CN114763579B - Molecular marker related to melon fruit shape, and method and application thereof - Google Patents
Molecular marker related to melon fruit shape, and method and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to melon fruit shape, a method and application thereof. The invention detects the application of the substance containing the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4 in the genome of the melon to be detected in the following steps: identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected; breeding melon varieties with long fruit shapes in the generation of seeds; and breeding melon varieties with offspring being oblate fruits. Experiments prove that the molecular marker disclosed by the invention is related to the fruit shape character of the muskmelon, the accuracy of identifying the fruit shape of the muskmelon by using the molecular marker disclosed by the invention can reach 100%, and the molecular marker can be further applied to auxiliary breeding, gene polymerization breeding and transgenic breeding of the muskmelon. Can be screened and identified at any stage of melon growth, has high efficiency, good specificity and high accuracy, greatly saves time and cost, and has important significance for accelerating melon and fruit shape breeding.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a melon fruit shape-related molecular marker, a method and application thereof.
Background
Melon (cutemis melo l.) is an annual herb of the genus melo of the family cucurbitaceae, and is an important horticultural cash crop at home and abroad. The variety of melon fruit related characters is quite rich, in recent years, along with the increasing of the living standard of people, the demand degree of the melon is also larger and larger, and meanwhile, the commodity of the melon is also higher, and the commodity of the melon is directly influenced by the melon fruit characters, so that the improvement of the internal and external quality of the melon is an important working content of the current fine variety breeding of the melon. Researches show that the related traits of melon fruits are controlled by a plurality of Quantitative Trait Loci (QTLs) and are affected differently by environmental effects. Therefore, the genetic mode of melon fruit related characters is deeply known, so that favorable reference and help can be provided for genetic improvement of fruit characters and improvement of fruit commodity, and a theoretical basis is also provided for further deep research.
In recent years, a great deal of genetic research on melon fruit-related traits is carried out by scholars at home and abroad. The same recombinant inbred line group (81 lines) is used as a mapping group by Zalapa and Paris and the like, and QTL analysis is carried out on melon yield-related traits, fruit diameter, seed cavity diameter, fruit proportion and other fruit-related traits. In 2002, perin et al performed QTL analysis on fruit shape by using two RIL populations obtained by crossing two kinds of elongated melon varieties 'PI 161375' and 'PI 414723' with the same circular melon variety 'velrantais', respectively, and showed that melon sex type gene a was closely linked with one major QTL of fruit length in linkage group No. 2. Wang Xianlei genetic localization of melon shape by using Japanese 'Annong No. two' and Xinjiang Hami melon 'K413' as materials, and found that QTLs for controlling melon shape exist on C2 linkage group of FS2.1 and FS 2.2. The QLFL 2.1 for controlling the fruit length and seven QTLs for controlling the fruit shape index are only found by carrying out QTL analysis on the related characters of melon fruits and seeds by using the muskmelon line ' ms-5 and the muskmelon line ' HM-1' as materials by the QNQI et al: FS2.1, FS4.1, FS6.1, FS6.2, FS7.1, FS7.2, FS7.3.Aurora Diaz et al, F obtained by crossing an Indian wild melon variety ' Trigonus ' with an European and American elite cultivar ' pixel de Sapo 2 QTL analysis of the fruit acclimation-related traits was performed on the population, and four QTLs were found that controlled fruit length: flqts controlling fruit shape are three QTLs, flqt2.1, flqt4.1, flqt6.1, flqt 8.1: fsqt2.1, fsqt4.1, fsqt6.1, wherein flqt2.1 and fsqt2.1, flqt4.1 and fsqt4.1, flqt6.1 and fsqt6.1 are all located in the same linkage group and in the same region. In 2018, marios et al have shown that melon fruit shape and fruit width are controlled by different genetic controls, fruit shape is mainly controlled by fruit length, fruit width is mainly determined by fruit transverse diameter, QTLs controlling fruit shape are mainly concentrated on chromosome 1, chromosome 2, chromosome 8 and chromosome 11, and QTLs controlling fruit width are mainly concentrated on chromosome 2, chromosome 3, chromosome 8 and chromosome 11.
