresh weight, seed kernel fresh weight, dry fruit weight, seed dry weight, seed kernel dry weight, and fruit moisture content, so 835-12 was determined to be an important high-yield closely associated gene loci of common oil-tea camellia.

[0036] Relevance Fruit High Fruit width Fruit shape index Peel thickness Number of seeds Fresh fruit weight Fresh weight of peel Seed fresh weight Seed kernel fresh weight Dried fruit weight Peel dry weight Seed dry weight Kernel dry weight Fruit moisture content Kernel moisture content Fresh seed rate Yield per plant Fruit High 1 Fruit width 0.81** 1 Fruit shape index 0.70** 0.20 1 Peel thickness 0.77** 0.83** 0.29 1 Number of seeds 0.43 0.44 -0.02 0.47* 1 Fresh fruit weight 0.85** 0.89** 0.28 0.74** 0.52* 1 Fresh weight of peel 0.85** 0.90** 0.33 0.84** 0.37 0.91** 1 Seed fresh weight 0.60** 0.62** 0.12 0.40 0.57* 0.83** 0.53* 1 Seed kernel fresh weight 0.57* 0.50* 0.18 0.30 0.49* 0.76** 0.45 0.96** 1 Dried fruit weight 0.73** 0.71** 0.28 0.53* 0.58** 0.86** 0.64** 0.92** 0.87** 1 Peel dry weight 0.87** 0.86** 0.39 4.74** 0.51* 0.93** 0.87** 0.74** 0.68** 0.91** 1 Seed dry weight 0.47* 0.44 0.12 0.24 0.54* 0.65** 0.29 0.93** 0.90** 0.91** 0.65** 1 Kernel dry weight 0.48* 0.43 0.21 0.26 0.47* 0.59** 0.30 0.81** 0.80** 0.90** 0.70** 0.93** 1 Fruit moisture content -0.01 0.08 -0.05 0.19 -0.29 -0.04 0.24 -0.42 -0.45 -0.54* -0.26 -0.71** -0.78** 1 Kernel moisture content -0.22 -0.18 -0.20 -0.14 -0.24 -0.14 -0.06 -0.21 -0.16 -0.50* -0.45 -0.46 -0.70** 0.76** 1 Fresh seed rate -0.53* -0.50* -0.43 -0.64** 0.11 -0.33 -0.68** 0.23 0.28 -0.01 -0.41 0.40 0.24 -0.53* 0.04 1 Yield per plant 0.72** 0.53* 0.44 0.40 0.54* 0.69** 0.47* 0.80** 0.79** 0.78** 0.70** 0.75** 0.74** -0.39 -0.37 0.06 1 Results showed that the yield per plant was significantly positively correlated with fruit height, seed fresh weight, kernel fresh weight, dry fruit weight, seed dry weight and kernel dry weight, among which the greatest correlation was found between seed fresh weight and kernel dry weight (Table 2). Therefore, fruit height can be used as a fruit trait, and seed fresh weight and kernel dry weight can be used as seed traits to evaluate the yield per plant of ordinary oil-tea camellia. It can be concluded that in production practice, combined with the principle of operational convenience, the seeds of high-yield oil-tea superior plants can be screened out through fruit height and seed fresh weight in hybrid fruits, that is, the longer the height of the oil-tea camellia fruit, the heavier the seed fresh weight, and the higher the yield of the cultivated plants.

[0037] (III) The dominant and recessive gene types and genetic biases of the gene locus 815-11 of the molecular marker ISSR815, the gene locus 835-12 of the molecular marker ISSR835, and the molecular markers ISSR858-2 and 858-3 were analyzed. The experimental results are shown in Table 3:

[0038] Significantly associated loci 815-11 835-12 858-3 858-2 Genetic maternal rate% 0 42.86 66.67 50 Genetic paternal rate% 0 57.14 50 0 Results showed that gene loci 835-12, 858-2 and 858-3 were dominant genes for high yield, and gene loci 815-11 was recessive gene for high yield; gene loci 835-12 tended to be paternally inherited, and gene loci 858-2 and 858-3 tended to be maternally inherited. Example 2

[0039] A method for high-yield breeding of common camellia oleifera comprises the following steps:

