A high-yield molecular marker and breeding method combining the traits of common Camellia oleifera seedlings

Abstract

The present invention provides a high-yield molecular marker and breeding method combining the traits of common Camellia oleifera seedlings, belonging to the technical field of gene breeding. The present invention has developed two high-yield molecular markers, ISSR815 and ISSR820, which are closely linked to the traits of one-year-old seedlings cultivated from hybrid seeds of common Camellia oleifera, and obtained four gene loci, 815-6, 815-3, 820-7, and 820-8. Combining the three indexes of seedling height, height-diameter ratio, and water use efficiency of one-year-old seedlings cultivated from hybrid seeds, high-yield excellent families and plants of common Camellia oleifera are selected from 19 hybrid families in the natural population, with a selection efficiency of 75% and a correct rate of 100%. According to this method, high-yield families of common Camellia oleifera are obtained, with high yields. The average yield per unit crown area is 2.91 kg / m 2 , which is much higher than the national industry standard of 1.2 kg / m for the average yield per unit crown area 2 . In each hybrid generation, high-yield varieties can be successfully screened out within two years, greatly shortening the breeding cycle compared with the more than ten or twenty years of the existing technology, and solving the problems of long breeding cycle, poor stability, and low efficiency in the existing Camellia oleifera cross-breeding

Description

Technical Field

[0001] The present invention relates to the technical field of gene breeding, and more specifically, to a high-yield molecular marker and breeding method combining the traits of common Camellia oleifera seedlings. Background Art

[0002] Camellia oleifera Abel. is a unique woody oil tree species in China and an important woody edible oil species in the south of China. Camellia oleifera is mainly planted in the subtropical regions of the south and is widely distributed in the hilly areas of the south, with a large cultivation area, among which the planting area of common Camellia oleifera is the largest. At present, the yield of Camellia oleifera forests is relatively low, which is in great contradiction with the rapidly growing market demand for camellia oil, becoming one of the most important dilemmas faced by the development of the Camellia oleifera industry. Therefore, cultivating new high-yield Camellia oleifera varieties is the key to the stable development of the Camellia oleifera industry.

[0003] Quantitative traits show continuous variation, are controlled by multiple minor genes, and are easily affected by the environment. Traditional breeding techniques mainly focus on phenotypic selection, and the results of selection and identification are easily biased by environmental factors. With the development of molecular marker technology, molecular marker-assisted selection (MAS) provides an effective means for the efficient selection of quantitative traits. After identifying beneficial alleles of candidate genes with significant phenotypic effects through association analysis, allele-specific PCR primers are designed for marker development and verified in germplasm resources with a wide genetic basis for MAS in a wide genetic background, thereby improving the selection efficiency. Using functional markers for MAS can, on the one hand, ensure the accuracy and efficiency of selection by directly selecting the gene itself, and on the other hand, using association analysis can target specific functional domains affecting trait variation to ensure the selection effect.

[0004] In genetic breeding, yield is a quantitative trait. To improve the accuracy of selection, it is necessary to carry out planting and selection over many years. As a perennial woody plant, the traditional cross-breeding cycle of Camellia oleifera is long, taking more than ten to twenty years, with low efficiency and unable to meet the rapid development of modern industrialization of Camellia oleifera. In addition, there are also problems such as occupying land resources and high labor costs, and it cannot meet the need to quickly and accurately select according to breeding goals. Molecular marker-assisted selection (MAS) technology is a new technology that uses molecular markers tightly linked to target genes to screen for superior genotypes and obtain target individuals, which can improve the accuracy of selection, shorten the breeding period, and improve breeding efficiency. The long growth cycle of forest trees is the main reason for the late effectiveness of Camellia oleifera breeding, but premature selection is prone to the risks of missing selection and misselection. The traits of progeny are stable at the seedling stage. Combining molecular marker-assisted selection will reduce the risk of misselection. ISSR (Inter-Simple Sequence Repeats), the inter-simple sequence repeat region, is a dominant marker with simple operation, simple primer design, good polymorphism, high repeatability, low experimental cost, and high experimental stability, and is a good DNA fingerprint marker.

[0005] However, currently in Camellia oleifera, molecular marker technology is mainly applied in the fields of genetic diversity analysis, variety identification, genetic relationship analysis, and hybrid identification of Camellia oleifera. Some molecular markers related to the quality of camellia oil have been developed, but molecular markers tightly linked to yield have not been developed, and there are no research reports on the application of the developed molecular markers in cross-breeding.

