CN115058536A - InDel molecular marker related to etiolation character of watermelon leaves and application thereof - Google Patents

InDel molecular marker related to etiolation character of watermelon leaves and application thereof Download PDF

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CN115058536A
CN115058536A CN202210669198.7A CN202210669198A CN115058536A CN 115058536 A CN115058536 A CN 115058536A CN 202210669198 A CN202210669198 A CN 202210669198A CN 115058536 A CN115058536 A CN 115058536A
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watermelon
molecular marker
etiolation
indel molecular
leaves
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朱迎春
孙德玺
王一帆
袁高鹏
刘君璞
李卫华
安国林
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Zhengzhou Fruit Research Institute CAAS
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention belongs to the technical field of molecular markers, and discloses an InDel molecular marker related to the etiolation trait of watermelon leaves, wherein a linkage gene locus of the InDel molecular marker starts at the 16217471bp base of the No.2 chromosome of a 97103V2 version of a watermelon reference genome, the amplification length of the InDel molecular marker is 122bp and/or 142bp, if the length of an amplification gene fragment is 122bp, the InDel molecular marker is a watermelon leaf etiolation homozygous single plant, if the length of the amplification gene fragment is 142bp, the InDel molecular marker is a watermelon leaf orthonormally green homozygous single plant, and if the length of the amplification gene fragment is 122bp and 142bp, the InDel molecular marker is a watermelon leaf orthogreen heterozygous single plant. The InDel molecular marker developed by the invention can be used for carrying out initial screening on varieties, the detection method is correct and reliable, the aim of molecular marker-assisted breeding is fulfilled, the breeding period can be greatly shortened, and the method has important theoretical and practical significance.