[0040] (1) Parent selection: Select parents with a peak production period of more than 10 years and a unit crown area yield of more than 1.2 kg / m 2 The oil-tea tree was used as the candidate parent, and the molecular markers ISSR815, ISSR835 and ISSR858 were used to assist in screening the parents, and the high-yield and superior strains showing one or more gene loci of 835-12, 858-2 or 858-3 were selected as the parents;

[0041] (2) Parent configuration and hybridization: select the high-yield superior strain with 858-2 and / or 858-3 gene loci in step (1) as the female parent, and the high-yield superior strain with 835-12 gene loci as the male parent, and hybridize them to obtain the F1 generation;

[0042] (3) Screening of high-yield and superior plants of the F1 generation: Based on the fruit traits of the F1 generation of ordinary oil tea and the genotype of the seedlings of the F1 generation of fruits, high-yield and superior plants were screened in the hybrid population, as follows:

[0043] ① Screening excellent families based on phenotype: In the fruit population of the F1 generation, screen the families whose hybrid fruit height is higher than the population average, or whose seed fresh weight is higher than the population average. 27, 46 and 51 are selected as excellent families;

[0044] ② Select superior individual plants in superior families by combining phenotypes: Based on superior families and combined with the fruit traits of the F1 generation, select:

[0045] A: In the F1 population, select the top 10 hybrid fruits with the highest fruit height;

[0046] B: In the F1 generation population, the top 10 hybrid fruits with the highest seed freshness were selected;

[0047] C: In the F1 generation population, excellent hybrid fruits requiring the presence of both A and B were selected and cultivated into seedlings 27-5, 46-2 and 51-2;

[0048] ③ Selecting excellent individual plants expressing 815-11 gene loci and at least one of 835-12, 858-2 or 858-3 from the seedlings cultivated in step ② using molecular markers ISSR815, ISSR835 and ISSR858, the specific process is as follows:

[0049] Molecular markers ISSR815, ISSR835 and ISSR858 primers were used to perform PCR amplification on the materials of step F1. The ISSR-PCR reaction system was (see Table 4). The amplification process used a Master-Cycler 05 gradient PCR instrument (eppendorf, Germany). The PCR cover temperature was set to 105°C, pre-denaturation was performed at 94°C for 5 min, denaturation was performed at 94°C for 45 s, annealing temperature was 54°C, extension was performed at 72°C for 1.5 min, 39 cycles were performed, extension was performed at 72°C for 7 min, and storage was performed at 4°C.

[0050] Components factory Initial concentration Volume (μl) <![CDATA[Mg 2+ (10 mmol / L)]]> TaKaRa 2 mmol / L 4 Primer (10 μmol / L) Shanghai Bioengineering 0.8 μmol / L 1.6 dNTPs (2.5 mmol / L) TaKaRa 0.3 mmol / L 2.4 DNA (20 ng / μl) TaKaRa 30 ng / μl 1.5 Taq enzyme (5 U / μl) TaKaRa 2.5 U / μl 0.5 <![CDATA[ddH2O]]> — 10 With the ISSR815 primer, the material amplified a single band of 1600 bp, which was the 815-11 gene locus; with the ISSR835 primer, the material amplified a single band of 850 bp, which was the 835-12 gene locus ( Figure 1 ); In the ISSR858 primer, the material amplified a single band of 300bp as the 858-2 gene locus, and a single band of 350bp as the 858-3 gene locus

[0051] Result analysis: Through the screening of molecular markers, the 815-11 gene locus appeared in the F1 generation seedlings, and at least one of the superior plants of 835-12, 858-2 or 858-3 appeared, that is, 27-5, 46-2 and 51-2 were high-yield and superior plants of the F1 generation.