In conclusion, melon shapes are regulated by multiple genes, and artificial breeding of fruit shapes is difficult to develop through conventional sexual hybridization in breeding, so that development of specific molecular markers according to gene control sites of the fruit shapes is urgently needed to carry out molecular marker assisted selection of the fruit shapes, so that the breeding period is shortened, the breeding process is accelerated, and the requirement of melon shape breeding is met.
Disclosure of Invention
It is an object of the present invention to provide the use of a substance for detecting whether the genome of a melon to be detected contains a nucleotide represented by sequence 3 or a nucleotide represented by sequence 4.
The invention provides an application of a substance for detecting whether the genome of a melon to be detected contains a nucleotide shown in a sequence 3 or a nucleotide shown in a sequence 4 in at least one of the following 1) to 10):
1) Identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
2) Preparing and identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
3) Breeding melon varieties carrying long-fruit shape genotypes;
4) Preparing and breeding a product of melon varieties carrying long round fruit genotypes;
5) Breeding melon varieties carrying oblate fruit genotypes;
6) Preparing and breeding products of melon varieties carrying oblate fruit genotypes;
7) Breeding melon varieties with long fruit shapes in the generation of seeds;
8) Preparing a product of melon varieties with long fruit shapes in seed selection sub-generations;
9) Selecting melon varieties with offspring of oblate fruits;
10 Preparing a product of melon varieties with flat round fruit-shaped seed generations;
the fruit genotype is oblate fruit genotype A2A2, long fruit genotype A1A1 or long fruit genotype A1A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in sequence 3, and the other homologous chromosome contains nucleotide shown in sequence 4.
In the above application, the substance is 1) or 2) as follows:
1) A set of primers, the set of primers satisfying: the DNA fragment obtained by PCR amplification with muskmelon genome DNA as a template contains a sequence shown as a sequence 3 or a sequence 4, wherein the difference between the sequence 3 and the sequence 4 is in a region of nucleotides 64-83 of the sequence 4 in a sequence table (shown in figure 3).
The embodiment of the invention specifically comprises a single-stranded DNA molecule or a derivative thereof shown in a sequence 1 in a sequence table and a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in the sequence table;
2) PCR reagents or kits containing the set of primers.
It is another object of the invention to provide a product.
The product provided by the invention comprises the substances;
the product has at least one of the following functions 1) -5):
1) Identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
2) Breeding melon varieties carrying long-fruit shape genotypes;
3) Breeding melon varieties carrying oblate fruit genotypes;
4) Breeding melon varieties with long fruit shapes in the generation of seeds;
5) And breeding melon varieties with offspring being oblate fruits.
The invention also provides a biological material, which comprises the DNA molecule shown in the sequence 3 and/or the DNA molecule shown in the sequence 4.
At least one function of the above mentioned biological material in the following 1) -5) is also within the scope of the present invention:
1) Identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
2) Breeding melon varieties carrying long-fruit shape genotypes;
3) Breeding melon varieties carrying oblate fruit genotypes;
4) Breeding melon varieties with long fruit shapes in the generation of seeds;
5) And breeding melon varieties with offspring being oblate fruits.
It is an object of the invention to provide the following method.
The invention provides a method for identifying or assisting in identifying the genotype of the fruit shape of a melon to be detected, which comprises the following steps:
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 3, the fruit genotype of the melon to be detected is a long fruit genotype A1A1;
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3 and the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is a long fruit genotype A1A2;
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is oblate fruit genotype A2A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in sequence 3, and the other homologous chromosome contains nucleotide shown in sequence 4.
Or, the invention provides a method for identifying or assisting in identifying the fruit shape of the melon or progeny thereof to be detected, which comprises the following steps:
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3, the melon to be detected or the progeny fruit shape thereof is a long fruit,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 4 and does not contain the nucleotide shown in the sequence 3, the melon to be detected or the progeny thereof has a oblate shape.
In the method, whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4 is detected as follows:
1) Directly sequencing the genome DNA to judge;
2) Amplifying the melon to be detected by using the set of primers, sequencing amplified products, and judging.
