CN115058536B - InDel molecular marker related to yellowing trait of watermelon leaves and application thereof - Google Patents
InDel molecular marker related to yellowing trait of watermelon leaves and application thereof Download PDFInfo
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- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 title claims abstract description 76
- 238000004383 yellowing Methods 0.000 title claims abstract description 56
- 239000003147 molecular marker Substances 0.000 title claims abstract description 42
- 241000219109 Citrullus Species 0.000 title claims abstract 19
- 241000196324 Embryophyta Species 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 25
- 238000009395 breeding Methods 0.000 claims abstract description 21
- 230000001488 breeding effect Effects 0.000 claims abstract description 20
- 230000003321 amplification Effects 0.000 claims abstract description 14
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 14
- 239000012634 fragment Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 8
- 210000000349 chromosome Anatomy 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 238000001962 electrophoresis Methods 0.000 claims description 7
- 108700028369 Alleles Proteins 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 4
- 244000241235 Citrullus lanatus Species 0.000 description 59
- 239000000463 material Substances 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 6
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000005204 segregation Methods 0.000 description 3
- 241001464837 Viridiplantae Species 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 1
- 235000009831 Citrullus lanatus Nutrition 0.000 description 1
- 241000219104 Cucurbitaceae Species 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention belongs to the technical field of molecular markers, and discloses an InDel molecular marker related to the yellowing property of watermelon leaves, wherein a linkage gene locus of the InDel molecular marker starts at a 16217471bp base of chromosome 2 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 yellowing homozygous single plant, if the length of the amplification gene fragment is 142bp, the InDel molecular marker is a watermelon leaf normal 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 normal green 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 purpose of molecular marker assisted breeding is achieved, the breeding period can be greatly shortened, and the method has important theoretical and practical significance.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and relates to an InDel molecular marker related to yellowing traits of watermelon leaves and application thereof.
Background
As an important cucurbitaceae 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 gardening crops in the world. In China, which is the first major country of world watermelon production and consumption, leaf color is often mutated in production and cultivation, and the leaf color shows the phenotypes of albino, yellowish green, delayed green, mottled, speckled and the like. Leaf color mutant is an ideal material for researching photosynthesis, chlorophyll synthesis and chloroplast development, and is also an important research material in inheritance and breeding.
In traditional selective breeding, because it is difficult to determine the genotype of the offspring, the basis of selection is typically 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 and inefficient selection. Molecular marker assisted breeding screens target characters by taking molecular markers closely linked with the target characters as tools, and the molecular markers screen germplasm resources through genotypes, so that the molecular marker assisted breeding 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 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 a great development prospect in the current molecular marker-assisted breeding work.
The yellowing property of the watermelon leaves is the most ideal marking property for identifying the purity of the hybridized watermelon seeds in the seedling stage. The full growth period of the watermelon leaf yellowing plant leaf is the yellowing leaf, and the full growth period of the normal green plant leaf is green. Therefore, yellowing is clearly distinguished from normal green seedlings or plants, with the earliest stage of identification being 7-10 days after sowing during the period from emergence of the cotyledons to 1 true leaf of the seedling. Through identifying the yellowing gene and developing the molecular marker closely linked with the yellowing gene for initial variety screening, the aim of molecular assisted breeding is fulfilled, the breeding period can be greatly shortened, and the breeding efficiency is improved.