[0006] Therefore, how to develop a molecular marker and breeding method tightly linked to the yield of common Camellia oleifera is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a high-yield molecular marker and breeding method combined with the traits of common Camellia oleifera seedlings. Two high-yield molecular markers, ISSR815 and ISSR820, tightly linked to the traits of one-year-old seedlings cultivated from common Camellia oleifera hybrid seeds have been developed, and four gene loci, 815-6, 815-3, 820-7, and 820-8, have been obtained. High-yield excellent families and plants of common Camellia oleifera are selected from 19 hybrid families in the natural population, with a selection efficiency of 75% and a correct rate of 100%. According to this method, high-yield families of common Camellia oleifera are obtained, with high yields. The average yield per unit crown area is 2.91 kg / m 2 , far higher than the average yield per unit crown area of 1.2 kg / m 2National industry standards; in each hybrid generation, high-yielding varieties can be successfully screened out within 2 years, greatly shortening the breeding cycle compared with the existing more than a decade or two decades, and solving the problems of long breeding cycle, poor stability and low efficiency in the existing oil tea cross-breeding.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A high-yield molecular marker combining the traits of common oil tea seedlings, including closely linked molecular markers ISSR815 and ISSR820; the molecular marker ISSR815 includes two gene loci 815-3 and 815-6; the molecular marker ISSR820 includes two gene loci 820-7 and 820-8; the primer sequence amplified by the molecular marker ISSR815 is 5'-CTCTCTCTCCTCCTCTG-3', SEQ ID NO: 1; the primer sequence amplified by the molecular marker ISSR820 is 5'-GTGTGTGTGTGTGTGTG-3', SEQ ID NO: 2.

[0010] Preferably, the gene loci 815-3 and 815-6 of the molecular marker ISSR815 are significantly correlated with the water use efficiency of the seedlings cultivated from 1-year-old common oil tea hybrid seeds; the gene loci 820-7 and 820-8 of the molecular marker ISSR820 are significantly correlated with the seedling height and height-diameter ratio of the seedlings cultivated from 1-year-old common oil tea hybrid seeds.

[0011] Preferably, the single-plant yield of common oil tea is extremely significantly positively correlated with the seedling height of the seedlings cultivated from 1-year-old common oil tea hybrid seeds, significantly positively correlated with the height-diameter ratio, and significantly negatively correlated with the water use efficiency.

[0012] Preferably, the gene loci 815-6 and 820-7 are high-yield dominant genes, and the gene loci 815-3 and 820-8 are high-yield recessive genes; the gene locus 815-6 shows paternal inheritance, and the gene locus 820-7 shows maternal inheritance.

[0013] Another object of the present invention is to provide the application of the above high-yield molecular marker combining the traits of common oil tea seedlings in the high-yield breeding of common oil tea.

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

[0015] (1) Parent selection: Select the yield per unit crown area of more than 1.2 kg / m in the full fruit-bearing period of more than 10 years 2The Camellia oleifera trees are used as alternative parents. Combining molecular markers ISSR815 and ISSR820 to assist in screening parents, select high-yield and excellent plants that show the 815-6 or 820-7 gene loci and do not show the 815-3 and 820-8 gene loci as parents;

[0016] (2)Parent configuration and hybridization: Select the high-yield and excellent plants with the 820-7 gene locus in step (1) as the female parent; select the high-yield and excellent plants with the 815-6 gene locus as the male parent, and hybridize to obtain the F1 generation;

[0017] (3)Backcross: Select the high-yield and excellent plants of the F1 generation as the female parent, and select the male parent with the 815-6 gene locus in step (2) as the backcross male parent for backcross; select the high-yield and excellent plants in the backcross offspring as the recurrent female parent, and backcross multiple times with the male parent with the 815-6 gene locus in step (2) to obtain the backcross offspring;

[0018] (4)Screening of high-yield and excellent plants in the backcross offspring: Combining the growth traits and genotypes of the seedlings cultivated from the backcross offspring of the common Camellia oleifera hybrid seeds, screening high-yield and excellent plants in the backcross offspring population, including the following steps:

[0019] ① Screening of excellent families by combining phenotypes: In the backcross offspring population, screen families where the height or stem height ratio of the backcross offspring seedlings is 30% higher than the population average, or the water use efficiency is 30% lower than the population average as excellent families;

[0020] ② Using molecular markers, select excellent families of backcross offspring that show the 815-6 or / and 820-7 gene loci and do not show the 815-3 and 820-8 gene loci in the excellent families screened in step ①;

[0021] ③ Screening of excellent individual plants within excellent families by combining phenotypes: Based on the excellent families screened in step ②, combining the growth traits of the seedlings cultivated from the backcross offspring, screen out:

[0022] A. In the family population, screen out the top 10 plants with the highest height and stem height ratio of the 1-year-old seedlings cultivated from hybrid seeds;

[0023] B. In the family population, screen out the last 10 plants with the lowest water use efficiency of the 1-year-old seedlings cultivated from hybrid seeds;

[0024] C. In the family population, screen out plants that meet both requirements A and B as high-yield and excellent plants of the backcross offspring.

[0025] (5)The high-yield and excellent plants of the backcross offspring screened in step (4)-③ are the high-yield varieties of common Camellia oleifera.

[0026] Preferably, in step (1), when a single band of 800 bp is amplified, it indicates that the tested plant has the 820-7 gene; when a single band of 900 bp is amplified, it indicates that the tested plant has the 820-8 gene; when a single band of 650 bp is amplified, it indicates that the tested plant has the 815-6 gene; when a single band of 250 bp is amplified, it indicates that the tested plant has the 815-3 gene.

[0027] Preferably, in step (3), the total number of backcrosses ≥ 2 times.

[0028] Preferably, the screening process of high-yield superior plants in the F1 generation in step (3) is the same as that of high-yield superior plants in the backcross progeny in step (4).

[0029] Another object of the present invention is to provide a set of amplification primers for combining the traits of high-yield molecular markers of common Camellia oleifera seedlings, including amplification primers for molecular markers ISSR815 and ISSR820. The primer sequences for amplifying the molecular marker ISSR815 are shown in SEQ ID NO: 1; the primer sequences for amplifying the molecular marker ISSR820 are shown in SEQ ID NO: 2.

[0030] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) Through the construction of a hybrid population, the present invention has completed the accumulation and homozygosity of high-yield quantitative genes in common Camellia oleifera, and at the same time, combined with ISSR-PCR and the growth traits of one-year-old seedlings of common Camellia oleifera hybrid seeds, developed and screened molecular markers ISSR815 and ISSR820 that are closely linked to high yield in common Camellia oleifera, obtained four gene loci of 815-6, 815-3, 820-7, and 820-8, selected high-yield excellent families and plants of common Camellia oleifera in 19 hybrid families, with a selection efficiency of 75% and a correct rate of 100%. According to this method, high-yield families of common Camellia oleifera are obtained, with high yields. The average yield per unit crown area is 2.91 kg / m 2 ; the average yield per unit crown area of the selected high-yield excellent plants is 3.69 kg / m 2 , which is much higher than the national industry standard of 1.2 kg / m for the average yield per unit crown area. 2

[0032] (2) The breeding method of the present invention can obtain superior plant offspring with stable traits and high yields for variety tests. In each hybrid generation, high-yield excellent varieties can be successfully obtained in two years. Compared with the prior art that requires more than a decade or two decades, the breeding cycle is greatly shortened, solving the problems of long breeding cycle, poor stability, and low efficiency in the existing Camellia oleifera cross-breeding. Description of the Drawings

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the provided drawings.

[0034] Figure 1 : Correlation coefficient diagram of single-plant yield and growth traits of 1-year-old seedlings. Detailed implementation manners

[0035] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0036] Example 1

[0037] (1) A high-yield molecular marker combined with the traits of common oil-tea camellia seedlings, including closely linked molecular markers ISSR815 and ISSR820; the molecular marker ISSR815 includes two gene loci, 815-3 and 815-6; the molecular marker ISSR820 includes two gene loci, 820-7 and 820-8;

[0038] The primer sequence amplified by the molecular marker ISSR815 is 5'-CTCTCTCTCCTCCTCTG-3', SEQ ID NO: 1; the primer sequence amplified by the molecular marker ISSR820 is 5'-GTGTGTGTGTGTG

[0039] TGTG-3', SEQ ID NO: 2.

