CN114292954B - Molecular marker closely linked with green petal gene Clgf of watermelon and application thereof - Google Patents
Molecular marker closely linked with green petal gene Clgf of watermelon and application thereof Download PDFInfo
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Abstract
The invention discloses two pairs of molecular markers closely linked with a watermelon green flower gene Clgf and application thereof, and belongs to the technical field of biology. The invention utilizes parent and F 2 And carrying out phenotype identification on the population, and carrying out preliminary positioning on the green flower character related genes by utilizing SSR markers according to the BSA principle. SSR and Indel markers are developed by combining with the resequencing of parents, polymorphism screening is carried out on the parents, and primers with polymorphism are used in F 2 Genotyping was performed in the population, positioning the watermelon green gene Clgf between markers ClSSR30082 and Indel 186. The molecular marker can provide a new means for identifying the color of the watermelon, accelerate the improvement process of the breeding of the watermelon and improve the accuracy and the selection efficiency of the breeding.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a molecular marker closely linked with a green petal gene Clgf of a watermelon and application thereof.
Background
Flower color is an important trait inherited by plants, mainly attracting insects for pollination. The flower color can influence aphid feeding, and the crop varieties with different flower colors are bred in production, so that the effect of reducing aphid disease transmission is achieved, the energy balance of petals can be maintained, and damage to flower organs is avoided. The flower colors are important appearance characters of ornamental plants, and various flower colors can be used for decorating and beautifying life and working environments and enriching the mental life of people.
In cruciferous crops, the flower color of 38-color rape is cultivated by Fudonghui and the like. Yang et al constructed an F derived from the Y640-288 (white flower) and Y641-87 (yellow flower) lines 2 In the population, the white flower gene BrWF3 of Chinese cabbage is positioned in the 105.6kb interval of chromosome 2, and the loss of protein function is caused by one SNP deletion of a third exon, so that the formation of PGs is interfered, and the activity of carotenoid metabolism and the carotenoid content are reduced. Jia et al have analyzed gene expression involved in carotenoid biosynthesis by transcriptomics and metabonomics using the rape yellow cultivar 'Zhongshuang 11' and the White self-line 'White Petal', and found that BnNCED4b encodes a protein involved in carotenoid degradation with abnormally high levels of expression in White petals, suggesting that it may play a key role in White Petal formation. In Solanaceae plants Wang et al found that the purple-flower trait of eggplant was controlled by a single dominant gene and located this gene encoding anthocyanin synthase SmFAS within a region of about 165.6kb on chromosome 8, a single base mutation resulted in premature termination of FAS resulting in white petals.
There is little research on flower color in cucurbitaceae crops, watermelon petals are usually yellow, but Young-seak Kwo et al found pale green flowers in watermelon line Kw-695. The research shows that the heredity of Kw-695 light green flower character is controlled by a recessive gene Clgf. Kw-695 plants have large vines and large pale green leaves, oval, yellowish green fruits with irregular dark green streaks, yellow to orange, inedible pulp, very low sugar content, pale yellow seeds. The genetic rules of Young-Seok Kwo et al were analyzed and no study was made at the molecular level. We have found from previous studies that no genes have been targeted to control the color of the petals of watermelons.
Disclosure of Invention
The invention aims to provide two pairs of molecular markers closely linked with a green petal gene Clgf of the watermelon, namely an SSR molecular marker and an Indel molecular marker, respectively, and respectively adopting two marker forms to position the green petal gene Clgf of the watermelon, thereby providing assistance for molecular marker-assisted breeding of the color of the watermelon.
In order to achieve the above purpose, the present invention provides the following technical solutions:
according to the invention, through a segregating group grouping analysis (BSA), 12 pairs of SSR primers with polymorphism differences on chromosome 11 are obtained, through analysis, one SSR molecular marker is closely linked with a green petal gene Clgf of watermelon, the upstream primer sequence of the primer pair for amplifying the molecular marker is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO. 2.
The invention also adopts another marking form to find Indel molecular marking closely linked with the green petal gene Clgf of the watermelon, the upstream primer sequence of a primer pair for amplifying the molecular marking is shown as SEQ ID NO.5, and the downstream primer sequence is shown as SEQ ID NO. 6.
The invention is mainly characterized in that the application of the molecular marker closely linked with the green petal gene Clgf of the watermelon in molecular breeding of the watermelon comprises the steps of identifying or assisting in identifying the color character of the petals of the watermelon and judging and identifying the genotype of the colors of the petals of the watermelon.
