CN113186325A - Apple rootstock dwarfing auxiliary screening molecular marker and application thereof - Google Patents
Apple rootstock dwarfing auxiliary screening molecular marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses an apple rootstock dwarfing auxiliary screening molecular marker and application thereof, wherein the apple rootstock dwarfing auxiliary screening molecular marker is a1, a2 or a3, and a1 is a DNA molecule shown in a sequence 1; the a2 is a Deletion polymorphism 11pb upstream 339bp of the MdLBD3 transcription start site in the apple genome; the a3 is a DNA molecule which is derived from apple and has more than 95%, 98% or 99% of homology with the sequence 1; the application of the apple rootstock dwarfing auxiliary screening molecular marker comprises the following steps: identifying the dwarfing character of the apple rootstock; predicting the dwarfing character of the apple rootstock; and (4) breeding apple rootstocks with different dwarfing characters. The invention develops and applies the molecular marker related to the dwarfing character, and the molecular marker assists in breeding, thereby being beneficial to directional selective breeding, shortening the breeding period and improving the screening efficiency.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an apple rootstock dwarfing auxiliary screening molecular marker and application thereof.
Background
At present, apples are the first big fruits in China, the economic yield of the apples in China in 2019 is nearly 4300 ten thousand t, which accounts for more than half of the total yield of the world, and the apples are rich in nutrition, high in yield and storage-resistant, and are a prop industry which meets the living consumption level of people and promotes the development of agriculture and rural economy. Currently, with the acceleration of urbanization progress in China and the aging of population structures in rural areas, the fruit industry is gradually exposed to the production situation of insufficient labor force. Therefore, mechanical operation and labor-saving cultivation become a necessary way for fruit industry development, and dwarfing cultivation has the advantages of close planting operation, uniformity, standardized management and the like, and is the premise of mechanical popularization and labor saving; and dwarfing cultivation of early fruits, high yield and high quality is the trend of development of fruit industry in the world and even China. The proper dwarfing stock is the basis for popularizing the modern orchard, the dwarfing mechanism of the stock is complex, the identification and screening period is longer, and the development of the stock process is restricted. Therefore, the research on the dwarfing gene variation of the apple rootstock has great significance for exploring a rootstock dwarfing mechanism, assisting screening and identifying in advance, accelerating a breeding process and promoting high-yield and high-quality cultivation.
The apple has the characteristics of complicated genetic background, long childhood period, incompatibility in selfing and the like, so that the genetic improvement and germplasm innovation of apple rootstocks are severely restricted.
Through the above analysis, the problems and defects of the prior art are as follows: the existing apple rootstock dwarfing mechanism is complex, the identification and screening period is long, and the development of the rootstock process is restricted.
The difficulty in solving the above problems and defects is: the apple (Malus domestica Mill) has good flavor and rich nutrition, and is one of the tree species with wide cultivation range and high yield in China and the world. The planting mode of the method is changed from traditional vigorous planting to dwarf close planting, and the dwarf close planting has the advantages of economic land utilization, high yield of early fruits, excellent quality, suitability for machinery, light weight, simplicity, labor saving and the like, and is a necessary way for the development of the apple industry in China. Dwarfing stock trees are the basis for realizing dwarfing close planting cultivation, and genetic improvement of dwarfing of the stock trees is the requirement of current industrial development.
And the Malus plants have 17 pairs (34) of chromosomes, and the genome sequencing shows that the Malus plants contain about 4 ten thousand genes, the genetic structure of the Malus plants is highly heterozygous, and the heterosis of a plurality of agronomic traits is polymerized. The selection and identification of the dwarfing character of the rootstock are research hotspots and difficulties at home and abroad, and generally, scientific researchers need to cultivate tens of thousands of hybrid seedlings and can screen out 1-2 new strains of promising excellent rootstocks after years of selection and identification. For example: the breeding period of the M-line rootstock in the United kingdom is 25-38 years, the breeding period of the O-line rootstock in the Canada is 23-28 years, and the CG-line rootstock in the United states is screened for 25-30 years; the self-breeding apple rootstock GM line rootstock is used for 24-27 years in China, the breeding age of SH line rootstock is near 30 years, the breeding of Liaoning stock line rootstock also lasts for 20-30 years, the dwarfing regulation mechanism of the apple rootstock is complex, the identification is completed by means of a rootstock-ear combination test, and the self-breeding apple rootstock GM line rootstock is a technical bottleneck for restricting the breeding of the apple rootstock and the dwarfing cultivation of fruits.