[0040] (2) Analyze the correlation between the gene loci 815-3 and 815-6 of the molecular marker ISSR815 and the gene loci 820-7 and 820-8 of the molecular marker ISSR820 and the growth traits of 1-year-old seedlings. The experimental results are shown in Table 1:

[0041] Table 1: Correlation analysis of ISSR molecular markers and growth traits of 1-year-old seedlings

[0042] Growth traits Associated site Significance test value Phenotypic variation% Seedlings 820-7 0.0005 39.87 Water use efficiency 815-3 0.0081 22.69 815-6 0.0081 22.69 Height-to-diameter ratio (seedling height / ground diameter) 820-7 0.0002 59.54 820-8 0.0014 48.44 average value - - 38.65

[0043] Result analysis: As can be seen from the data in Table 1, for the correlation analysis between the molecular marker ISSR and the growth traits of 1-year-old seedlings, the average variation explanation rate is 38.65%. Among them, the gene loci 815-3 and 815-6 of the molecular marker ISSR815 are significantly associated with the water use efficiency of the seedlings of the first-generation hybrid seeds of common Camellia oleifera, and the variation explanation rate is 22.69%; the gene loci 820-7 and 820-8 of the molecular marker ISSR820 are significantly associated with the seedling height and height-diameter ratio of the seedlings of the first-generation hybrid seeds of common Camellia oleifera, and the average variation explanation rate is 49.28%.

[0044] (3) Analyze the correlation coefficient between the yield per plant and the growth traits of 1-year-old seedlings. The experimental results are as Figure 1 shown;

[0045] Result analysis: As Figure 1 can be seen, the yield per plant is extremely significantly positively correlated with the seedling height of the seedlings cultivated from the first-generation hybrid seeds of common Camellia oleifera, significantly positively correlated with its height-diameter ratio, and significantly negatively correlated with its water use efficiency.

[0046] (4) Analyze the dominant and recessive gene types and genetic biases of the gene loci 815-3 and 815-6 of the molecular marker ISSR815 and the gene loci 820-7 and 820-8 of the molecular marker ISSR820. The experimental results are shown in Table 2:

[0047] Table 2 Genetic bias analysis of ISSR significantly associated loci

[0048] Significantly associated loci 820-7 820-8 815-3 815-6 Genetic maternal rate% 100 0 0 0 Genetic paternal rate% 0 0 0 42.86

[0049] Result analysis: The gene loci 815-6 and 820-7 are high-yield dominant genes, and the gene loci 815-3 and 820-8 are high-yield recessive genes; the 815-6 gene locus shows paternal inheritance bias, and the 820-7 gene locus shows maternal inheritance bias.

[0050] In summary, the greater the values of the seedling height and height-diameter ratio of Camellia oleifera, the lower the water use efficiency, and the higher the yield of the cultivated plants. By screening for high-yield dominant genes 815-6 and 820-7, high-yield plants can be identified. Among them, plants with the 815-6 gene locus can be used as female parents for hybridization, and plants with the 820-7 gene locus can be used as male parents for hybridization.

[0051] Example 2

[0052] A method for high-yield breeding of common Camellia oleifera includes the following steps:

[0053] (1) Selection of parents: Select plants with a yield per unit crown area greater than 1.2 kg / m in the full fruit-bearing period of more than 10 years 2The oil tea trees are used as alternative parents. Combining molecular markers ISSR815 and ISSR820 to assist in screening parents, select high-yield and excellent strains that show the 815-6 or 820-7 gene loci and do not show the 815-3 and 820-8 gene loci as parents;

[0054] (2)Parent configuration and hybridization: Select the high-yield and excellent strains with the 820-7 gene locus in step (1) as the female parent (F0 generation); select the high-yield and excellent strains with the 815-6 gene locus as the male parent (F0 generation), and hybridize to obtain the F1 generation;

[0055] (3)Screening of high-yield and excellent strains in the F1 generation: Combining the growth traits of the seedlings of the F1 generation cultivated from the hybrid seeds of common oil tea, screen the high-yield and excellent strains in the F1 generation population, as follows:

[0056] ① Screen excellent families by combining phenotypes: In the F1 generation population, screen the families where the height or stem height ratio of the F1 generation seedlings is 30% higher than the population average (the screening results are 56, 51, and 27), or the water use efficiency is 30% lower than the population average (the screening results are 46 and 41). Therefore, 51, 46, 27, 56, and 41 are used as high-yield and excellent families of common oil tea in the F1 generation;

[0057] ② Further screen excellent high-yield families for high-yield genes: Use molecular markers ISSR815 and ISSR820 primers to perform PCR amplification on 5 high-yield and excellent families in the F1 generation. The ISSR-PCR reaction system (as shown in Table 3), and the amplification process is as follows: Use the Master-Cycler 05 gradient PCR instrument (Eppendorf, Germany), set the PCR hot lid temperature to 105°C, pre-denature at 94°C for 5 min, denature at 94°C for 45 s, anneal at 54°C, extend at 72°C for 1.5 min, 39 cycles, extend at 72°C for 7 min, and store at 4°C.

[0058] Table 3 ISSR-PCR reaction system

[0059] 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

[0060] Use molecular marker ISSR820 primer to perform PCR amplification on 5 high-yield and excellent families in the F1 generation.

[0061] Gene locus judgment: With the ISSR815 primer, when the material amplifies a single band of 650 bp, it is the 815-6 gene locus; when it amplifies a single band of 250 bp, it is the 815-3 gene locus. With the ISSR820 primer, when the material amplifies a single band of 800 bp, it is the 820-7 gene locus, and when it amplifies a single band of 900 bp, it is the 820-8 gene locus.

[0062] It can be seen from the PCR amplification results that: using the molecular markers ISSR815 and ISSR820, families that showed the 815-6 or / and 820-7 gene loci and did not show the 815-3 and 820-8 gene loci were selected. The results are shown in Table 4:

[0063] Table 4 Analysis of closely linked gene loci of high-yield families in the F1 generation of Camellia oleifera

[0064] Significantly associated loci 27 41 46 51 56 820-7 - - - √ - 820-8 - - - - - 815-3 - - - - - 815-6 √ - √ - -

[0065] Result analysis: According to the characteristics of the gene loci, families 27, 46, and 51 were screened out. Among them, 27 and 46 had the 815-6 gene locus, and their male parents were Min 56 and Min 62 respectively (Table 5). Therefore, Min 56 and Min 62 can be used as backcross male parents.

[0066] After 10 years of growth of the F1 hybrid germplasm materials and continuous yield measurement for 3 years, the families with the single-plant yield of Camellia oleifera higher than 30% of the population average were 51, 46, 27, 56, and 41. Compared with the families 27, 46, and 51 screened by the breeding method of the present invention, the high-yield selection efficiency of the excellent families in the F1 generation reached 60%, the correct rate reached 100%, and the average yield per unit crown area was 2.41 kg / m 2 .

[0067] ③ Screening of excellent high-yield plants. Based on the excellent families 27, 46, and 51, combined with the growth traits of the cultivated seedlings, the following were screened out

[0068] The top 10 plants with the highest height and stem height ratio of the 1-year-old seedlings cultivated from A hybrid seeds,

[0069] The last 10 plants with the lowest water use efficiency of the 1-year-old seedlings cultivated from B hybrid seeds,

[0070] C Plants that meet the requirements of both A and B, namely 27-5, 46-2, and 51-2, were selected as excellent single plants, which can be used as high-yield lines or as backcross or hybrid parents. The average yield per unit crown area was 3.47 kg / m 2 , and the breeding cycle was greatly shortened.

[0071] The specific Camellia oleifera hybrid population in Example 2 is shown in Table 5 as follows:

[0072] Table 5 Camellia oleifera hybrid population

[0073] 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

[0074] Analysis of the molecular genetic parameters of each generation of Camellia oleifera, and the experimental results are shown in Table 6:

[0075] Table 6 Analysis of molecular genetic parameters of each generation of Camellia oleifera

[0076] 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 F1-F1 genetic gain (%) - 9.05 7.80 22.34 21.77

[0077] Result analysis: The genetic gain of the backcross of F1 is significantly higher than that of the F1 generation. Therefore, the number of backcrosses can reach at least 2 times.