Description

InDel molecular marker related to etiolation character of watermelon leaves and application thereof
Technical Field
The invention belongs to the technical field of molecular markers, and relates to an InDel molecular marker related to the etiolation character of watermelon leaves and application thereof.
Background
As an important cucurbit crop, watermelon (Citrulluslanatus) is an important fruit for people to relieve summer heat and quench thirst in summer and plays a very important role in the world of horticultural crops. In China, which is the first major country for watermelon production and consumption in the world, the color of leaves is often mutated in production and cultivation, and the leaves show the phenotypes such as albino, yellow-green, delayed green-turning, mottle and the like. The leaf color mutant is an ideal material for researching photosynthesis, chlorophyll synthesis and chloroplast development, and is also an important research material in heredity and breeding.
In conventional selective breeding, because it is difficult to determine the genotype of progeny, the selection is usually based on the phenotype of the plant rather than the genotype, the selection time is long, and the phenotype is susceptible to environmental factors, resulting in inaccurate selection and low efficiency. The molecular marker assisted breeding screens the target characters by using a molecular marker which is tightly linked with the target characters as a tool, and the molecular marker screens germplasm resources by using genotypes, so that the method has the advantages of accuracy, rapidness and no interference from environmental conditions, avoids blindness of character selection in the traditional breeding process, and improves the breeding efficiency. The InDel marker is a molecular marker for analyzing an amplification product, has the characteristics of high flux, simplicity, stability, high sensitivity and the like, and is a molecular marker with great development prospect in the current molecular marker-assisted breeding work.
The etiolation character of the watermelon leaves is the most ideal marker character for identifying the purity of the watermelon hybrid seeds in the seedling stage. The full growth period of the watermelon leaf etiolation character plant leaves is etiolation leaves, and the full growth period of the normal green plant leaves is green. Therefore, yellowing is obviously different from normal green seedlings or plants, and the earliest identification period is from the emergence of cotyledons to 1 true leaf of the seedlings and is 7-10 days after sowing. By identifying the etiolation gene and developing the closely linked molecular markers for initial screening of varieties, the purpose of molecular assisted breeding is achieved, the breeding period can be greatly shortened, and the breeding efficiency is improved.
The etiolation character of watermelon leaves can accurately identify the hybrid at the seedling stage, and has the advantages of accuracy, intuition, simplicity, rapidness, low consumption and the like. However, the character is recessive, the first filial generation is not expressed, and great difficulty is brought to the breeding work. The color of the watermelon leaves is an important agronomic trait of the watermelon and is closely related to the photosynthesis capability of watermelon plants, so that the research on the color of the watermelon leaves is urgently needed to be deeply developed, and a molecular marker for breeding the watermelon is developed so as to shorten the breeding process and improve the breeding efficiency.
Disclosure of Invention
The invention aims to provide an InDel molecular marker related to the etiolation character of watermelon leaves and application thereof, which can be used for carrying out initial screening on watermelon varieties, have reliable detection method and can greatly shorten the breeding period.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an InDel molecular marker related to the etiolation trait of watermelon leaves, wherein the linkage gene locus of the InDel molecular marker starts from the 16217471bp base position of No.2 chromosome of a 97103V2 version of a watermelon reference genome, the amplification length of the InDel molecular marker is 122bp and/or 142bp, if the length of an amplification gene fragment is 122bp, the InDel molecular marker is a watermelon leaf etiolation homozygous single plant, if the length of the amplification gene fragment is 142bp, the InDel molecular marker is a watermelon leaf normal evergreen homozygous single plant, and if the length of the amplification gene fragment is 122bp and 142bp, the InDel molecular marker is a watermelon leaf normal evergreen heterozygous single plant.
The invention also provides a primer pair for amplifying the InDel molecular marker related to the etiolation trait of the watermelon leaves, wherein the forward primer sequence of the primer pair is shown as SEQ ID NO.3, and the reverse primer sequence is shown as SEQ ID NO. 4.
The invention also provides a method for identifying whether the color of the watermelon leaves is yellowed by using the primer pair, which comprises the following steps: extracting the genome DNA of a watermelon sample to be detected; carrying out PCR amplification on watermelon genome DNA by using a primer pair; judging the allele type of the etiolation character of the watermelon leaves according to the electrophoresis bands of the PCR amplification products, if only 122bp bands are displayed, the watermelon is an etiolation homozygous single plant, if only 142bp bands are displayed, the watermelon is a normal green homozygous single plant, and if two bands of 122bp and 142bp are displayed, the watermelon is a normal green heterozygous single plant.
The invention also provides application of the primer pair in identifying whether the color of the watermelon leaves is yellowed.
The invention also provides application of the primer pair in molecular marker assisted breeding of whether the color of watermelon leaves is yellowed or not.
The invention also provides a kit containing a primer pair for amplifying the InDel molecular marker related to the etiolation character of the watermelon leaves.
Compared with the prior art, the invention has the beneficial effects that:
the invention carries out the identification of the yellowing of leaves on F2 population, BC1 segregation population and natural population, discovers that the InDel locus starts at the 16217471bp base of No.2 chromosome of 97103V2 version of watermelon reference genome, the polymorphism shows 20bp base difference, and develops InDel molecular markers related to the yellowing character of watermelon leaves; the specific InDel molecular marker designed by the invention can be used for carrying out initial screening on varieties, the detection method is correct and reliable, the aim of molecular marker-assisted breeding is fulfilled, the breeding period can be greatly shortened, and the method has important theoretical and practical significance.
Drawings
FIG. 1 is an electrophoresis diagram of PCR amplification products of an InDel molecular marker of the present invention versus an F2 isolate population.
FIG. 2 is an electrophoresis diagram of PCR amplification products of the InDel molecular marker of the present invention against a natural population.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.
Example one