The amplification is performed using the set of primers described above, and the concentration of both the FS-F and the FS-R in the reaction system may be 0.5. Mu. Mol/L. Specifically, the following reaction system can be adopted for PCR amplification: 2. Mu.L Buffer, 1.6. Mu.L dNTPs (each concentration of the four dNTPs is 2 mmol/L), the FS-F and the FS-R, 1.5. Mu.L of the melon genomic DNA to be tested (50 ng/. Mu.L), 0.2DNA Polymerase for PAGE，ddH 2 O was added to 20. Mu.L. Buffer and->DNA Polymerase for PAGE can be Beijing all gold biotechnology limited company.
The annealing temperature at the time of PCR amplification using the primer pair may be 59 ℃. The PCR amplification by using the primer pair can be specifically carried out by adopting the following reaction conditions: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 5min.
Or, the invention provides a method for breeding melon varieties carrying long fruit shape genotypes or breeding melon varieties with long fruit shapes or melon varieties with long fruit shapes selected by seed generations, which is used for breeding melons to be detected, wherein the fruit shape genotypes in the first method are the long fruit shape genotypes A1A2 or A1A 1.
Or, the invention provides a method for breeding melon varieties carrying oblate fruit genotypes or breeding oblate fruit varieties or breeding melon varieties with oblate fruit generations, which is to select melon to be detected, wherein the fruit genotypes in the first method are oblate fruit genotypes A2A2.
The long fruits are in the shape of length-width ratio >1; the round or oblate fruit shape has an aspect ratio of less than or equal to 1.
The melon fruit-shaped molecular marker also belongs to the protection scope of the invention.
In the invention, the melon can be a long fruit melon variety B8 or a flat round fruit melon variety HP22 or a filial generation of the long fruit melon variety B8 or the flat round fruit melon variety HP 22.
The offspring of the long fruit melon variety B8 comprise various generations obtained by hybridization and/or backcrossing of the long fruit melon variety B8 serving as a parent and other melons.
The offspring of the oblate fruit melon variety HP22 comprise various generations obtained by crossing and/or backcrossing the oblate fruit melon variety HP22 serving as a parent with other melons.
In an embodiment of the invention, the progeny of the cross is the progeny of the cross of the long fruit melon variety B8 with the oblate fruit melon variety HP 22.
Experiments prove that the melon fruit-shaped molecular marker (FS) is related to the fruit shape of melon, and the melon with the A2A2 genotype, wherein both chromosomes of the melon are nucleotide sequences shown as sequence 4 in the sequence table, is circular or oblate melon. The accuracy of identifying the shape of the melon can reach 100% by utilizing the melon shape molecular marker, the melon shape molecular marker can be further used for auxiliary breeding of the melon shape molecular marker, screening and identifying can be carried out at any stage of melon growth, the efficiency is high, the specificity is good, the accuracy is high, the time and the cost are greatly saved, and the method has important significance for accelerating the breeding of the melon shape.
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FIG. 1 shows the melon parent and F 1 Fruit substitute shape; a: melon parent and F 1 A fruit-bearing phenotype; b: melon parent and F 1 Fruit length statistics.
FIG. 2 is a labeled FS pair parent and F thereof 1 Part BC 1 F 1 The detection result of the single plant is replaced; m is 100bp DNA ladder; p (P) 1 Is HP22; p (P) 2 B8; f (F) 1 Hybridization F with B8 as male parent and HP22 as female parent 1 Substitution; 1-31 is BC 1 F 1 The order of the generation of single plants is as follows: no.2, no.3, no.4, no.5, no.6, no.7, no.13, no.14, no.15, no.17, no.18, no.19, no.10, no.11, no.12, no.20, no.21, no.22, no.31, no.32, no.33, no.42, no.43, no.44, no.33, no.34, no.35, no.61, no.62, no.63, no.64.
FIG. 3 shows the alignment of the sequences of the fragments of interest of the parental B8, HP22 and reference genome.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples are only illustrative of the present invention and are not intended to limit the invention. In the embodiment, if no special description exists, the adopted experimental methods are all conventional methods; the reagents and the like used are all commercially available.