The significance of solving the problems and the defects is as follows: the traditional dwarfing identification methods such as field investigation, tissue dissection, physiology and biochemistry and the like have long screening period, poor reliability and low accuracy; if the dwarfing of apple rootstocks can be identified on the gene level, molecular markers of dwarfing genes are designed and developed, so that the research on dwarfing mechanisms of fruit trees can be enriched, the breeding period can be shortened, the breeding efficiency can be improved, and the modernized development of fruit industry can be promoted. On the basis of a large amount of preliminary work, a continuous 11bp (5'-TCAACAACATA-3') Deletion characteristic sequence is found in the promoter region of the major gene MdLBD3 through prospective research methods such as genetic analysis, gene localization, omics analysis and the like, and the sequence is completely linked with the dwarfing character. By taking the sequence as a target spot, the molecular marker of the dwarfing gene is developed and designed, and the dwarfing characters of seedlings and resources can be accurately and effectively identified and predicted in advance; only 1 leaf of a 1-year-old seedling needs to be extracted, and the dwarfing character of the strain can be identified through electrophoresis detection of a PCR amplification product; the dwarfing identification time of 8-10 years is shortened to 1-2 years by the conventional method, which is equivalent to the identification period of 7-8 years, so that the screening efficiency is greatly improved, the reliability and the stability are good, and the management cost of the seedling stage and the utilization efficiency of the land are saved. The technical means are enriched in the rootstock breeding discipline, the selection tool is optimized in the rootstock breeding practice, and the breeding period is shortened; provides theoretical guidance and technical innovation for cultivating excellent stocks, and has great significance for promoting the dwarf cultivation development of apples and the upgrading of fruit industry structure in China.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an apple rootstock dwarf auxiliary screening molecular marker and application thereof.
The invention is realized in such a way that the dwarf property auxiliary screening molecular marker of the apple rootstock is marked as a1, a2 or a 3;
a1 a Deletion polymorphism of 11bp existing upstream of a transcription initiation site of a lateral growth tissue development gene MdLBD3 in a genome of an apple rootstock, wherein the Deletion polymorphism is tightly linked with rootstock dwarfing; the characteristic DNA molecular sequence of the a1 is 5'-TCAACAACATA-3'.
a2 designing a pair of primers according to the 11bp Deletion polymorphism:
(upstream primer) MdLBD 3-d-F: 5'-GTCGTTGGCCTCTATTCTTCTGCAG-3', respectively;
(downstream primer) MdLBD 3-d-R: 5'-TCAAACAGACACCAAGTAGTTCCGT-3', respectively;
the a2 is the 326 th and 349 th nucleotides of the lateral growth tissue development gene MdLBD3 in the apple genome, starting from the start codon to the front (5' end).
The method is characterized in that DNA of apple rootstock seedlings or germplasm resources is taken as a template, a reaction system for 20 mu LPCR amplification is adopted, and the reaction procedure of PCR amplification is as follows: 5min at 94 ℃; 36 cycles of 94 ℃ for 30s, 59 ℃ for 30s, 72 ℃ for 50 s; storing at 72 deg.C for 7min and 4 deg.C; after the PCR generation under the reaction program is detected by 1.5% agarose electrophoresis, if a strip exists at the position of 550bp, the material is indicated to be a dwarfing stock; if no PCR product exists or no band exists at 550bp, the material is an arborization stock.
a311bp insertion polymorphism and development of DNA molecules designed to have a homology of more than 95%, 98% or 99% to the designed primer sequence.
Further, the reaction system for PCR amplification consists of (20. mu.L): 2 XPCR Mix 10. mu.L, MdLBD3-d-F solution 1.0. mu.L, MdLBD3-d-R solution 1.0. mu.L, template solution 2.0. mu.L, ddH2O6.0. mu.L. The concentration of MdLBD3-d-F in the MdLBD3-d-F solution is 10 mu M; MdLBD3-d-R solution, MdLBD3-d-R concentration is 10 u M. In the template solution, the DNA concentration was 500 ng/. mu.L.
The invention also aims to provide an application of the apple rootstock dwarfing auxiliary screening molecular marker, which comprises the following steps:
identifying the dwarfing character of the apple rootstock; predicting the dwarfing character of the apple rootstock; and breeding apple rootstocks with different dwarfing traits.
Further, the method for predicting the dwarfing character of the apple rootstock comprises the following steps:
detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than that of the LiLi genotype apple rootstock;
if two chromosomes of the apple rootstock to be detected have the Deletion molecular marker, the genotype of the apple rootstock to be detected is an LdLd genotype; if the Deletion molecular marker exists in one chromosome of the to-be-detected apple, the genotype of the to-be-detected apple is an LdLi genotype; and if the Deletion molecular marker does not exist in the two chromosomes of the apple to be detected, determining that the genotype of the apple to be detected is the LiLi genotype.
Further, the method for breeding apple rootstocks with different dwarfing traits comprises the following steps:
detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than that of the LiLi genotype apple rootstock;
if the two chromosomes of the apple to be detected are both lack of the molecular marker, the genotype of the apple to be detected is LdLd genotype; if one chromosome of the to-be-detected apple has the molecular marker, the genotype of the to-be-detected apple is the LdLi genotype.
The dwarfing property is the dwarfing property of apple rootstocks, namely the dwarfing property of the apple rootstocks, namely the dwarfing property of scions grafted on the rootstocks, and is characterized by dwarfing of plants, thinner trunk diameter, smaller plant crowns and the like.