[0078] (4) Screening of high-yield superior plants in backcross progeny

[0079] ① Screening of excellent high-yield families: In step (4), among the F2 (12 families) and the backcross of F1 (7 families) generations C1-C19, by combining phenotypes, the families with the seedling height of the seedlings cultivated from hybrid seeds greater than 30% of the population average are C13 and C14; the families with the stem height ratio of the seedlings cultivated from the hybrid seeds of common Camellia oleifera for 1 year higher than 30% of the population average are C8, C13 and C14; the families with the water use efficiency of the seedlings cultivated from the hybrid seeds of common Camellia oleifera for 1 year lower than 30% of the population average are C8, C14, C16, C17 and C19 (Table 7); In summary, through phenotypic selection, the excellent high-yield families obtained are 6 F2-generation hybrid families, namely C8, C13, C14, C16, C17, and C19, among which 5 families, namely C13, C14, C16, C17, and C19, are F1 backcross families. The results of Table 7 are shown as follows:

[0080] Table 7 Analysis of the traits of 1-year-old seedlings and the yield traits in the full fruit stage of each family of F2 and the backcross of F1 of common Camellia oleifera

[0081] F2 family number Seedling height (cm) Aspect Ratio Water use efficiency <![CDATA[Unit crown area yield (Kg / m 2 )]]> C1 11.65 4.96 7.89 1.03 C2 12.83 5.46 10.42 3.02 C3 11.63 5.59 8.85 1.28 C4 11.83 4.75 7.97 2.88 C5 10.83 4.38 11.70 1.14 C6 10.25 4.42 9.68 2.18 C7 13.35 5.54 8.37 1.74 C8 14.88 7.25 2.98 3.08 C9 13.22 5.27 8.58 1.39 C10 12.10 5.15 4.87 1.67 C11 12.37 5.83 6.24 1.12 C12 13.20 4.41 6.23 1.64 C13 17.77 7.53 5.60 1.80 C14 17.75 7.33 3.61 2.73 C15 11.88 4.85 6.28 2.22 C16 12.92 4.68 4.49 2.84 C17 13.88 4.55 3.60 3.16 C18 15.18 5.93 5.42 1.74 C19 12.07 4.13 3.93 3.02 average value 13.14 5.32 6.82 2.07 30% above average 17.08 6.91 - 2.70 30% below average - - 4.77

[0082] ② Further screening of excellent high-yield families with high-yield genes: Use molecular marker ISSR815 and ISSR820 primers to perform PCR amplification on 31 materials from F0-F2 generations. The ISSR-PCR reaction system is as shown in Table 3. The amplification process is as follows: Use the Master-Cycler 05 gradient PCR instrument (Eppendorf, Germany), set the PCR hot lid temperature to 105°C, pre-denature at 94°C for 5 min, denature at 94°C for 45 s, anneal at 54°C, extend at 72°C for 1.5 min, perform 39 cycles, extend at 72°C for 7 min, and store at 4°C.

[0083] Perform PCR amplification on 30 materials from F0-F2 generations using molecular marker ISSR815 primers, and perform PCR amplification on 30 materials from F0-F2 generations using ISSR820 primers.

[0084] Result analysis: Gene locus determination. For the ISSR815 primer, when a single band of 650 bp is amplified in the material, it is the 815-6 gene locus; when a single band of 250 bp is amplified, it is the 815-3 gene locus. For the ISSR820 primer, when a single band of 800 bp is amplified in the material, it is the 820-7 gene locus; when a single band of 900 bp is amplified, it is the 820-8 gene locus.

[0085] It can be seen from the PCR amplification results that: using the molecular markers ISSR815 and ISSR820, individual plants that show the 815-6 or / and 820-7 gene loci and do not show the 815-3 and 820-8 gene loci are selected. The results are shown in Table 8:

[0086] Table 8 Analysis of tightly linked gene loci of high-yield families in the F1 backcross of Camellia oleifera

[0087] Tightly linked loci C13 C14 C16 C17 C19 820-7 - √ √ √ - 820-8 - - - - - 815-3 - - - - - 815-6 - √ √ √ -

[0088] Result analysis: According to the characteristics of the gene loci, the backcross families C14, C16, and C17 are selected as high-yield families.

[0089] After 10 years of growth of the F1 backcross germplasm materials and continuous yield measurement for 3 years, the families C14, C16, C17, and C19 have a single plant yield of Camellia oleifera higher than 30% of the population average. Compared with the families C14, C16, and C17 selected by the breeding method of the present invention, the high-yield selection efficiency of the present invention reaches 75%, the correct rate reaches 100%, and the average yield per unit crown area is 2.91 kg / m 2 .