1) Selecting a watermelon material to be tested, wherein the watermelon material to be tested comprises a male parent, a female parent, an F1 generation, an F2 population, a BC1 population and a natural population. The male parent is a watermelon material ZK with normal green leaves; the female parent is a watermelon material w-yl with etiolated leaves, and the leaves of the watermelon material are etiolated in the whole growth period; f1 is watermelon material obtained by hybridization of male parent and female parent; the F2 group is watermelon material obtained by F1 selfing; the BC1 population is the watermelon material obtained by the F1 and the female parent; the natural population is watermelon material randomly selected from a resource library, and comprises 38 watermelon materials with etiolated leaves and normal green leaves, which are shown in table 1. All the above-mentioned test materials are germplasm resource materials preserved in diploid watermelon subject group of Zhengzhou fruit tree institute of Chinese academy of agricultural sciences.
TABLE 1 watermelon material varieties and phenotypes selected by Natural groups
Figure BDA0003694172820000031
2) And (4) determining the yellowing property of the leaves of the test material.
The direct observation method is adopted to determine the leaf etiolated plants in the watermelon material to be tested. The etiolation character of the watermelon leaves is that the newborn cotyledon and the leaves are etiolation leaves, and the normal green plant leaves are green. Therefore, the yellowing of leaves is obviously different from that of normal green seedlings and plants.
Leaf yellowing trait identification was performed on the normally green parent ZK (male parent), the etiolated parent (female parent), individuals of the F1 population, 1834 individuals of the F2 isolate population and 233 BC1 isolate population. The results show that: the etiolation traits of the leaf of the (male parent), the etiolation parent and the F1 are normal green, etiolation and normal green respectively. The F2 population is obtained after the F1 plant is selfed, and the result of the leaf yellowing trait identification shows that: of the 634 individuals, 480 normally green leaves and 154 plants showed leaf yellowing, x2 ═ 0.17 and P ═ 0.68 according to the chi-square test, with no significant difference, consistent with the theoretical segregation ratio of 3: 1. Identification is carried out through a BC1 population yellowing trait, and as a result, the following results are found: 32 of the 70 individuals showed normal green and 38 plants with etiolated leaves, and x2 ═ 0.51 and P ═ 0.47 by the chi-square test were not significantly different and matched the theoretical segregation ratio of 1: 1. And (3) combining the results of the individual plant etiolation trait identification of the parents, the F1, the 634F 2 segregating populations and the 70 BC1 segregating populations to obtain the recessive trait of the watermelon leaf etiolation gene controlled by a single gene.
3) And obtaining candidate InDel gene loci.
Constructing an extreme mixed pool for plants with normally green leaves and yellowing leaves in an F2 population, carrying out genome sequencing, analyzing the difference of allele frequencies, preliminarily positioning a target gene interval and obtaining candidate InDel gene loci; the target region was located within a 6.78Mb region of 11890000-18670000bp of the No.2 chromosome of the 97103V2 version of the watermelon reference genome.
4) Obtaining of InDel molecular marker
Designing an InDel molecular marker aiming at a candidate InDel locus by using http:// cucurbitangenomics.org/published watermelon reference genome 97103V2 version data, verifying the candidate InDel gene locus in male parent, female parent, F1 generation and F2 population, and obtaining the InDel molecular marker linked with watermelon leaf etiolation gene wyl. The linkage gene locus of the InDel molecular marker starts from the 16217471bp base of the No.2 chromosome of a watermelon reference genome 97103V2 version, the length of the InDel molecular marker is 122bp and/or 142bp, namely, the polymorphism shows 20bp difference, the gene sequence of 122bp is shown as SEQ ID NO.1, and the gene sequence of 142bp is shown as SEQ ID NO. 2.
5) And carrying out PCR amplification reaction on the respective genome DNA of the male parent, the female parent, the F1 generation and the F2 population to obtain respective PCR amplification products. The sequences of the amplification primer pairs are as follows:
wyl-F, forward primer: 5'-TCGTACGTAAGAGCCACACA-3' (SEQ ID NO. 3);
wyl-R, reverse primer: 5'-CTTGGGTAACTGGGGTTGCG-3' (SEQ ID NO. 4).
In the PCR amplification reaction, the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 58 ℃ and 30s at 72 ℃, and 30 times of cycle; extending for 5min at 72 ℃; keeping at 4 ℃.
In the PCR amplification reaction, the PCR amplification reaction system is 20. mu.L, and comprises 10. mu.L of 2 XTaq PCRMasterMix, 2. mu.L of template DNA, 1. mu.L of each of the forward and reverse primers and 6. mu.L of ddH 2 O。
Polymorphism detection is performed by polyacrylamide gel electrophoresis, and pictures are taken in a gel imaging system. FIG. 1 is an electrophoresis diagram of PCR amplification products of InDel molecular Marker vs. F2 isolate, wherein lane 1 is molecular weight Marker, lane 2 is the etiolation parental PCR product, lane 3 is the PCR product of the green parental, lane 4 is the PCR product of F1 generation, and the other lane is the PCR product of F2 isolate part strain (strain 33). And (3) judging the allelic gene type of the watermelon leaf etiolation character according to the band type of the PCR product, wherein as shown in figure 1, the amplified fragment of the watermelon leaf etiolation homozygous single plant obtains a 122bp band, the watermelon leaf normal evergreen homozygous single plant obtains a 142bp band, and the watermelon leaf normal evergreen heterozygous single plant shows two bands of 122bp and 142 bp. The result shows that the leaf yellowing identification result and the marking detection result show coseparation.
6) And verifying the natural population by using the InDel molecular marker
And further verifying the linkage relation between the InDel molecular marker and the watermelon leaf etiolation gene wyl by using a natural population. Carrying out PCR amplification reaction by taking genome DNA of the natural population as a template to obtain a PCR specific fragment of the natural population; and then carrying out electrophoresis detection on the PCR specific fragment. The specific operation is the same as that in step 5).
FIG. 2 is an electrophoresis diagram of PCR amplification products of the natural population by InDel molecular Marker, wherein lane 1 is molecular weight Marker, lane 2 is yellowing parent PCR product, lane 3 is green parent PCR product, lane 4 is F1 generation PCR product, and the other lanes are 36 natural population materials.
The result shows that the marker detection of 36 natural population materials is consistent with the identification of leaf yellowing, and the coincidence rate of the InDel molecular marker on resource identification is counted to be 100%. Further proves that the designed InDel molecular marker is closely linked with the watermelon leaf etiolation gene wyl; the InDel locus and the InDel molecular marker designed by the InDel locus have very high utilization value in identifying the etiolation character of the watermelon leaves, and can be effectively applied to molecular assisted breeding of the watermelon.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.
SEQUENCE LISTING
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> InDel molecular marker related to etiolation traits of watermelon leaves and application thereof
<130> 2022
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 122
<212> DNA
<213> Artificial sequence
<400> 1
tcgtacgtaa gagccacaca accatgttac accacaatac agaaactagt ggttgtcaaa 60
acatgggagt tattttgagg ataatatata ttcctccctc atcgcaaccc cagttaccca 120
ag 122
<210> 2
<211> 142
<212> DNA
<213> Artificial sequence
<400> 2
tcgtacgtaa gagccacaca accatgttac accacaatac agaaactagt ggttgtcaaa 60
acatgggagt tattttgagg ataatataca tcacttagta agtaacacta ttcctccctc 120
atcgcaaccc cagttaccca ag 142
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (wyl-F)
<400> 3
tcgtacgtaa gagccacaca 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (wyl-R)
<400> 4
cttgggtaac tggggttgcg 20