The invention also aims to provide a specific primer pair for screening a molecular marker by using the apple rootstock dwarfing assistance, wherein the specific primer pair is designed based on the molecular marker and consists of a primer F and a primer R;
the primer F is b1 or b 2:
(upstream primer) MdLBD 3-d-F: 5'-GTCGTTGGCCTCTATTCTTCTGCAG-3', respectively;
b1 is a single-stranded DNA molecule shown in a sequence 2 of the sequence table;
b2 is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2;
the primer R is b3 or b 4:
(downstream primer) MdLBD 3-d-R: 5'-TCAAACAGACACCAAGTAGTTCCGT-3', respectively;
b3 is a single-stranded DNA molecule shown in a sequence 3 of the sequence table;
b4 is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
The invention also aims to provide an application of the specific primer pair, which comprises the following steps:
identifying the dwarfing character of the apple rootstock; predicting the dwarfing character of the apple rootstock; and breeding apple rootstocks with different dwarfing traits.
Further, the method for predicting the dwarfing character of the apple rootstock comprises the following steps:
taking the genome DNA of the apple rootstock to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only one DNA molecule and the size is 550bp, the plant is of the LdLi genotype; if the PCR amplification has no product, the plant is in the LiLi genotype;
the LdLi genotype is apple dwarfing stock, and the dwarfing property of the tree body of the LdLi genotype is greater than that of the apple stock of the LiLi genotype.
Further, the method for breeding apple rootstocks with different dwarfing traits comprises the following steps:
taking the genome DNA of the apple to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only one DNA molecule and the size is 550bp, the plant is of the LdLi genotype; if the PCR amplification has no product, the plant is in the LiLi genotype;
dwarfing of LdLi genotype apple rootstocks > dwarfing of LiLi genotype apple rootstocks.
Further, the dwarfing property is dwarfing property of the stock to the scion; the shorter the dwarfing property is, the larger the height of the tree is.
If the molecular marker exists in one of the two chromosomes of the apple to be detected, the genotype of the apple to be detected is an LdLi genotype, namely the apple to be detected is a dwarfing rootstock; and if the two chromosomes of the to-be-detected apple do not have the molecular marker, the genotype of the to-be-detected apple is a LiLi genotype which is an arbor stock.
Further, the apple dwarf rootstock is a plant which grows for more than 5 years after the rootstock is grafted with the variety, the height of the tree body is less than or equal to 3.5m, the diameter of a position 20cm close to the ground of a trunk is less than or equal to 6.0cm, and the diameter of a crown is less than or equal to 2.5 m.
By combining all the technical schemes, the invention has the advantages and positive effects that: the apple rootstock dwarfing auxiliary screening molecular marker and the application thereof provided by the invention have the advantages that candidate genes MdLBD3 related to apple rootstock dwarfing are obtained through fine positioning, and a Deletion polymorphism based on 11bp is developed and can be used as a molecular marker; further, the inventor verifies in the next generation extreme phenotype (vigorous/dwarf) and germplasm resources, and confirms that the molecular marker can be used for apple rootstock dwarf preselection.
The method can identify the genotype of the apple rootstock hybrid progeny and the rootstock resource only by PCR amplification with or without a strip and can judge the dwarfing property of the coming rootstock through genotype prediction, and is simple, rapid, convenient and effective. The method can predict dwarfing of the apple rootstock in the early seedling stage, can be used for early screening of the dwarfed apple rootstock, can screen dwarfing or vigorous phenotype of offspring in the early hybrid breeding stage, meets the requirements of various requirements (fruit tree scientific research, production and cultivation), and can improve the screening efficiency of apple rootstock breeding to a great extent and shorten the breeding period of the rootstock in view of the characteristics of long childhood period, complicated genetic background and the like of the apple rootstock.
The invention develops the molecular marker related to the dwarfing character, applies the molecular marker to assist breeding, compensates the defects of conventional hybrid breeding, is beneficial to directional selective breeding, shortens the breeding period, and provides a research method and a selection tool for the early screening and identification of apple rootstock breeding.
Drawings
FIG. 1 is an electrophoretogram (at 550bp, characteristic band) obtained by performing step two on 30 dwarf populations provided in the examples of the present invention.
FIG. 2 is an electrophoretogram obtained by performing step two on 30 georgette populations provided by the example of the present invention (no characteristic band at 550 bp).
FIG. 3 is a schematic diagram showing the comparison of tree heights of apple rootstock hybridization populations of different MdLBD3 genotypes (108 hybrid progeny).
FIG. 4 is a schematic diagram of the comparison of tree heights of apple rootstock germplasm resources of different MdLBD3 genotypes (64 resources) provided by the embodiment of the present invention.
FIG. 5 is a schematic diagram of the validation of MdLBD3 promoter monoclonal Deletion sequence differences in (vigorous/dwarf) rootstock resources and segregating populations.