The method for judging the green petal variety or genotype of the watermelon by utilizing the SSR molecular markers comprises the following steps:
(1) Extracting genome DNA of the watermelons to be detected;
(2) Taking the genomic DNA extracted in the step (1) as a template, carrying out PCR amplification by utilizing primer pairs (SEQ ID NO.1 and SEQ ID NO. 2) of SSR molecular markers, and carrying out electrophoresis detection and/or sequencing on PCR amplification products;
(3) Judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has a characteristic band with the length of 248bp as shown in SEQ ID NO.4, the variety to be detected is a watermelon green petal character variety (genotype Clgf/Clgf); if the PCR amplification product only has a characteristic band with the length of 268bp as shown in SEQ ID NO.3, the variety to be detected is a homozygous yellow petal character variety (genotype CLGF/CLGF) of the watermelon; if the PCR amplified product has a characteristic band with the length of 248bp as shown in SEQ ID NO.3 and a characteristic band with the length of 268bp as shown in SEQ ID NO.4, the variety to be detected is a heterozygous watermelon yellow petal character variety (genotype CLGF/Clgf).
The method for judging the green petal variety or genotype of the watermelon by utilizing Indel molecular markers is characterized by comprising the following steps of:
(1) Extracting genome DNA of the watermelons to be detected;
(2) Taking the genome DNA extracted in the step (1) as a template, carrying out PCR (polymerase chain reaction) amplification by using a primer pair of a molecular marker, and carrying out electrophoresis detection and/or sequencing on a PCR amplification product;
(3) Judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has one characteristic band with the length of 82bp shown as SEQ ID NO.7, the variety to be detected is a watermelon yellow petal character variety (genotype Clgf/Clgf); if the PCR amplification product only has one characteristic band with the length of 76bp shown as SEQ ID NO.8, the variety to be detected is a homozygous green petal character variety (genotype CLGF/CLGF) of the watermelon; if the PCR amplified product has a characteristic band with the length of 76bp as shown in SEQ ID NO.7 and a characteristic band with the length of 82bp as shown in SEQ ID NO.8, the variety to be detected is a heterozygous watermelon yellow petal character variety (genotype CLGF/Clgf).
In addition, the kit containing the primer pair can be used for identifying the petal color character of the watermelon material, and the kit can be prepared by selecting the kit containing the molecular marker primer pair when the kit is applied specifically.
The application of the reagent for detecting the existence of the SSR molecular marker or Indel marker in the positioning of the watermelon green flower gene Clgf can be carried out by utilizing the molecular marker of the invention to position the watermelon green flower gene Clgf, and the application can be carried out according to a conventional method.
The invention has the advantages that:
the research utilizes the hybridization of the green flower material PI482276 and the yellow flower materials WT2 and WT15, utilizes the molecular marking technology to finely position the green flower character of the watermelon, finds out SSR and Indel molecular markers closely linked with the green flower character, has stable variation and easy detection, and can be directly used for molecular marker assisted breeding of the green petal material of the watermelon. The molecular marker has the advantages of simplicity, convenience, rapidness and high flux in an auxiliary breeding system, so that the molecular marker provided by the invention has good application value in cultivation of new varieties of watermelon green flowers.
Drawings
FIG. 1 is a photograph of different petals of different watermelon materials, wherein A is watermelon material PI482276, B is watermelon material WT2, and C is watermelon material WT15;
FIG. 2 is a fine positioning map of the watermelon green flower gene Clgf;
FIG. 3 shows that the C1SSR30082 molecular markers used in the invention are used in the parent PI482276 and WT2, F1 and F 2 Polymorphism electrophoresis pattern in the generation plant;
FIG. 4 shows the Indel186 molecular markers used in the present invention in the parent PI482276 and WT2, F1 and F 2 Polymorphism electrophoresis pattern in the generation plants.
Detailed Description
The following is a detailed description of the invention
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting 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. The reagents and materials employed, unless otherwise indicated, are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Biological material
Watermelon material PI482276, the color of which is green;
watermelon material WT2 is a high-generation inbred line bred by the inventor, the color is yellow, the material can be obtained through commercial paths or provided by a genetic breeding subject group of melon crops of Henan agricultural university (it is to be explained that the adoption of the material as a research basis is only the convenience reason for obtaining experimental materials, and the implementation of the related technical scheme of the application is not to be understood as depending on the experimental materials);
the watermelon material WT2 is consistent with the normal common tendril-containing watermelon material WT2 adopted in the Chinese patent with publication number CN1l0938706A, which is a molecular marker closely linked with the tendril-free gene Clnt of watermelon plants and application.