[0052] (4) Backcrossing: Based on the high-yield superior plant of the F1 generation selected in step (3) as the female parent, the male parents with the 835-12 gene locus in step (2), namely Min 62, Min 56 and Min 53, are selected as the backcross male parents for backcrossing, and the high-yield superior plant among the backcross progeny is selected as the recurrent female parent, and backcrossed with the male parent with the 835-12 gene locus in step (2) for multiple times to obtain backcross progeny;

[0053] The molecular genetic parameters of each generation (F0-F1) of common oil-tea tea were analyzed. The experimental results are shown in Table 5:

[0054] generations Family coefficient Observed allele number Effective number of alleles Nei genetic diversity Shannon Index F0 7 1.613 1.444 0.251 0.366 F1 5 1.746 1.489 0.282 0.418 F1 back 7 1.904 1.605 0.345 0.509 F0-F1 genetic gain (%) - 8.25 3.12 12.35 14.21 Genetic gain of F1-F1 cycle (%) - 9.05 7.80 22.34 21.77 Result analysis: The genetic gain of F1 backcross generation was significantly higher than that of F2 generation, therefore, the number of backcrossing can reach at least 2 times.

[0055] (5) Screening of high-yield and superior plants of backcross progeny: Based on the fruit traits of the backcross progeny of ordinary tea oil and the genotype of the seedlings produced by the backcross fruit, high-yield and superior plants of the backcross progeny are screened from the backcross progeny population, as follows:

[0056] ① Screening excellent families based on phenotype: In the fruit population of backcross progeny, families with fruit height higher than the population average (screening results: C2, C8, C10, C13, C14, C15, C16 and C17) or families with seed fresh weight higher than the population average (screening results: C8, C13, C14, C15, C16, C17 and C19) were screened. In summary, the excellent families screened were as follows: C2, C8, C10, C13, C14, C15, C16, C17 and C19, a total of 9 families, of which C13, C14, C15, C16, C17 and C19 were backcross families, i.e., the screened excellent families of backcross progeny (as shown in Table 6);

[0057] F2 hybrid family Fruit height (mm) Seed fresh weight (g) <![CDATA[Unit crown area yield (Kg / m 2 )]]> C1 23.11 8.09 1.03 C2 33.01 7.28 3.02 C3 20.61 6.22 1.28 C4 29.54 7.34 2.88 C5 30.69 7.05 1.14 C6 30.24 8.14 2.18 C7 30.76 8.54 1.74 C8 32.11 13.60 3.08 C9 30.91 7.57 1.39 C10 34.02 8.52 1.67 C11 29.23 8.11 1.12 C12 30.84 8.68 1.64 C13 40.35 16.72 1.80 C14 34.57 12.82 2.73 C15 32.41 13.30 2.22 C16 34.16 9.80 2.84 C17 32.08 11.77 3.16 C18 30.25 7.19 1.74 C19 29.80 9.99 3.02 average value 31.00 9.41 2.07 30% above average - - 2.70 ② Select superior plants in superior families by combining phenotypes: Based on superior families and combining the fruit traits of backcross progeny, select:

[0058] A: In the backcross progeny population, select the top 10 hybrid fruits with the highest fruit height;

[0059] B: In the backcross progeny population, the top 10 hybrid fruits with the highest seed freshness were selected;

[0060] C: In the backcross progeny population, select the excellent hybrid fruits that require both A and B to exist, and cultivate them into seedlings;

[0061] ③ Using molecular markers ISSR815, ISSR835 and ISSR858, the seedlings cultivated in step ② are selected to show the 815-11 gene locus, and at least one of 835-12, 858-2 or 858-3, which are high-yield ordinary tea varieties. The specific process is as follows:

[0062] Molecular markers ISSR815, ISSR835 and ISSR858 primers were used for backcrossing materials for PCR amplification. The ISSR-PCR reaction system (as shown in Table 4) was used. The amplification process was as follows: the Master-Cycler 05 gradient PCR instrument (Germany, eppendorf) was used, the PCR hot cover temperature was set to 105°C, pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing temperature at 54°C, extension at 72°C for 1.5 min, 39 cycles, extension at 72°C for 7 min, and storage at 4°C.

[0063] The results of the analysis of tightly linked gene loci in the high-yield families of the F1 backcross generation of common tea oil are shown in Table 7.

[0064] F2 generation family 835-12 858-2 858-3 815-11 C13 - √ √ - C14 - - √ √ C15 - - √ - C16 √ - √ √ C17 √ - √ - C19 √ - √ √ Result analysis: The F1 backcross families that showed the recessive gene locus 815-11 by using molecular markers ISSR815, ISSR835 and ISSR858, and at least one dominant gene locus (835-12, 858-2 and 858-3) were C14, C16 and C19, which are the common high-yield tea cultivars screened by the method of this application.