The yellowing property of the watermelon leaves can accurately identify the hybrid in the seedling stage, and has the advantages of accuracy, intuitiveness, simplicity, convenience, rapidness, low consumption and the like. However, the character is recessive, the first filial generation is not represented, and great difficulty is brought to breeding work. Leaf color is an important agronomic trait of watermelons and is closely related to photosynthesis ability of watermelon plants, so that research on the leaf color of watermelons is urgently needed to be developed deeply, and molecular markers which can be used for watermelon breeding are 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 yellowing traits of watermelon leaves and application thereof, which can be used for carrying out initial screening on watermelon varieties, and a detection method is reliable and can greatly shorten a breeding period.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The invention provides an InDel molecular marker related to the yellowing property of watermelon leaves, wherein the linkage gene locus of the InDel molecular marker starts at the 16217471bp base of chromosome 2 of a 97103V2 version of a watermelon reference genome, the amplification length of the InDel molecular marker is 122bp and/or 142bp, if the amplification gene fragment length is 122bp, the InDel molecular marker is a watermelon leaf yellowing homozygous single plant, if the amplification gene fragment length is 142bp, the InDel molecular marker is a watermelon leaf normal green homozygous single plant, and if the amplification gene fragment length is 122bp and 142bp, the InDel molecular marker is a watermelon leaf normal green heterozygous single plant.
The invention also provides a primer pair for amplifying InDel molecular markers related to yellowing traits of 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 yellow or not by using the primer pair, which comprises the following steps: extracting genome DNA of a watermelon sample to be detected; carrying out PCR amplification on the genomic DNA of the watermelon by using a primer pair; judging the allele type of the yellowing property of the watermelon leaf according to the electrophoresis band of the PCR amplification product, wherein the watermelon is a yellowing homozygous single plant if only the 122bp band is displayed, is a normal green homozygous single plant if only the 142bp band is displayed, and is a normal green heterozygous single plant if both the 122bp band and the 142bp band are displayed.
The invention also provides application of the primer pair in identifying whether the color of watermelon leaves is yellow.
The invention also provides application of the primer pair in molecular marker assisted breeding of whether the color of the watermelon leaves is yellow or not.
The invention also provides a kit containing a primer pair for amplifying InDel molecular markers related to yellowing traits of watermelon leaves.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, leaf yellowing identification is carried out on an F2 population, a BC1 segregation population and a natural population, and InDel loci are found to start at the 16217471bp basic group of chromosome 2 of a 97103V2 version of a watermelon reference genome, polymorphism is represented by 20bp basic group difference, and InDel molecular markers related to watermelon leaf yellowing characteristics are developed; 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 purpose of molecular marker assisted breeding is achieved, the breeding period can be greatly shortened, and the method has important theoretical and practical significance.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of the InDel molecular markers of the present invention versus F2 isolate.
FIG. 2 is an electrophoretogram of the InDel molecular markers of the present invention versus PCR amplification products of natural populations.
Detailed Description
The following examples are illustrative of the present invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified.
Example 1
1) Selecting a watermelon material to be tested, wherein the watermelon material to be tested comprises male parent, female parent, F1 generation, F2 population, BC1 population and natural population. The male parent is the evergreen watermelon material ZK of the leaf; the female parent is leaf yellowing watermelon material w-yl, and the leaf of the material is uniformly yellowing in the whole growth period; the F1 generation is the watermelon material obtained by hybridization of male parent and female parent; the F2 population is watermelon material obtained by F1 selfing; the BC1 population is watermelon material obtained by the F1 and a female parent; the natural population is randomly selected watermelon material in the resource pool, including leaf yellowing watermelon material and leaf normal green watermelon material, and total of 38 are shown in table 1. All the tested materials are germplasm resource materials stored by a diploid watermelon subject group of Zhengzhou fruit tree institute of Chinese academy of agricultural sciences.
TABLE 1 watermelon material varieties and phenotypes selected from natural populations
2) Determination of yellowing behavior of the leaves of the tested materials.
And determining leaf yellowing plants in the watermelon material to be tested by adopting a direct observation method. The yellowing character of the watermelon leaves is that the new cotyledons and leaves are yellowing leaves, and the leaves and leaves of the normal green plants are green. Thus, leaf yellowing is clearly distinguished from normal green seedlings and plants.