FIG. 6 is an example of the use of MdLBD3 dwarf gene markers to identify dwarf phenotypes in apple rootstock resources 'M9' and 'Malus spectabilis'; a: 'M9' and 'Malus spectabilis' field dwarf phenotypes and corresponding genotypes; b: statistics and analysis were investigated on the ` M9 ` and ` Malus spectabilis ` field dwarf phenotype data.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the technical problems of high difficulty and long period of apple stock dwarf identification, a research team firstly takes important core parents such as Malus glabra ' Malus robusta (alias Malus glabra, Malus asiatica and Malus prunifolia, which are originally produced in China and belong to Malus wild species, are mainly distributed in northern China plain and have strong cold resistance and disease resistance), M9 ' Malus Mill (original Yellow merzpark Yellow Metz, seedling randomly selected in French merzsch in 1908, published in the British Dong forest test station in 1939, Chinese introduced in 1958 and have good dwarf property and early fruiting property) as materials (the 2 plant materials are stored in apple stock resources of a research institute of fruit trees and trees in Ningning province of ' province-level public welfare type research unit), a double-parental group heavy database is excavated, the dwarf gene is subjected to serialized logic sequencing, and the characteristic sequence difference of dwarf gene starter regions is screened, the molecular marker tightly linked with the dwarfing gene is developed and designed to be applied to apple rootstock dwarfing auxiliary screening, has the advantages of high identification accuracy, strong operability, low application cost and the like, has the value of being developed into a special kit for large-scale and commercialized popularization and application and the like, has originality, and has no same or similar reports at home and abroad before.
The present invention is described in detail below with reference to the attached drawings.
The invention firstly protects a molecular marker which is a1, a2 or a3 as follows:
a1 characteristic DNA molecule sequence SEQ ID NO: 1 is 5'-TCAACAACATA-3';
TABLE 1 analysis of differential sites in resource sequencing databases of "Malus Robusta", "M9' (Maluskuliula Mill) parental and rootstock
As shown in Table 1, parental re-sequencing shows that 11bp of deletion polymorphism (5'-TCAACAACATA-3') exists in dwarf rootstock resources 'M9' (Maluspalmila Mill M9) and 'B9' (Malus pumila Mill B9) at the 5 th linkage group 4.851Mb physical position and 339bp upstream from the transcription starting position, and the 11bp of polymorphism deletion does not exist in arbor rootstock resources 'Malus versicolor' (Malus robusta) and 'Malus baccata' (Malus baccata), and the deletion is a characteristic sequence of the dwarf rootstock resources.
a2 nucleotide 326-349 th from the start codon to the front (5' end) of the lateral growth tissue development gene MdLBD3 in the apple genome;
a3 is derived from DNA molecules with homology of more than 95%, 98% or 99% in the apple genome.
The invention also protects the application of the molecular marker, which is (c1), (c2) or (c 3):
(c1) identifying the dwarfing character of the apple rootstock;
(c2) predicting the dwarfing character of the apple rootstock;
(c3) and (4) breeding apple rootstocks with different dwarfing characters.
The invention also provides a method for predicting dwarfing of apple rootstocks, which comprises the following steps: detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than the dwarfing property of the LiLi genotype apple rootstock;
if two chromosomes of the apple rootstock to be detected have the Deletion molecular marker, the genotype of the apple rootstock to be detected is the LdLd genotype; if the Deletion molecular marker exists in one chromosome of the apple to be detected, the genotype of the apple to be detected is LdLi genotype (figure 5), and an application example shows that: dwarfing rootstock resources 'M9', 'B9', dwarfing types 4-54 and 5-38 in the segregation population all have the Deletion sequence polymorphism (figure 5); if the Deletion molecular marker does not exist in the two chromosomes of the apple to be detected, the genotype of the apple to be detected is the LiLi genotype, such as: the Deletion sequence polymorphisms do not exist in the arbor stock resources 'begonia octagon' and 'statio mountain', the arbor types 8-43, 8-36, 7-57, 6-125, 9-35, 11-43, 2-135, 9-55, 3-67 and the like in the segregation population (figure 5), and further show that the Deletion sequence and the genotypes corresponding to the molecular markers thereof completely accord with the dwarfing phenotype of the rootstock.
The invention also provides a method for breeding apple rootstocks with different dwarfing characters, which comprises the following steps: detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than that of the LiLi genotype apple rootstock;
the dwarfing property is the dwarfing property of apple rootstocks, namely the dwarfing property of the apple rootstocks, namely the dwarfing property of scions grafted on the rootstocks, and is characterized by dwarfing of plants, thinner trunk diameter, smaller plant crowns and the like.
The invention also protects a specific primer pair which is designed based on the molecular marker and consists of a primer F and a primer R;
the primer F is b1 or b2 as follows:
SEQ ID NO：2：
(upstream primer) MdLBD 3-d-F: 5'-GTCGTTGGCCTCTATTCTTCTGCAG-3', respectively;
b1 single-stranded DNA molecule shown in sequence 2 of the sequence table;
b2 DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function with the sequence 2;
the primer R is b3 or b4 as follows:
SEQ ID NO：2：
(downstream primer) MdLBD 3-d-R: 5'-TCAAACAGACACCAAGTAGTTCCGT-3' are provided.
b3 single-stranded DNA molecule shown in sequence 3 of the sequence table;
b4 DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
The invention also protects the application of the specific primer pair, which is (c1), (c2) or (c 3):
(c1) identifying the dwarfing character of the apple rootstock;
(c2) predicting the dwarfing character of the apple rootstock;
(c3) and (4) breeding apples with different dwarfing characters.