[0065] After 10 years of growth and 3 consecutive years of yield testing of F1 backcross germplasm materials, the families with an average per-plant yield of 30% higher than the average of the hybrid population were C14, C16, C17 and C19 (Table 6). This proved that the efficiency of high-yield selection reached 75%, the correct rate reached 100%, and the average unit crown width yield was 2.86 kg / m 2 , and greatly shortened the breeding cycle.

[0066] The 19 common tea oil hybrid populations in Example 2 are specifically shown in Table 8:

[0067] Number name generations ♀x♂ Number name generations ♀x♂ 1 Fujian 43 F0 - 16 C4 F2 27-5× 41-4 2 Min 62 F0 - 17 C5 F2 46-2× 51-2 3 Fujian 56 F0 - 18 C6 F2 46-2× 27-5 4 Fujian 54 F0 - 19 C7 F2 46-2× 41-4 5 Fujian 53 F0 - 20 C8 F2 46-2× 56-2 6 Min 50 F0 - 21 C9 F2 51-2× 46-2 7 Fujian 49 F0 - 22 C10 F2 51-2× 27-5 8 27-5 F1 Min 62×Min 56 23 C11 F2 51-2× 56-2 9 41-4 F1 Min 54×Min 43 24 C12 F2 51-2× 41-4 10 46-2 F1 Min 50×Min 62 25 C13 F1 back 27-5× Min 62 11 51-2 F1 Min 43×Min 53 26 C14 F1 back 27-5× Min 56 12 56-2 F1 Min 43×Min 49 27 C15 F1 back 46-2×Min56 13 C1 F2 27-5× 51-2 28 C16 F1 back 46-2×Min62 14 C2 F2 27-5× 46-2 29 C17 F1 back 51-2× Min 56 15 C3 F2 27-5× 56-2 30 C18 F1 back 51-2×Min62 31 C19 F1 back 51-2×Min53 Example 3

[0068] A method for high-yield breeding of common camellia oleifera comprises the following steps:

[0069] (1) Min 62 and Min 56, the unit crown area yield of tea oil trees in the peak production period of more than 10 years is greater than 1.2 kg / m 2 , while Min 62 has dominant gene loci 858-2 and 835-12; Min 56 has dominant gene loci 835-12, so high-yield and superior plants Min 62 and Min 56 were selected as parents;

[0070] (2) Using high-yield and superior strain Min 62 as the female parent and high-yield and superior strain Min 56 as the male parent, the hybrids were used to obtain the F1 generation;

[0071] (3) Screening of F1 high-yield superior strains: the screening result is 27-5;

[0072] (4) Backcrossing: The high-yield plant 27-5 of the F generation was used as the female parent, and the high-yield plant Min 56 was used as the male parent to obtain the F1 backcross generation. The high-yield ordinary tea oil plant C14 was obtained after screening. The average unit crown area yield of its 10-year-old tea oil tree is 2.73 kg / m 2 . Example 4

[0073] A method for high-yield breeding of common camellia oleifera comprises the following steps:

[0074] (1) Min 50 and Min 62, the yield per unit crown area of ​​tea oil trees in the peak production period of more than 10 years is greater than 1.2 kg / m 2 , while Min 50 has the dominant gene locus 858-3; Min 62 has the dominant gene loci 835-12 and 858-2, so the high-yield and superior plants Min 50 and Min 62 were selected as parents;

[0075] (2) Using high-yield superior plant Min 50 as the female parent and high-yield superior plant Min 62 as the male parent, hybridization was performed to obtain the F1 generation;

[0076] (3) Screening of high-yield and superior strains of the F1 generation: the screening result was 46-2;

[0077] (4) Backcrossing: The high-yield and superior plant 46-2 of the F1 generation was used as the female parent, and the high-yield and superior plant Min 62 was used as the male parent to obtain the F1 backcross generation. The high-yield ordinary tea oil plant C16 was obtained after screening. The average unit crown area yield of its 10-year-old tea oil tree is 2.84 kg / m 2 . Example 5

[0078] A method for high-yield breeding of common camellia oleifera comprises the following steps:

[0079] (1) Min 43 and Min 53, the unit crown area yield of tea oil trees in the peak production period of more than 10 years is greater than 1.2 kg / m 2 , while Min 43 has dominant gene loci 858-3 and 835-12; Min 53 has dominant gene loci 835-12, so high-yield and superior plants Min 43 and Min 53 were selected as parents;

[0080] (2) Using high-yield and superior strain Min 43 as the female parent and high-yield and superior strain Min 53 as the male parent, the hybrids were used to obtain the F1 generation;

[0081] (3) Screening of high-yield and superior strains of the F1 generation: the screening result is 51-2;

[0082] (4) Backcrossing: The high-yield superior plant 51-2 of the F1 generation was used as the female parent, and the high-yield superior plant Min 53 was used as the male parent to obtain the F1 backcross generation. The common high-yield tea plant C19 was obtained after screening. The characteristics are: the average unit crown area yield of 10-year-old tea trees is 3.02 Kg / m 2 .

[0083] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0084] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for high-yield breeding of common camellia oleifera, characterized in that: The steps include: (1) Parent selection: Select parents with a peak production period of more than 10 years and a yield per unit crown area of ​​more than 1.2 kg / m 2 The oil-tea tree was used as the candidate parent, and the closely linked molecular markers ISSR815, ISSR835 and ISSR858 were used to assist in screening the parents, and the high-yield superior plants showing one or more gene loci of 835-12, 858-2 or 858-3 were selected as the parents; (2) Parent configuration and hybridization: select the high-yield superior strain with 858-2 and / or 858-3 gene loci in step (1) as the female parent, and the high-yield superior strain with 835-12 gene loci as the male parent, and hybridize to obtain the F1 generation; (3) Backcrossing: selecting a high-yield superior plant of the F1 generation as the female parent, selecting the male parent having the 835-12 gene locus in step (2) as the backcross male parent for backcrossing, selecting a high-yield superior plant among the backcross progeny as the recurrent female parent, and backcrossing it with the male parent having the 835-12 gene locus in step (2) for multiple times to obtain backcross progeny; (4) Screening of high-yield and superior plants in backcross progeny: Based on the fruit traits and genotypes of the backcross progeny of ordinary Camellia oleifera, high-yield and superior plants were screened in the backcross progeny population, as follows: ① Screening excellent families based on phenotype: In the fruit population of backcross progeny, families with fruit height higher than the population average, or seed fresh weight higher than the population average, are selected as excellent families; ② Select superior individual plants in superior families by combining phenotypes: Based on superior families and combining the fruit traits of backcross progeny, select superior seeds with high fruit height and high seed fresh weight, and cultivate them into seedlings; ③ Using molecular markers, select the high-quality individual plants showing the 815-11 gene locus and at least one of 835-12, 858-2 or 858-3 from the seedlings cultivated in step ② using the tightly linked molecular markers ISSR815, ISSR835 and ISSR858, which are the common high-yield varieties of Camellia oleifera; The primer sequence for amplifying the tightly linked molecular marker ISSR815 is 5'-CTCTCTCTCCTC CTCTG-3', SEQ ID NO.1; the primer sequence for amplifying the tightly linked molecular marker ISSR835 is 5'-AgAgAgAgAgAgAgAgYC-3', SEQ ID NO.2; the primer sequence for amplifying the tightly linked molecular marker ISSR858 is 5'-TGTGTGTGTGTGTGTGTGRT-3', SEQ ID NO.3; During the high-yield superior plant screening process of steps (1) and (4), when a single band of 1600 bp is amplified, it means that the detected plant has the 815-11 gene locus; When a single band of 850 bp was amplified, it indicated that the tested plant had the 835-12 gene locus; When a single band of 300 bp was amplified, it indicated that the tested plant had the 858-2 gene locus; When a single band of 350 bp is amplified, it means that the detected plant has the 858-3 gene locus.

2. The method for high-yield breeding of common camellia oleifera according to claim 1, characterized in that: The total number of backcrossings in step (3) is ≥ 2.

3. The method for high-yield breeding of common camellia oleifera according to claim 1, characterized in that: The screening process of the F1 generation high-yield superior plants in step (3) is the same as the screening process of the backcross generation high-yield superior plants in step (4).

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