Watermelon material WT15 is a high-generation inbred line bred by the inventor, the color is yellow, the material can be obtained through commercial paths or provided by a genetic breeding subject group of melon crops of Henan agricultural university (it is to be explained that the adoption of the material as a research basis is only the convenience reason for obtaining experimental materials, and the implementation of the related technical scheme of the application is not to be understood as depending on the experimental materials);
the above biological materials are stored in laboratories of applicant units and can be distributed to the public for verification tests within twenty years from the filing date or can be obtained by purchase.
In the experimental process, the parents and the constructed groups are planted in a sunlight greenhouse of a Mao Zhuang science education park of Henan agricultural university, and the planting process is as follows: and after germination acceleration, plug seedling is carried out, and the color type of individual petals is counted in the day of flowering by adopting a normal watermelon cultivation management mode.
Experimental reagents and apparatus:
in the experimental process, PCR Taq-Mix for PCR amplification is purchased from Nanjinouzan gene technology Co., ltd; other electrophoresis and silver staining related reagents such as acrylamide, methylene acrylamide, agNO 3 Reagents such as NaOH and formaldehyde were purchased from beijing solebao technologies limited;
primers for PCR amplification (artificial synthesis) and gene sequencing in the experimental process are provided by the Soaceae genome research center Co.
The PCR instrument is a Hema9600 gene amplification instrument of the medical instrument of Pinctada martensii;
the electrophoresis apparatus is JY300HC universal electrophoresis apparatus, which is produced by Beijing junyi Oriental electrophoresis apparatus Limited company;
the electrophoresis tank is an HT-SCZ04A high-flux vertical electrophoresis tank and is produced by Beijing hong Tao Ji Zhou Ji Zhi Ji.
Example 1
The method for determining the green flower gene locus of the watermelon comprises the following specific steps:
(1) Construction of genetically isolated populations
The hybrid composition wt2×pi482276 (QT 1), wt15×pi482276 (QT 2) was prepared from the yellow petals of watermelon by using the yellow petals of watermelon materials WT2 and WT15 as female parent and the green petals of watermelon PI482276 as male parent. The results show that F is obtained 1 The petals of the plant are all yellow. (phenotype of parent Material is shown in FIG. 1.)
From QT1-F 1 、QT2-F 1 10 single plants are selected from the generation plants, and F is obtained by selfing 2 And seed generation for genetic analysis and gene localization.
For F 2 The petal color phenotype of the individuals of the generation is identified and verified by using chi-square test. The results show that: 441 QT1-F plants planted in spring 2021 2 The color phenotype investigation analysis of the population shows that: the yellow petals have 334 plants, the green petals have 107 plants, and the separation ratio of the yellow petals to the green petals is 3:1 after chi-square test. For 358 strain QT2-F 2 The population phenotype survey shows that 258 plants are yellow petals, 100 plants are green petals, and the separation ratio of 3:1 is also met through chi-square analysis.
From the analysis of the results, the green petal shape of the watermelon is controlled by a recessive single gene.
(2) Preliminary localization of watermelon green flower gene
The parent PI482276, WT2 and WT15 are used as templates to carry out preliminary screening on the SSR primers synthesized by the subject group, and the primers with polymorphism between parents are screened out. According to the principle of separation group analysis (BSA), from F 2 Randomly selecting 20 green flower plants and 20 yellow flower plants in the population to construct a mixed pool, and secondarily screening the polymorphic primers obtained by primary screeningAnd (3) screening 12 pairs of polymorphic markers. According to the information of SSR markers (synthesized before the present laboratory), 12 markers with polymorphism between parents and pools were all located on chromosome 11.