The invention also provides a method for predicting the dwarfing character of the apple rootstock, which comprises the following steps:
taking the genome DNA of the apple rootstock to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only one DNA molecule and the size is 550bp, the plant is LdLi genotype; if no product is obtained by PCR amplification, the plant is in the LiLi genotype;
the LdLi genotype is apple dwarfing stock, and the dwarfing property of the tree body of the LdLi genotype is greater than that of the apple stock of the LiLi genotype.
The invention also provides a method for breeding apple rootstocks with different dwarfing characters, which comprises the following steps:
taking the genome DNA of the apple to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only a DNA molecule and the size is 550bp, the plant is LdLi genotype; if the PCR amplification has no product, the plant is in the LiLi genotype;
dwarfing of LdLi genotype apple rootstocks > dwarfing of LiLi genotype apple rootstocks.
The dwarfing property is dwarfing property of the stock to the scion; the more dwarfing the tree, the shorter the tree, and the weaker the dwarfing the tree, the taller the tree.
If the molecular marker exists in one of the two chromosomes of the apple to be detected, the genotype of the apple to be detected is an LdLi genotype, namely the apple to be detected is a dwarfing rootstock; and if the two chromosomes of the to-be-detected apple do not have the molecular marker, the genotype of the to-be-detected apple is a LiLi genotype which is an arbor stock.
The apple dwarfing rootstock: the height of the tree body is less than or equal to 3.5m, the diameter of the position, 20cm, close to the ground, of the trunk is less than or equal to 6.0cm, and the diameter of the crown is less than or equal to 2.5m, which are plants with the age of more than 5 years after the stock is grafted.
The reaction system for any of the above PCR amplifications consists of (20. mu.L): 2 XPCR Mix 10. mu.L, MdLBD3-d-F solution 1.0. mu.L, MdLBD3-d-R solution 1.0. mu.L, template solution 2.0. mu.L, ddH2O6.0. mu.L. The concentration of MdLBD3-d-F in the MdLBD3-d-F solution is 10 mu M; MdLBD3-d-R solution, MdLBD3-d-R concentration is 10 u M. In the template solution, the DNA concentration was 500 ng/. mu.L.
The reaction procedure of any one of the above PCR amplifications may specifically be: 5min at 94 ℃; 36 cycles of 94 ℃ for 30s, 59 ℃ for 30s, and 72 ℃ for 50 s; storing at 72 deg.C for 7min and 4 deg.C.
Any one of the apples is a filial generation of an apple rootstock 'Malus octandra' (Malus robusta) and an apple rootstock 'M9' (Malus coluria Mill).
Any apple is pollen of apple rootstock 'eight-edge crabapple' (Malus robusta) and is a hybrid progeny obtained by pollinating emasculated apple rootstock 'M9' (Maluspalma Mill).
Any of the above apples may be an existing apple rootstock.
Any of the above apples is an apple plant.
Any one of the molecular markers, the specific primer pairs or the method can be used for apple rootstock breeding.
The technical solution of the present invention is further described with reference to the following examples.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, which were set up in triplicate, were averaged.
Example 1: screening of target Gene
Dwarf phenotypes such as plant height, trunk diameter, crown diameter and the like of a colony 1032 line are separated by hybridization of Malus octagona ' (Malus robusta) × ' M9 ' (Malusspuliu Mill) for investigation and statistics, and a high-density genetic map is used for QTL positioning to position the dwarf trait in a 0.86Mb interval of chromosome 5. According to the InDel mark of the re-sequencing, the QTL interval is shortened through a genetic method, and finally a gene related to the apple dwarf trait, namely the MdLBD3 gene, is obtained through cloning by combining gene functions.
Further research shows that a Deletion polymorphism of 11bp (5'-TCAACAACATA-3') exists in the promoter region of the MdLBD3 gene, and is 339bp upstream of the transcription start site of the gene. Defining a haploid with the 11bp Deletion nucleic acid as Ld and a haploid without the 11bp Deletion nucleic acid as Li; the apple is 2 ploid, parental sequencing shows that the Deletion of the gene is a heterozygous locus, and based on the Deletion polymorphism, the stock seedling and the resource are LdLi genotype or LiLi genotype.
A pair of primers is designed based on the 11bp Deletion polymorphism as follows:
(upstream primer) MdLBD 3-d-F: 5'-GTCGTTGGCCTCTATTCTTCTGCAG-3', respectively;
(downstream primer) MdLBD 3-d-R: 5'-TCAAACAGACACCAAGTAGTTCCGT-3' are provided.
Example 2: identification of dwarfing traits of progeny of dwarf and arbor parents by molecular markers
First, create the hybrid segregation population
1. Pollen of Malus spectabilis (Malus robusta) apple is taken, emasculated M9 (Malus pumila Mill) apple is pollinated, and hybrid seeds are harvested;
2. cultivating the hybrid seeds to obtain apple seedlings;
the planting place is a stock breeding test area of the fruit tree scientific research institute of Liaoning province, 1032 lines of apple seedlings are obtained, 3 plants of each line of apple seedlings are bred by cutting, and the seedlings are temporarily planted in a nursery garden for cultivation in the next year;
3. in spring, a hard branch grafting method is adopted, a 'Fuji' (Fuji) variety scion is grafted at a position 20cm close to the ground of a stock seedling, conventional management such as sprout removal, weeding, watering and the like is paid attention to in growing season, plant row spacing is fixedly planted according to 1m multiplied by 3m plant row spacing in spring next year, and normal operation management such as water fertilizing, weeding, pest control and the like is carried out.