The biological material was obtained from the applicant by the public of the long fruit seed B8 (described in Ma Jian, wang Jianshe. Molecular marker development and application of melon total-female regulatory gene g [ J ]. Plant genetic resource journal, 2019, 20 (4): 1080-1086), and was used only for repeated experiments related to the present invention, but was not used for other purposes.
The flat fruit seed HP22 (described in Ma Jian, li Congcong, zhang Meng, wang Jianshe. Genetic analysis of melon yellow seed coats, mark development [ J ]. Gardening journal, 2019, 46 (S1): 2700) in the examples described below) is publicly available to the applicant for this biological material, which was used only for repeated experiments related to the invention, and was not used for other purposes.
The long fruit in the following examples is in the form of an aspect ratio >1, and the oblate fruit is in the form of an aspect ratio +.1.
The aspect ratio is the length of the fruit/width of the fruit (transverse diameter of the fruit).
In the following examples, F1 generation was obtained by crossing with variety B8 as female parent and variety HP22 as male parent 1 Substitution;
BC in the following examples 1 F 1 The generation is hybridized by taking B8 as a female parent and HP22 as a male parent to obtain F 1 Then taking HP22 as recurrent parent backcross to obtain BC 1 F 1 And (5) generating a population.
Example 1 genetic analysis of melon shape
Melon B8 is a high-generation inbred line of the cantaloupe with crisp cantaloupe, the length of fruits is 27.7+/-1.24 cm, the width of the fruits is 7.5+/-0.8 cm, the length-width ratio of the fruits is 3.69, and the fruits are long fruits; melon HP22 is a high-generation inbred line of muskmelon, the length of the fruits in the mature period is about 10.1+/-0.59 cm, the width of the fruits is 11.5+/-0.6 cm, the length-width ratio of the fruits is 0.82, and the fruits are oblate fruits (figure 1A). Hybridization is carried out by taking B8 as male parent, HP22 as female parent or taking B8 as female parent and HP22 as male parent to obtain F 1 Instead of, forward and backward crossing F 1 The fruit length of the plant is 18.9+ -1.6 cm, the fruit width is 8.5+ -0.5 cm, the length-width ratio of the fruit is 2.22, and the fruit is medium-length fruit (figure 1B). These data phenotypically oblate fruit shapes are semi-dominant inheritance patterns.
By F 1 The generation (obtained by hybridization with B8 as female parent and HP22 as male parent) is used as female parent and HP22 as male parent to obtain BC 1 F 1 Population of generations, planting 262 BC strains 1 F 1 For the generation of single plants, each plant obtained one selfed fruit in an selfing mode, the length and width of each single plant fruit were measured by a ruler in the mature period, and the aspect ratio was calculated, and the statistical data are shown in Table 1. Wherein the fruit shape is long fruit shape (i.e. aspect ratio>1) The single plant 141 is a single plant 121 with a round or oblate fruit shape (namely, the aspect ratio is less than or equal to 1), and the separation ratio of the fruit shape is long fruit shape: round or oblate fruit = 141:121, exhibiting a mendelian genetic segregation ratio (χ 2 ＝1.37<3.84 Indicating that the round or oblate fruit trait is controlled by a single gene.
Table 1 shows parents and BC thereof 1 F 1 Statistical analysis of phenotype and genotype of generation individual plant
EXAMPLE 2 development, verification and establishment of method for detecting melon and fruit-shaped specific molecular marker FS
1. Development of melon and fruit shape specific molecular marker FS
In order to rapidly obtain a target region of the fruit-shaped control gene on a chromosome, a mixed pool sequencing method (BSA) is utilized to initially locate the target gene. Extracting parent genome DNA to construct two parent pools, at BC 1 F 1 And respectively selecting 30 long fruit-shaped single plants and round or oblate fruit-shaped single plants from the generation group to extract genome DNA to construct mixed pools L and S, and carrying out mixed pool sequencing on the constructed mixed pools by using a high-throughput method, wherein the parent sequencing depth is 20X, and the offspring mixed pool sequencing depth is 30X. Data correlation analysis was performed using ΔSNP-index and ΔInDel-index based on the Euclidean distance (Euclidean Distance, ED) algorithm, and finally the gene of interest was located within the interval of chromosome 8 24.88 Mb-28.44Mb (reference genome version V3.6.1), with a total length of 3.56Mb, where the correlation threshold was 0.49. According to the interval of the target gene, the parent B8 and HP22 genome sequences are found to have an InDel (InDel) site at the corresponding physical position 28262326 of the reference genome. Therefore, based on the difference site, the invention designs a molecular marker FS capable of specifically detecting the site, the markerConsists of the nucleotide sequences shown in the sequence 1 and the sequence 2:
FS-F: CTCGCCCCCACAGTTCTAAA (sequence 1);
FS-R: TGATAATGTCACACACACGCA (SEQ ID NO: 2).