(4) Fine localization of watermelon green flower gene
Three parents were subjected to 20 x depth high throughput resequencing using an IIlumina Hi-seq2000 high throughput sequencing platform. Combining the results of (2), by developing more SSR markers, increasing F 2 Population number of individuals we mapped the watermelon green gene Clgf between chromosome 11 cisr 30082 and cisr 30101. Combining the result of parent resequencing, taking the segment of the whole genome sequence of the watermelon between SSR markers CISSR30082 and CISSR30101 as a reference sequence, comparing the sequences, developing and designing 5 pairs of SSR markers and 3 pairs of Indel markers according to SNPs and Indels differences between the two parent sequences, detecting 3 SNP loci, and finally locking candidate genes in a 139.7kb region between SNP1 and CISSR30219 (see figure 2).
(4) Development of SSR Indel markers for candidate regions
The invention utilizes the CISSR30082 molecular marker to carry out PCR amplification distinction, and the primer sequence is designed as follows:
C1SSR30082-F：5’-CGATTAGAGGACCTGCTTCG-3’(SEQ ID NO.1)；
C1SSR30082-R：5’-TGATTTTCGCATATCAACTGC-3’(SEQ ID NO.2)。
a characteristic band of 268bp is obtained by PCR amplification, and the specific base sequence of the characteristic band of 268bp is shown as follows:
a248 bp characteristic band is obtained through PCR amplification, and the specific base sequence of the 248bp characteristic band is shown as follows:
the amplification product of the primer in the female parent (yellow flower) has the size of 268bp, the amplification product in the male parent (green flower) has the size of 248bp, and the primer is used for amplifying the DNA in the F 2 The accuracy of the mark in the method reaches 99.49 percent.
The invention uses the Indel186 molecular marker to carry out PCR amplification, and the primer sequence is designed as follows:
Indel186-F：5’-CATATCCTTGAACATTTCTG-3’(SEQ ID NO.5)；
Indel186-R：5’-AGTTTTCCATGCTTCTATTT-3’(SEQ ID NO.6)。
a characteristic band of 82bp is obtained through PCR amplification, and the specific base sequence of the characteristic band of 82bp is shown as follows:
a76 bp characteristic band was obtained by PCR amplification, and the specific base sequence of the 76bp characteristic band was as follows:
the amplified product of the primer in the female parent (yellow flower) has the size of 82bp, the amplified product in the male parent (green flower) has the size of 76bp, and the primer is used for amplifying the DNA in the F 2 The accuracy of the mark in the method reaches 97.7 percent.
Example 2 application of molecular markers in breeding
(1) DNA extraction
Extracting parent strain, F by CTAB method 1 F (F) 2 Total DNA of leaves of the isolated population: 3-5g of tender leaves (or frozen leaves) are put into a vacuum freeze dryer for dehydration and drying, immediately ground into powder on a high-flux tissue grinder after drying, put into a 2ml centrifuge tube, added with 800 mul of preheated CTAB extraction buffer solution, fully and uniformly mixed with the buffer solution, and subjected to water bath at 65 ℃ for 0.5-2h;adding 700 μl chloroform/isoamyl alcohol (24:1) mixture, slightly turning over, mixing, and extracting parent strain with CTAB method, F 1 F (F) 2 Total DNA of leaves of the isolated population: taking the tender leaves at the upper part, rapidly grinding the tender leaves into powder in liquid nitrogen, and placing the powder into a centrifuge tube with the volume of 1.5 ml; adding 800 μl of preheated CTAB extraction buffer, and water-bathing at 65deg.C for 30min; adding equal volume of chloroform isoamyl alcohol, wherein the volume ratio of chloroform to isoamyl alcohol is 24:1, after uniform mixing, centrifuging for 15min at 12000 r/min; transferring the supernatant into a new centrifuge tube, adding isopropyl alcohol with equal volume, gently mixing, and ice-bathing for more than 1 h; centrifuging at 12000r/min for 15min; pouring out the supernatant, washing the precipitate twice with ethanol with the volume fraction of 75%, drying, adding 200 mu l of TE buffer solution for dissolution, adding 10 mu g/ml of RNase for RNA removal, and carrying out water bath at 37 ℃ for 30min; the concentration of the obtained DNA was estimated by electrophoresis on a 0.8% agarose gel using 50 ng/. Mu.l of DNA as a standard; diluting with TE to final concentration of 100 ng/. Mu.l, and storing at-20deg.C;
(2) PCR amplification
The reaction system: master Mix 5. Mu.L, DNA template (30 ng/. Mu.L) 1. Mu.L, F-Primer (5. Mu. Mol/. Mu.L) 0.5. Mu.L, R-Primer (5. Mu. Mol/. Mu.L) 0.5. Mu.L, dd H 2 O 3 Mu L; the reaction procedure is: 95℃for 5min (pre-denaturation); 94℃45S (denaturation); 6 cycles 68-58 ℃ (annealing); 1min (extension) at 72 ℃; 30S at 94 ℃ (denaturation); 8 cycles 58-50 ℃ (annealing); 1min (extension) at 72 ℃; 30S at 94 ℃ (denaturation); 30S (annealing) at 50 ℃;20 cycles at 72℃for 1min (extension); 7min at 72 ℃ (extension); 4 ℃ (heat preservation).