Second, determination of dwarfing of apple rootstock seedlings
Dwarfing phenotypes such as plant height, trunk diameter, crown diameter and the like are investigated from the 4 th to 6 th year of permanent planting of stock-ear grafting separation groups, wherein the dwarfing contrast is 'M9', and the arbor contrast is 'malus octagona'. The investigation method is as follows:
plant height: the measurement is vertically carried out from the base to the tip of the tree body by a metric ruler to the accuracy of cm.
Dry diameter: the diameter is measured by calipers in two crossed directions at the position 10cm above the grafting opening of the anvil segment, and the accuracy is 0.1 cm.
3. Crown diameter: and measuring the maximum diameter of the crown in east-west direction and south-north direction to be accurate to cm.
Third, genotype identification
1. Extracting the genome DNA of the root system of the isolated colony.
2. And (3) performing PCR amplification by using the genomic DNA extracted in the step 1 as a template and adopting a primer pair consisting of MdLBD3-d-F and MdLBD 3-d-R.
Reaction system for PCR amplification (20 μ L): 2 XPCR Mix 10. mu.L, MdARF6-i-F solution 1.0. mu.L, MdARF6-i-R solution 1.0. mu.L, template solution 2.0. mu.L, ddH2O6.0. mu.L. MdARF6-i-F solution at a concentration of 10. mu.M; MdARF6-i-R solution, the concentration of which is 10. mu.M. In the template solution, the DNA concentration was 500 ng/. mu.L.
Reaction procedure for PCR amplification: 5min at 94 ℃; 36 cycles of 94 ℃ for 30s, 59 ℃ for 30s, 72 ℃ for 50 s; storing at 72 deg.C for 7min and 4 deg.C.
3. And (3) carrying out 1.5% agarose gel electrophoresis on the PCR amplification product obtained in the step (2).
If the PCR amplification product is a DNA molecule and the size is 550bp, the plant is LdLi genotype; if the PCR amplification product has no band at 550bp, the plant is of the LiLi genotype.
Genotype of dwarf type plant and arbor type plant
If the plant height of the apple rootstock-ear grafting complex of a certain mature plant is about 3.5m, the apple rootstock is a dwarfing type rootstock; if the plant height of the apple stock-ear grafting complex of a certain mature plant is about 4.7m, the apple plant is an arbor type stock.
30 plants which are identified as extreme dwarfing type and 30 plants which are identified as extreme vigorous type are picked from 1032 lines of apple rootstock seedlings for three consecutive years.
30 of the 30 extremely dwarf plants are LdLi genotypes, and 0 is LiLi genotype; of the 30 extremely vigorous plants, 30 were of the LiLi genotype and 0 was of the LdLi genotype. In the 60 segregating groups, the LdLi genotype exists only in extremely dwarf plants, and the LiLi genotype exists only in extremely vigorous plants.
The electrophoresis chart obtained by the step two of partial plants in the 30 extremely dwarf plants is shown in figure 1, and different lanes correspond to different plants; and (3) electrophoresis images obtained by the step two of partial plants in the 30 extremely vigorous plants are shown in the figure 2, and different lanes correspond to different plants.
Average tree dwarfing of five, different genotype groups
108 plants were randomly selected from 1032 apple seedlings.
108 plants are divided into two groups according to genotypes, and the average value of the malic acid content of fruits in each group is counted, wherein the results are as follows:
plants of LdLi genotype, 52, with an average plant height of 3.45 + -0.37 m (see FIG. 3);
the average plant height of the LiLi genotype, 56 plants, was 4.75 ± 0.41m (see fig. 3).
The significant correlation is shown at the level of P <0.01, which shows that the 11bp Deletion polymorphism genotype discovered in example 1 has better significance on the dwarfing of rootstock seedlings.
Comparison of tree heights (64 resources) for different MdLBD3 genotype apple rootstock germplasm resources is shown in fig. 4.
Combining the results, the LdLi genotype is completely associated with the dwarfing character of the stock, and the LiLi genotype is completely associated with the arbor dwarfing character; for the height trait of the apple scion combined tree body, the LdLi genotype plant height is greater than the LiLi genotype plant height. The Deletion polymorphism of 11bp found in example 1 can be used for early screening of dwarfing of seedlings in apple rootstock crossbreeding.
Sequencing verification is carried out on the plants in the fourth step and the plants in the fifth step: the PCR amplification product of the LdLi genotype plant is only one DNA molecule with the size of 550bp, and the LiLi genotype plant has no 550bp characteristic fragment or PCR amplification product.
Example 3: identification of dwarfing of Malus rootstock germplasm resources by using molecular marker
The following operations are respectively carried out on 64 existing malus stock germplasm resources:
and (3) detecting dwarfing of the apple rootstock germplasm resources, wherein the method is the same as the second step of the example 2.