Leaf yellowing trait identification is carried out on a normal green parent ZK (male parent), a yellowing parent (female parent), single plants of an F1 group, 1834 single plants of an F2 segregating group and 233 BC1 segregating groups. The results show that: the leaf yellowing traits of (male parent), yellowing parent and F1 are normal green, yellowing and normal green, respectively. F2 colony is obtained after the F1 plant is selfed, and the leaf yellowing character identification result shows that: of the 634 plants, 480 plants were normally green and 154 plants showed leaf yellowing, with the card square test x2 = 0.17, p = 0.68, the difference was not significant, conforming to a theoretical separation ratio of 3:1. Identification through BC1 colony yellowing character, and found out: among the 70 plants, 32 plants showed normal green and 38 plants showed yellowing of the leaves, the square of the card test x2 = 0.51, the p = 0.47, the difference was not significant, and the theoretical separation ratio of 1:1 was met. And combining the identification results of the single plant yellowing traits of the parents, F1, 634F 2 segregations and 70 BC1 segregations to obtain the recessive trait that the watermelon leaf yellowing genes are controlled by single genes.
3) And obtaining candidate InDel gene loci.
Constructing an extreme mixed pool of plants with normal green leaves and yellowing leaves in the F2 population, performing genome sequencing, analyzing the difference of allele frequencies, preliminarily positioning a target gene interval and obtaining candidate InDel gene loci; the target interval was located within a stretch of 6.78Mb of 11890000-18670000bp of chromosome 2 of version 97103V2 of the watermelon reference genome.
4) Obtaining InDel molecular markers
InDel molecular markers are designed aiming at candidate InDel loci by utilizing https:// cucurbstgenomics.org/published data of version 97103V2 of watermelon reference genome, and the candidate InDel loci are verified in male parent, female parent, F1 generation and F2 population to obtain the InDel molecular markers linked with watermelon leaf yellowing genes wyl. The linkage gene locus of the InDel molecular marker starts at the 16217471bp base of chromosome 2 of the 97103V2 version of the watermelon reference genome, the length of the InDel molecular marker is 122bp and/or 142bp, namely the polymorphism shows 20bp difference, the 122bp gene sequence is shown as SEQ ID NO.1, and the 142bp gene sequence is shown as SEQ ID NO. 2.
5) And (3) performing PCR amplification reaction on the genomic DNA of each male parent, female parent, F1 generation and F2 population to obtain each PCR amplification product. 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: pre-denaturation at 94℃for 5min;94 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 30s, and the total circulation is 30 times; extending at 72 ℃ for 5min; maintained at 4 ℃.
In the PCR amplification reaction, the PCR amplification reaction system was 20. Mu.L in terms of volume, and it included 10. Mu.L of 2X Taq PCRMasterMix, 2. Mu.L of template DNA, 1. Mu.L of forward and reverse primers, and 6. Mu. LddH 2 O each.
Polymorphism detection was performed by polyacrylamide gel electrophoresis, and photographs were taken in a gel imaging system. FIG. 1 shows the electrophoresis pattern of the PCR amplified product of InDel molecular Marker on F2 isolated population, wherein lane 1 shows the molecular weight Marker, lane 2 shows the yellow parent PCR product, lane 3 shows the green parent PCR product, lane 4 shows the F1 generation PCR product, and the other lanes show the F2 isolated population partial strain PCR product (33 strains). And judging the allele type of the yellowing property of the watermelon leaves according to the banding pattern of the PCR product, wherein as shown in figure 1, 122bp bands are obtained by amplifying fragment watermelon leaf yellowing homozygous single plants, 142bp bands are obtained by watermelon leaf normal green homozygous single plants, and 122bp and 142bp bands are shown by watermelon leaf normal green heterozygous single plants. The results show that the leaf yellowing identification result and the mark detection result show coseparation.
6) Verification of natural populations using InDel molecular markers
And further verifying the linkage relation between the InDel molecular marker and the watermelon leaf yellowing gene wyl by using natural populations. Performing PCR amplification reaction by taking genomic DNA of 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 the step 5).