[0090] ③ Screening excellent individual plants within excellent families in combination with phenotypes: Screening of excellent high-yield plants. Based on the excellent families C14, C16, and C17, combined with the growth traits of the cultivated seedlings, the top 10 plants with the highest height and stem height ratio of the 1-year-old seedlings cultivated from hybrid seeds are selected, and the last 10 plants with the lowest water use efficiency of the 1-year-old seedlings cultivated from hybrid seeds are selected. The above plants are all excellent individual plants and can be selected as high-yield lines. The average yield per unit crown area is 3.69 kg / m 2 , and the breeding cycle is greatly shortened.

[0091] The various embodiments in the specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0092] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those 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. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for high-yield breeding of Camellia oleifera Abel., characterized in that, It includes the following steps: (1) Parent selection: Select Camellia oleifera trees with a yield per unit crown area greater than 1.2 kg / m 2 in the full fruit-bearing stage for over 10 years as candidate parents. Combine molecular markers ISSR815 and ISSR820 to assist in screening parents, and select high-yield and excellent plants that show the 815-6 or 820-7 gene loci and do not show the 815-3 and 820-8 gene loci as parents; (2) Parent configuration and hybridization: Select the high-yield superior plants with the 820-7 gene locus in step (1) as the female parent; select the high-yield superior plants with the 815-6 gene locus as the male parent, and hybridize to obtain the F1 generation; (3) Backcross: Select the high-yield superior plants of the F1 generation as the female parent, and select the male parent with the 815-6 gene locus in step (2) as the backcross male parent for backcross; select the high-yield superior plants in the backcross offspring as the recurrent female parent, and backcross multiple times with the male parent with the 815-6 gene locus in step (2) to obtain the backcross offspring; (4) Screening of high-yield superior plants in the backcross offspring: Combine the growth traits and genotypes of the seedlings cultivated from the backcross offspring of the common Camellia oleifera hybrid seeds, and screen high-yield superior plants in the backcross offspring population, including the following steps: ① Screening of excellent families by combining phenotypes: In the backcross offspring population, screen the families in which the height or stem height ratio of the seedlings from the backcross offspring is 30% higher than the population average, or the water use efficiency is 30% lower than the population average as excellent families; ② Using molecular markers, select the excellent families of the backcross offspring that show the 815-6 or / and 820-7 gene locus and do not show the 815-3 and 820-8 gene loci in the excellent families screened in step ①; ③ Screening of excellent individual plants within the excellent families by combining phenotypes: Based on the excellent families screened in step ②, combine the growth traits of the seedlings cultivated from the backcross offspring to screen out: A. In the family population, screen out the top 10 plants with the highest height and stem height ratio of the 1-year-old seedlings cultivated from the hybrid seeds; B. In the family population, screen out the last 10 plants with the lowest water use efficiency of the 1-year-old seedlings cultivated from the hybrid seeds; C. In the family population, screen out the plants that meet both requirements A and B as the high-yield superior plants of the backcross offspring; (5) The high-yield superior plants of the backcross offspring screened in step (4)-③ are the high-yield varieties of common Camellia oleifera; The closely linked molecular marker ISSR815 includes two gene loci, 815-3 and 815-6; the molecular marker ISSR820 includes two gene loci, 820-7 and 820-8; the primer sequence amplified by the molecular marker ISSR815 is 5'-CTCTCTCTCCTCCTCTG-3', SEQ ID NO: 1; the primer sequence amplified by the molecular marker ISSR820 is 5'-GTGTGTGTGTGTGTGTG-3', SEQ ID NO: 2; In steps (1) and (4), when a single band of 800 bp is amplified, it represents that the tested plant has the 820-7 gene; When a single band of 900 bp is amplified, it represents that the tested plant has the 820-8 gene; When a single band of 650 bp is amplified, it represents that the tested plant has the 815-6 gene; When a single band of 250 bp is amplified, it represents that the tested plant has the 815-3 gene.

2. The method for high-yield breeding of Camellia oleifera Abel as claimed in claim 1, wherein In step (3), the total number of backcrosses ≥ 2 times.

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

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