(3) Gel plate preparation
Cleaning of the glass plate: and cleaning the glass plate, thoroughly airing, and then assembling and glue filling.
Working fluid (two, about 80 mL) was prepared: 29:1 (acrylic acid: bis-acrylic acid): 24ml 5 XTBE: 16ml ddH 2 O:40ml TEMED:60 μl 10% AP:700 μl of the solution was poured into a beaker and then mixed uniformly, and then immediately poured into the gap between the two glass plates, and after the glue solution was poured to the bottom, a comb was gently inserted between the upper and lower glass plates at the glue pouring port, and allowed to coalesce for 15-30min. After gelling, the electrode buffer 1xTBE was poured into the two tanks and the comb was pulled out slowly and in equilibrium.
Spotting: the PCR products were added to 1-2. Mu.l Loading dye per well, and the Loading amount was generally 0.8-1.2. Mu.l, and the electrophoresis was performed by connecting the electrophoresis line immediately after the completion of spotting.
Electrophoresis: the electrophoresis time is estimated according to the fragment size of the PCR product, and is generally 0.5-1.5h.
Dyeing: configuring AgNO 3 Dyeing liquid, weighing 1.2g AgNO 3 1000m1 ddH was added 2 O. Shaking the mixture on a shaking table for 3-5min.
Color development: a color-developing solution was prepared, 15g of NaOH was weighed and 1000ml of ddH was added thereto 2 O, adding 15ml of formaldehyde solution (now available) and adding ddH after dyeing 2 O is washed for 2 times, and the color development liquid is added. The shaker is gently shaken for 3-5min until DNA bands appear.
(4) Band type interpretation: the developed gel was placed on a reader table and the difference in the positions of the two parent bands was observed, some of which are shown in FIGS. 3 and 4.
Specifically, the Indel186 molecular marker is applied to breeding, and the application steps are as follows:
PCR reactions were performed using Indel186 molecular markers to determine the presence of the molecular markers at F 2 Genotype in population. Detection of the two genotypes of the Indel186 molecular marker at 787F 2 If the distribution condition in the population is that only one PCR amplification product has a size of 76bp, the variety to be detected is a variety with the green petal character of the watermelon; if the PCR amplification product only has a characteristic band with the length of 82bp, the variety to be detected is a homozygous watermelon yellow petal character variety; if the PCR amplified product has a characteristic band with the length of 76bp and a characteristic band with the length of 82bp, the variety to be detected is a heterozygous watermelon yellow petal-shaped variety, the Indel186 molecular marker primer (SEQ ID NO.5 and SEQ ID NO. 6) is utilized for carrying out PCR amplification, different varieties are identified, and the detection is carried out in F 2 The identification accuracy of the marker reaches 97.7 percent.
The application of the C1SSR30082 molecular marker in breeding comprises the following application steps:
PCR reactions using the C1SSR30082 molecular marker to determine the molecular marker at F 2 Genes in a populationType (2). Detection of ClSSR30082 molecular markers with two genotypes of 787F 2 If the distribution condition in the population is that only one PCR amplification product has 248bp, the variety to be detected is a variety with the green petal character of the watermelon; if the PCR amplification product only has a characteristic band with the length of 268bp, the variety to be detected is a homozygous yellow petal character variety of the watermelon; if the PCR amplified product has a characteristic band with the length of 248bp and a characteristic band with the length of 268bp, the variety to be detected is a heterozygous watermelon yellow petal character variety, and different varieties are identified by PCR amplification by using the CISSR30082 molecular marker primers (SEQ ID NO.1 and SEQ ID NO. 2) of the invention, and the identification is carried out on the different varieties in F 2 The identification accuracy of the marker reaches 99.49 percent.