2. Application of melon and fruit shape specific molecular marker FS
Two parents B8, HP22 and their hybridization F using the above primer pairs FS-F and FS-R 1 And carrying out PCR amplification instead of the above to obtain a PCR amplification product.
The reaction system for PCR amplification is as follows: 2. Mu.L Buffer, 1.6. Mu.L dNTPs (each of the four dNTPs at a concentration of 2 mmol/L), 0.5ul FS-F (each of the four dNTPs at a concentration of 0.5. Mu. Mol/L in the system), 0.5ul FS-R (each of the four dNTPs at a concentration of 0.5. Mu. Mol/L in the system), 1.5. Mu.L of the genomic DNA of the melon to be tested (50 ng/. Mu.L), 0.2DNA Polymerase for PAGE，ddH 2 O was added to 20. Mu.L.
The reaction procedure for the PCR amplification is as follows: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 59℃for 30s, extension at 72℃for 30s, and running for 35 cycles; extending at 72℃for 5min.
Detecting PCR amplification products by 8% polyacrylamide gel electrophoresis, wherein the detection result is shown in figure 2, and M is 100bp DNA ladder; p (P) 1 Is HP22; p (P) 2 B8; f (F) 1 Hybridization F with B8 as female parent and HP22 as male parent 1 Substitution; 1-31 is BC 1 F 1 The order of the generation of single plants is as follows: no.2, no.3, no.4, no.5, no.6, no.7, no.13, no.14, no.15, no.17, no.18, no.19, no.10, no.11, no.12, no.20, no.21, no.22, no.31, no.32, no.33, no.42, no.43, no.44, no.33, no.34, no.35, no.61, no.62, no.63, no.64; the bands of the parents B8 (genotype A1A 1) and HP22 (genotype A2A 2) show polymorphism, and the hybridization F 1 The generation is two bands (genotype A1A 2).
Subsequently, the PCR products amplified by the two parents were submitted to a company for sequencing, and the sequencing result analysis is shown in FIG. 3, wherein the amplification product in B8 is 114bp (the nucleotide sequence is sequence 3) and the amplification product in HP22 is 122bp (the nucleotide sequence is sequence 4) compared with the 130bp of the reference genome sequence.
The genotype of a variety corresponding to a PCR product having a nucleotide sequence of sequence 3 and not sequence 4 was designated as a long-fruit genotype A1A1;
the fruit genotype of a variety corresponding to a PCR product containing a nucleotide sequence of sequence 4 and not sequence 3 is designated as oblate fruit genotype A2A2;
the fruit genotype of the variety corresponding to the PCR products containing nucleotide sequences 3 and 4 was designated as the long fruit genotype A1A2.
The oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain the nucleotide shown in the sequence 3 and all do not contain the nucleotide shown in the sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in sequence 3, and the other homologous chromosome contains nucleotide shown in sequence 4.
BC 1 F 1 In the generation of the single plant, the plant,
0 PCR products in 141 long fruit varieties are 114bp, and the fruit genotype is A1A1; there are 141 PCR products of 114bp and 122bp, and the genotype of the fruit shape is A1A2.
121 PCR products in 121 offspring single plants with oblate fruit shapes are 122bp, and the genotype of the fruit shape is A2A2.
The experimental result shows that the marker FS detection genome of the invention contains the sequence 3 or the sequence 4 which is related to the fruit shape genotype of the melon, and can be used for detecting the fruit shape genotype of the melon or the fruit shape of the offspring thereof.