Genotyping was performed in the same manner as in step three of example 2.
Dividing 64 varieties into two groups according to genotypes, and counting the average dwarfing value of each group, wherein the result is as follows:
25 LdLi genotypes, the average plant height is 3.50 +/-0.41 m;
39 LiLi genotypes are used, and the average plant height is 4.82 +/-0.45 m.
The significant correlation is shown at the level of P <0.01, which shows that the 11bp Deletion polymorphism discovered in example 1 has better significance on rootstock resource dwarfing.
Some results are shown in table 1.
Table 1 partial results
Sequencing verification is carried out on 64 rootstock resources: the PCR amplification product of the LdLi genotype plant is only a DNA molecule and the size is 550 bp; no product is generated by PCR amplification of plants of the LiLi genotype; the PCR amplification product of LdLi genotype plant has specific molecular mark, and the 550bp characteristic band may be used as molecular mark for identifying dwarfing of apple stock seedling and resource.
The technical effects of the present invention will be described in detail with reference to experiments.
FIG. 6 application of MdLBD3 dwarf gene markers in apple rootstock resources ` M9 ` (Maluskulium Mill.) and ` Malus glabra ` (Malus robusta) to identify dwarf phenotypes; a: 'M9' and 'Malus spectabilis' field dwarf phenotype and corresponding genotype; b: statistics and analysis were investigated on the ` M9 ` and ` Malus spectabilis ` field dwarf phenotype data. 'eight-edge crabapple' and 'M9' are apple rootstocks commonly used in production, wherein the 'eight-edge crabapple' is an arbor, the 'M9' is a dwarfing anvil, the field dwarfing of 'Fuji' (Malus domestica Fuji.) grafted on two rootstocks for 2016-2019 for three consecutive years is identified on three characters of plant height, trunk diameter and crown diameter, and the results show that: the dwarf genotype of M9 'is LiLi with plant height, stem diameter and crown diameter of 2.78M, 3.34cm and 1.45M respectively, and the dwarf genotype of malus spectabilis' is LiLi with large values of plant height, stem diameter and crown diameter of 4.26M, 5.64cm and 2.26M respectively (figure.6); the dwarf phenotype values (plant height, stem diameter and crown diameter) of 'M9' are 65.2%, 59.3% and 64.2% of 'eight-edge crab' respectively (figure 6); the data are stable in the year, the standard deviation is 0.10-0.22, and the average value is 0.15. The field investigation values of the rootstock resources 'M9' and 'Malus spectabilis' dwarf phenotype completely accord with respective genotypes, and the application example further proves that the developed and designed dwarf gene marker can accurately identify the field dwarf phenotype value of the rootstock resources.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.
Sequence listing
<110> research institute for fruit tree science in Liaoning province
<120> apple rootstock dwarfing auxiliary screening molecular marker and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcaacaacat a 11
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtcgttggcc tctattcttc tgcagtcaaa cagacaccaa gtagttccgt 50
Claims (10)
1. The molecular marker for apple rootstock dwarf assisted screening is characterized in that the molecular marker for apple rootstock dwarf assisted screening is a1, a2 or a 3;
a1 a Deletion polymorphism of 11bp existing at the upstream of the transcription initiation site of a lateral growth tissue development gene MdLBD3 in the genome of an apple rootstock, wherein the Deletion polymorphism is tightly linked with the dwarfing property of the rootstock; the characteristic DNA molecular sequence of the a1 is 5'-TCAACAACATA-3';
a2 designing a pair of primers according to the 11bp Deletion polymorphism:
MdLBD3-d-F：5′-GTCGTTGGCCTCTATTCTTCTGCAG-3′；
MdLBD3-d-R：5′-TCAAACAGACACCAAGTAGTTCCGT-3′；
the a2 is the 326 th and 349 th nucleotides of the lateral growth tissue development gene MdLBD3 'from the initial codon to the front 5' end in the apple genome;
the method is characterized in that DNA of apple rootstock seedlings or germplasm resources is taken as a template, a reaction system for 20 mu LPCR amplification is adopted, and the reaction procedure of PCR amplification is as follows: 5min at 94 ℃; 36 cycles of 94 ℃ for 30s, 59 ℃ for 30s, and 72 ℃ for 50 s; storing at 72 deg.C for 7min and 4 deg.C; after the PCR generation under the reaction program is detected by 1.5% agarose electrophoresis, if a strip exists at the position of 550bp, the material is indicated to be a dwarfing stock; if the PCR product does not exist or no band exists at 550bp, the material is the vigorous rootstock;
a311bp insertion polymorphism and development of DNA molecules designed to have a homology of more than 95%, 98% or 99% to the designed primer sequence.
2. The apple rootstock dwarf assisted selection molecular marker of claim 1, wherein the reaction system of PCR amplification comprises: 2 XPCR Mix 10. mu.L, MdLBD3-d-F solution 1.0. mu.L, MdLBD3-d-R solution 1.0. mu.L, template solution 2.0. mu.L, ddH2O6.0 μ L; the concentration of MdLBD3-d-F in the MdLBD3-d-F solution is 10 mu M; the concentration of MdLBD3-d-R in the MdLBD3-d-R solution is 10 mu M; in the template solution, the DNA concentration was 500 ng/. mu.L.