Sequence listing
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Claims (9)
1. Green petal gene of watermelonClgfThe closely linked SSR molecular marker primer is characterized in that the upstream primer sequence is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO. 2.
2. Green petal gene of watermelonClgfThe closely linked Indel molecular marker primer is characterized in that the upstream primer sequence is shown as SEQ ID NO.5, and the downstream primer sequence is shown as SEQ ID NO. 6.
3. A watermelon green petal gene according to claim 1 or claim 2ClgfThe application of the closely linked molecular marker primer in molecular breeding of color traits of watermelon petals.
4. The use according to claim 3, wherein the molecular marker primer is used for identification or assisted identification of the colour trait of watermelon petals.
5. The method for judging the green petal variety of the watermelon is characterized by comprising the following steps of:
(1) Extracting genome DNA of the watermelons to be detected;
(2) Performing PCR amplification by using the molecular marker primer of claim 1 and performing electrophoresis detection and/or sequencing on the PCR amplification product by using the genomic DNA extracted in the step (1) as a template;
(3) Judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has one characteristic band with the length of 248bp as shown in SEQ ID NO.4, the variety to be detected is a variety with the green petal character of the watermelon; if the PCR amplification product only has one characteristic band with the length of 268bp as shown in SEQ ID NO.3, the variety to be detected is a homozygous watermelon yellow petal character variety; if the PCR amplified product has both a characteristic band with the length of 248bp as shown in SEQ ID NO.3 and a characteristic band with the length of 268bp as shown in SEQ ID NO.4, the variety to be detected is a heterozygous watermelon yellow petal character variety.
6. The method for judging the green petal variety of the watermelon is characterized by comprising the following steps of:
(1) Extracting genome DNA of the watermelons to be detected;
(2) Using the genome DNA extracted in the step (1) as a template, performing PCR amplification by using the molecular marker primer of claim 2, and performing electrophoresis detection and/or sequencing on a PCR amplification product;
(3) Judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has one characteristic band with the length of 82bp as shown in SEQ ID NO.7, the variety to be detected is a watermelon yellow petal-shaped variety; if the PCR amplification product only has one characteristic band with the length of 76bp as shown in SEQ ID NO.8, the variety to be detected is a homozygous green petal character variety of the watermelon; if the PCR amplified product has a characteristic band with the length of 82bp as shown in SEQ ID NO.7 and a characteristic band with the length of 76bp as shown in SEQ ID NO.8, the variety to be detected is a heterozygous watermelon yellow petal character variety.
7. A kit for identifying the green petal trait of watermelons, comprising the primer pair of claim 1 or claim 2.
8. The application of the reagent for detecting the existence of the SSR molecular marker in the color gene positioning of the watermelon petals is characterized in that the reagent comprises a primer pair for amplifying the SSR marker, wherein the upstream primer sequence of the primer pair is shown as SEQ ID No.1, and the downstream primer sequence is shown as SEQ ID No. 2.
9. The application of the reagent for detecting the presence of the Indel marker in the color gene localization of the watermelon petals is characterized in that the reagent comprises a primer pair for amplifying the Indel marker, wherein the upstream primer sequence of the primer pair is shown as SEQ ID No.5, and the downstream primer sequence is shown as SEQ ID No. 6.
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| CN202210105137.8A CN114292954B (en) | 2022-01-26 | 2022-01-26 | Molecular marker closely linked with green petal gene Clgf of watermelon and application thereof |
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| CN202210105137.8A CN114292954B (en) | 2022-01-26 | 2022-01-26 | Molecular marker closely linked with green petal gene Clgf of watermelon and application thereof |
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| CN114292954B true CN114292954B (en) | 2023-06-30 |
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| WO2015192566A1 (en) * | 2014-06-16 | 2015-12-23 | 北京市农林科学院 | Cucumber target leaf spot resistance gene cca as well as linked molecular markers and applications thereof |
| CN107254542A (en) * | 2017-08-09 | 2017-10-17 | 中国农业科学院郑州果树研究所 | Watermelon color traits major gene loci and its InDel molecular labelings and application |
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| CN113322345A (en) * | 2021-07-01 | 2021-08-31 | 河南农业大学 | Molecular marker co-separated from watermelon peel grain covering gene ClGS and application |
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| CN107254542A (en) * | 2017-08-09 | 2017-10-17 | 中国农业科学院郑州果树研究所 | Watermelon color traits major gene loci and its InDel molecular labelings and application |
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