Claims (6)

1. The InDel molecular marker related to the watermelon leaf etiolation trait is characterized in that a linkage gene locus of the InDel molecular marker starts from the 16217471bp base of the No.2 chromosome of a 97103V2 version of a watermelon reference genome, the amplification length of the InDel molecular marker is 122bp and/or 142bp, if the length of an amplification gene fragment is 122bp, the InDel molecular marker is a watermelon leaf etiolation homozygous single plant, if the length of the amplification gene fragment is 142bp, the InDel molecular marker is a watermelon leaf orthonormally green homozygous single plant, and if the length of the amplification gene fragment is 122bp and 142bp, the InDel molecular marker is a watermelon leaf orthonormally green heterozygous single plant.
2. The primer pair for amplifying the InDel molecular marker related to the etiolation trait of the watermelon leaves is characterized in that the forward primer sequence of the primer pair is shown as SEQ ID NO.3, and the reverse primer sequence is shown as SEQ ID NO. 4.
3. The method for identifying whether the color of the watermelon leaves is yellowed by using the primer pair of claim 2, which is characterized by comprising the following steps: extracting the genome DNA of a watermelon sample to be detected; carrying out PCR amplification on watermelon genome DNA by using a primer pair; judging the allele type of the etiolation character of the watermelon leaves according to the electrophoresis bands of the PCR amplification products, if only 122bp bands are displayed, the watermelon is an etiolation homozygous single plant, if only 142bp bands are displayed, the watermelon is a normal green homozygous single plant, and if two bands of 122bp and 142bp are displayed, the watermelon is a normal green heterozygous single plant.
4. The primer pair of claim 2, for use in identifying whether the color of watermelon leaves is yellow.
5. The primer pair of claim 2 is applied to molecular marker-assisted breeding for determining whether the color of watermelon leaves is yellowed.
6. The kit contains a primer pair for amplifying InDel molecular markers related to the etiolation trait of watermelon leaves.
CN202210669198.7A 2022-06-14 2022-06-14 InDel molecular marker related to etiolation character of watermelon leaves and application thereof Pending CN115058536A (en)

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