3. The application of the apple rootstock dwarf auxiliary screening molecular marker according to any one of claims 1-2, wherein the application of the apple rootstock dwarf auxiliary screening molecular marker comprises the following steps: identifying the dwarfing character of the apple rootstock; predicting the dwarfing character of the apple rootstock; and breeding apple rootstocks with different dwarfing characters.
4. The application of the apple rootstock dwarf assisted molecular marker in claim 3, wherein the method for predicting the dwarf trait of the apple rootstock comprises the following steps: detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than that of the LiLi genotype apple rootstock;
if two chromosomes of the apple rootstock to be detected have the Deletion molecular marker, the genotype of the apple rootstock to be detected is an LdLd genotype; if the Deletion molecular marker exists in one chromosome of the to-be-detected apple, the genotype of the to-be-detected apple is an LdLi genotype; and if the Deletion molecular marker does not exist in the two chromosomes of the apple to be detected, determining that the genotype of the apple to be detected is the LiLi genotype.
5. The application of the apple rootstock dwarf assisted molecular marker in the claim 3, wherein the method for breeding the apple rootstocks with different dwarf traits comprises the following steps: detecting the genotype of the apple rootstock to be detected based on the molecular marker, wherein the dwarfing property of the LdLi genotype apple rootstock is greater than that of the LiLi genotype apple rootstock;
if the two chromosomes of the apple to be detected are both lack of the molecular marker, the genotype of the apple to be detected is LdLd genotype; if one chromosome of the to-be-detected apple has the molecular marker, the genotype of the to-be-detected apple is an LdLi genotype;
the dwarfing property is the dwarfing property of apple rootstocks, namely the dwarfing property of the apple rootstocks, namely the dwarfing property of scions grafted on the rootstocks, and is characterized by dwarfing of plants, thinner trunk diameter and smaller plant crowns.
6. The specific primer pair for screening the molecular marker by using the apple rootstock dwarfing assistance of any one of claims 1-2, is characterized in that the specific primer pair is designed based on the molecular marker and consists of a primer F and a primer R;
the primer F is b1 or b 2:
MdLBD3-d-F：5′-GTCGTTGGCCTCTATTCTTCTGCAG-3′；
b1 is a single-stranded DNA molecule shown in a sequence 2 of the sequence table;
b2 is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2;
the primer R is b3 or b 4:
MdLBD3-d-R：5′-TCAAACAGACACCAAGTAGTTCCGT-3′；
b3 is a single-stranded DNA molecule shown in a sequence 3 of the sequence table;
b4 is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
7. Use of the specific primer pair according to claim 6, wherein the use of the specific primer pair comprises: identifying the dwarfing character of the apple rootstock; predicting the dwarfing character of the apple rootstock; and breeding apple rootstocks with different dwarfing characters.
8. The use of the specific primer pair of claim 7, wherein the method for predicting the dwarfing trait of apple rootstock comprises: taking the genome DNA of the apple rootstock to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only one DNA molecule and the size is 550bp, the plant is of the LdLi genotype; if the PCR amplification has no product, the plant is in the LiLi genotype;
the LdLi genotype is apple dwarfing stock, and the dwarfing property of the tree body of the LdLi genotype is greater than that of the apple stock of the LiLi genotype.
9. The use of the specific primer pair according to claim 7, wherein the method for breeding apple rootstocks with different dwarf traits comprises: taking the genome DNA of the apple to be detected as a template, and carrying out PCR amplification by adopting the specific primer pair; if the PCR amplification product is only one DNA molecule and the size is 550bp, the plant is of the LdLi genotype; if the PCR amplification has no product, the plant is in the LiLi genotype;
dwarfing of LdLi genotype apple rootstocks > dwarfing of LiLi genotype apple rootstocks.
10. The use of a specific primer pair according to claim 9, wherein the dwarfing is dwarfing of the scion by the rootstock; the more dwarfing the tree, the shorter the tree, and the more dwarfing the tree, the higher the tree;
if the molecular marker exists in one of the two chromosomes of the apple to be detected, the genotype of the apple to be detected is an LdLi genotype, namely the apple to be detected is a dwarfing rootstock; if the two chromosomes of the to-be-detected apple do not have the molecular marker, the genotype of the to-be-detected apple is a LiLi genotype which is an arbor stock;
the apple dwarfing rootstock is a plant which grows for more than 5 years after the rootstock is grafted with the variety, the height of a tree body is less than or equal to 3.5m, the diameter of a position, close to 20cm of the ground, of a trunk is less than or equal to 6.0cm, and the diameter of a crown is less than or equal to 2.5 m.
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| CN112322618A (en) * | 2020-12-02 | 2021-02-05 | 辽宁省果树科学研究所 | Molecular marker and application thereof in apple rootstock dwarfing screening |
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| CN112322618A (en) * | 2020-12-02 | 2021-02-05 | 辽宁省果树科学研究所 | Molecular marker and application thereof in apple rootstock dwarfing screening |
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