Therefore, by detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4, the genotype of the fruit shape of the melon to be detected or whether the child fruit shape of the melon to be detected is a long fruit shape or a oblate fruit shape is identified or assisted to be identified;
the method comprises the following steps:
1) A method for identifying or aiding in the identification of the genotype of the fruit shape of a melon to be tested, comprising in particular the following steps:
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 3, the fruit genotype of the melon to be detected is a long fruit genotype A1A1;
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3 and the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is a long fruit genotype A1A2;
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 4, the genotype of the melon to be detected is oblate genotype A2A2;
2) A method for identifying or assisting in identifying whether the melon or progeny thereof to be tested is in the form of a long or flat fruit;
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3, the melon to be detected or the progeny fruit shape thereof is a long fruit,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 4 and does not contain the nucleotide shown in the sequence 3, the melon to be detected or the progeny thereof has a oblate shape.
The method for detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4 comprises the following steps:
1) Direct sequencing;
2) Amplifying the melon to be detected by using the primer pair marked with FS, sequencing the amplified product, and judging whether the amplified product contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4.
EXAMPLE 3 melon shape specific molecular marker FS at BC 1 F 1 Verification in a population
262 BC are extracted by using CTAB method 1 F 1 Genomic DNA of the individual plants was first subjected to PCR using the above primer set FS-F and primer set FS-R for 262 individual plantsAmplifying to obtain PCR amplified product.
The reaction system for PCR amplification is as follows: 2. Mu.L Buffer, 1.6. Mu.L dNTPs (each of the four dNTPs at a concentration of 2 mmol/L), 0.5ul FS-F (each of the four dNTPs at a concentration of 0.5. Mu. Mol/L in the system), 0.5ul FS-R (each of the four dNTPs at a concentration of 0.5. Mu. Mol/L in the system), 1.5. Mu.L of the genomic DNA of the melon to be tested (50 ng/. Mu.L), 0.2DNA Polymerase for PAGE，ddH 2 O was added to 20. Mu.L. The reaction procedure for PCR amplification was: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 30s, and running for 35 cycles; extending at 72℃for 5min.
The PCR amplification products were detected by 8% polyacrylamide gel electrophoresis, the detection results are shown in FIG. 2, and the statistical results of the fruit shape genotype (FS) are shown in Table 1.
Wherein 141 fruits are long fruits (namely, the aspect ratio is more than 1), the genotype of the single plant is A1A2, 121 fruits are round fruits or oblate fruits (namely, the aspect ratio is less than or equal to 1), the genotype of the single plant is A2A2, and the genotype and the phenotype completely correspond.
Therefore, these data indicate that the molecular marker FS of the present invention can distinguish the genotypes of melon with long shape (i.e. aspect ratio > 1) and melon with round or oblate shape, and can identify the fruit carrying gene in early stage of melon growth by co-segregation with fruit shape character. The method provided by the invention is accurate, reliable and simple to operate, can be used for molecular marker assisted selection in fruit shape breeding, and can be used for rapidly breeding new varieties of round fruit shape or oblate fruit shape melons, thereby greatly improving breeding efficiency.
The foregoing examples are merely illustrative of the present invention, and embodiments of the present invention are not limited to the above-described examples, but are intended to be equivalent alternatives, modifications, substitutions, combinations and simplifications, which may be made without departing from the spirit and principles of the present invention.
Sequence listing
<110> academy of agriculture and forestry science in Beijing city
<120> a molecular marker related to melon shape, method and application thereof
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<170> PatentIn version 3.5
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ca 122
Claims (7)
1. Use of a substance comprising a nucleotide as set forth in sequence 3 or a nucleotide as set forth in sequence 4 in the genome of a melon to be tested for detection in any one of the following 1) to 10):
1) Identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
2) Preparing and identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
3) Breeding melon varieties carrying long-fruit shape genotypes;
4) Preparing and breeding a product of melon varieties carrying long round fruit genotypes;
5) Breeding melon varieties carrying oblate fruit genotypes;
6) Preparing and breeding products of melon varieties carrying oblate fruit genotypes;
7) Breeding melon varieties with long fruit shapes in the generation of seeds;
8) Preparing a product of melon varieties with long fruit shapes in seed selection sub-generations;
9) Selecting melon varieties with offspring of oblate fruits;
10 Preparing a product of melon varieties with flat round fruit-shaped seed generations;
the fruit genotype is oblate fruit genotype A2A2, long fruit genotype A1A1 or long fruit genotype A1A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in a sequence 3, and the other homologous chromosome contains nucleotide shown in a sequence 4;
the substances are 1) or 2) as follows:
1) The primer set comprises a single-stranded DNA molecule shown as a sequence 1 in a sequence table and a single-stranded DNA molecule shown as a sequence 2 in the sequence table;
2) PCR reagents or kits containing the set of primers.