FIG. 2 is an electrophoretogram of InDel molecular markers versus PCR amplified products of natural populations, wherein lane 1 is a molecular weight Marker, lane 2 is a yellowing parent PCR product, lane 3 is a green parent PCR product, lane 4 is an F1 generation PCR product, and the other lanes are 36 parts of natural population materials.
The results show that the mark detection of 36 natural population materials is consistent with the leaf yellowing identification, and the coincidence rate of the InDel molecular mark to the resource identification is counted to be 100%. Further proves that the designed InDel molecular marker is tightly linked with the watermelon leaf yellowing gene wyl; the InDel locus and the InDel molecular marker designed by using the InDel locus have high utilization value in identifying the yellowing trait of the watermelon leaves, and can be effectively applied to watermelon molecular assisted breeding.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> InDel molecular marker related to yellowing property of watermelon leaf 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 (5)
1. The InDel molecular marker related to the yellowing property of the watermelon leaves is characterized in that the linkage gene locus of the InDel molecular marker starts at the 16217471bp base of chromosome 2 of the 97103V2 version of the watermelon reference genome, the amplification length of the InDel molecular marker is 122bp and/or 142bp, if the amplification gene fragment length is 122bp, the InDel molecular marker is a watermelon leaf yellowing homozygous single plant, if the amplification gene fragment length is 142bp, the InDel molecular marker is a watermelon leaf normal green homozygous single plant, and if the amplification gene fragment length is 122bp and 142bp, the InDel molecular marker is a watermelon leaf normal green heterozygous single plant; the forward primer sequence of the primer pair for amplifying the InDel molecular marker is shown as SEQ ID NO.3, and the reverse primer sequence is shown as SEQ ID NO. 4.
2. The method for identifying whether the color of the watermelon leaves is yellow or not by using the primer pair is characterized by comprising the following steps: extracting genome DNA of a watermelon sample to be detected; carrying out PCR amplification on the genomic DNA of the watermelon by using a primer pair, wherein the forward primer sequence of the primer pair is shown as SEQ ID NO.3, and the reverse primer sequence of the primer pair is shown as SEQ ID NO. 4; judging the allele type of the yellowing property of the watermelon leaf according to the electrophoresis band of the PCR amplification product, wherein the watermelon is a yellowing homozygous single plant if only the 122bp band is displayed, is a normal green homozygous single plant if only the 142bp band is displayed, and is a normal green heterozygous single plant if both the 122bp band and the 142bp band are displayed.
3. The application of the primer pair in identifying whether the color of watermelon leaves is yellow 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.
4. The application of the primer pair in molecular marker assisted breeding of whether the color of watermelon leaves is yellow 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.
5. The kit containing the primer pair for amplifying InDel molecular markers related to yellowing traits of 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.
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| CN105284608A (en) * | 2015-06-01 | 2016-02-03 | 中国农业科学院郑州果树研究所 | Culture medium for watermelon flower callus induction and method for inducing watermelon flower callus by adopting culture medium |
| NL2015547B1 (en) * | 2015-10-02 | 2017-04-21 | Rijk Zwaan Zaadteelt En Zaadhandel Bv | Copy number variant leading to virus resistance. |
| US20180273972A1 (en) * | 2015-12-06 | 2018-09-27 | The State of Israel, Ministry of Agriculture & Rural Development, Argricultural Research Organiza | Methods of increasing virus resistance in cucumber using genome editing and plants generated thereby |
| CN111518945A (en) * | 2020-06-12 | 2020-08-11 | 中国农业科学院郑州果树研究所 | CAPS mark related to watermelon leaf color and application thereof |
| CN114381544B (en) * | 2022-01-12 | 2023-12-01 | 中国农业科学院郑州果树研究所 | Watermelon leaf yellowing lethal major gene, dCAPS molecular marker for identifying major gene and application |
| CN114292954B (en) * | 2022-01-26 | 2023-06-30 | 河南农业大学 | Molecular marker closely linked with green petal gene Clgf of watermelon and application thereof |
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