2. A product comprising the substance of claim 1;
the product has any one of the following functions 1) -5):
1) Identifying or assisting in identifying the genotype of the fruit shape of the melon to be detected;
2) Breeding melon varieties carrying long-fruit shape genotypes;
3) Breeding melon varieties carrying oblate fruit genotypes;
4) Breeding melon varieties with long fruit shapes in the generation of seeds;
5) And breeding melon varieties with offspring being oblate fruits.
3.A method for identifying or assisting in identifying the genotype of the fruit shape of a melon to be tested, comprising the steps of:
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 3, the fruit genotype of the melon to be detected is a long fruit genotype A1A1;
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3 and the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is a long fruit genotype A1A2;
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is oblate fruit genotype A2A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in sequence 3, and the other homologous chromosome contains nucleotide shown in sequence 4.
4. A method for identifying or assisting in identifying the shape of a melon or progeny thereof to be tested, comprising the steps of:
detecting whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3, the melon to be detected or the progeny fruit shape thereof is a long fruit,
if the genome of the melon to be detected contains the nucleotide shown in the sequence 4 and does not contain the nucleotide shown in the sequence 3, the melon to be detected or the progeny thereof has a oblate shape.
5. A method according to claim 3 or 4, characterized in that: the detection of whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4 is as follows:
1) Directly sequencing the genome DNA to judge;
2) Amplifying the melon to be detected by using the set of primers in claim 1, sequencing amplified products, and judging.
6. A method for breeding a melon variety carrying a long fruit shape genotype or a melon variety with a long fruit shape bred by seed substitution, comprising the following steps:
1) Amplifying the melon to be detected by using the set of primers according to claim 1, sequencing the amplified product, judging whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 3, the fruit genotype of the melon to be detected is a long fruit genotype A1A1;
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3 and the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is a long fruit genotype A1A2;
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is oblate fruit genotype A2A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in a sequence 3, and the other homologous chromosome contains nucleotide shown in a sequence 4;
2) And breeding melons to be detected, wherein the fruit shape genotype of each melon is a long fruit shape genotype A1A2 or A1A 1.
7. A method for breeding melon varieties carrying oblate fruit genotypes or breeding oblate fruit varieties or breeding melon varieties with oblate fruit generations, comprising the following steps:
1) Amplifying the melon to be detected by using the set of primers according to claim 1, sequencing the amplified product, judging whether the genome of the melon to be detected contains the nucleotide shown in the sequence 3 or the nucleotide shown in the sequence 4,
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 3, the fruit genotype of the melon to be detected is a long fruit genotype A1A1;
if the genome of the melon to be detected contains the nucleotide shown in the sequence 3 and the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is a long fruit genotype A1A2;
if the genome of the melon to be detected only contains the nucleotide shown in the sequence 4, the fruit genotype of the melon to be detected is oblate fruit genotype A2A2;
the oblate fruit genotype A2A2 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 4 and all do not contain nucleotides shown in a sequence 3;
the long fruit genotype A1A1 is that 2 homologous chromosomes of the muskmelon genome all contain nucleotides shown in a sequence 3 and all do not contain nucleotides shown in a sequence 4;
the long fruit genotype A1A2 is that 1 homologous chromosome of the muskmelon genome contains nucleotide shown in a sequence 3, and the other homologous chromosome contains nucleotide shown in a sequence 4;
2) And breeding melons to be detected, wherein the fruit genotype of each melon is oblate fruit genotype A2A2.
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