CN110607391A - Molecular marker closely linked with character control of Indian pumpkin red fruit peel, primer and application - Google Patents
Molecular marker closely linked with character control of Indian pumpkin red fruit peel, primer and application Download PDFInfo
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Abstract
A molecular marker, a primer and an application which are closely linked with the control of the property of the red fruit peel of the Indian pumpkin belong to the technical field of molecular marker assisted breeding. The molecular marker FC-01 consists of a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2. FC-02 is composed of the nucleotide sequence shown by SEQ ID NO.3 and the nucleotide sequence shown by SEQ ID NO.4 in the sequence list. The invention discloses a primer sequence for amplifying the molecular marker, which has higher stability. The molecular auxiliary selection of the Indian pumpkin red peel can be used for simply and quickly screening the red peel types in the pumpkin seedling stage, and the backcross transformation method can quickly culture the Indian pumpkin red peel variety improved strain.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a molecular marker closely linked with the property of controlling the red fruit peel of an Indian pumpkin, a primer and application.
Background
Molecular marker assisted breeding is a rapidly developing new technology, and direct selection of genotypes is realized by rapidly and accurately analyzing the genetic composition of individuals on the molecular level, so that molecular breeding is performed.
Pumpkin (Cucurbita maxima L.) is an annual vine herb of the Cucurbitaceae family (Cucurbitaceae) Cucurbita, originates from mexico to central and south america, is widely cultivated worldwide, and is also one of the important cucurbit crops in China. The fruit pulp and the seeds can be eaten, and the cultivation method has the advantages of easy cultivation, high yield, rich nutritive value and the like; the fruit is the most important part of the economic traits of the pumpkin, and the pumpkin fruit belongs to the bottle gourd and is developed by a lower ovary. An important quality character of the pumpkin fruits is the color of the peel, and the color of the peel is an important commodity character of the pumpkin. The color of the Indian pumpkin peel has rich diversity and has different colors such as orange, orange yellow, dark green, light green, gray and the like. The color of the peel is mainly regulated and controlled by carotenoid, chloroplasts are gradually changed into color bodies in the process of continuous fruit ripening, chlorophyll is continuously reduced, and carotenoids such as lutein and carotene are generated in large quantity and accumulated in the color bodies, so that the peel of the pumpkin is represented as orange or orange which mainly contains lutein. The bright color is one of important commodity characters of the pumpkins, so that the Indian pumpkin fruits with the tangerine peel character bred by utilizing the tangerine peel character of the pumpkins have wide application prospect and great economic value.
The traditional breeding method has the problems of long breeding period and the like in the selection process of the fruit peel of the Indian tangerine peel. The method is an effective method in pumpkin genetic breeding by using molecular markers closely linked with target traits for marker-assisted selection, can effectively broaden the application of the traits and promote the quality breeding process. Therefore, the development of the molecular marker of the indian pumpkin and the exocarpium citri rubrum with closely linked characters has very important significance for improving the quality of the indian pumpkin and disclosing the molecular mechanism of the exocarpium citri rubrum formation.
Disclosure of Invention
In order to rapidly and efficiently identify the red peel property of the pumpkin indicum and accelerate the selective breeding process of the pumpkin indicum, the invention provides a molecular marker which is closely linked with the property of controlling the red peel property of the pumpkin indicum, the molecular marker is FC-01 and FC-02, and the molecular marker FC-01 consists of a DNA fragment shown by SEQ ID NO.1 and a DNA fragment shown by SEQ ID NO. 2; the molecular marker FC-02 consists of a DNA fragment shown by SEQ ID NO.3 and a DNA fragment shown by SEQ ID NO. 4.
Further limited, the DNA fragment shown in SEQ ID NO.1 in the molecular marker FC-01 is linked with the red fruit peel trait gene R of the pumpkin indicum, and the DNA fragment shown in SEQ ID NO.2 is linked with the non-red fruit peel trait gene R of the pumpkin indicum.
Further limited, the DNA fragment shown in SEQ ID NO.3 in the molecular marker FC-02 is linked with the red fruit peel trait gene R of the pumpkin indicum, and the DNA fragment shown in SEQ ID NO.4 is linked with the non-red fruit peel trait gene R of the pumpkin indicum.
The invention also provides a primer for amplifying the molecular marker, wherein the sequence of the primer for amplifying the molecular marker FC-01 is shown as SEQ ID NO.5 and SEQ ID NO. 6; the primer sequence of the amplification molecular marker FC-02 is shown as SEQ ID NO.7 and SEQ ID NO. 8.
The invention also provides application of the molecular marker in breeding of the pumpkin with the peel character.
The invention also provides application of the primer in pumpkin breeding with the peel character.
The invention also provides a method for breeding the pumpkin with the peel character, which comprises the following steps:
1) taking the Indian pumpkin red peel line 2013-12 as a female parent and the pumpkin gray peel line 9-6 as a male parent, hybridizing the female parent and the male parent to obtain F1Backcrossing the generations with the male parent to obtain offspring BC1F1Respectively scanning BC by using the molecular marker FC-01 and the molecular marker FC-02 of the invention1F1Selecting plants which are scanned by the molecular marker FC-01 and have nucleotide fragments of 44nt, 396nt, 326nt and 722nt and scanned by the molecular marker FC-02 and have nucleotide fragments of 420nt, 421nt and 841nt, backcrossing the plants with the male parent to obtain the plant populationSubstituted BC2F1；
2) Scanning BC Using molecular marker FC-01 and molecular marker FC-02, respectively2F1Selecting plants which have nucleotide fragments of 44nt, 396nt, 326nt and 722nt after being scanned by a molecular marker FC-01 and have nucleotide fragments of 420nt, 421nt and 841nt after being scanned by a molecular marker FC-02, and backcrossing the plants with the male parent to obtain the backcross third generation BC3F1；
3) Scanning of BC Using molecular marker FC-01 and molecular marker FC-023F1Selecting plants with 44nt, 396nt, 326nt and 722nt nucleotide fragments after being scanned by the molecular marker FC-01 and 420nt, 421nt and 841nt nucleotide fragments after being scanned by the molecular marker FC-02 for selfing, and marking the progeny as BC3F2；
4) Scanning of BC Using molecular markers FC-01 and FC-023F2And selecting a plant which has 44nt and 722nt nucleotide fragments after being scanned by the molecular marker FC-01 and has 841nt nucleotide fragments at the marker FC-02, namely the improved red pericarp inbred line.
Preferably, the pumpkin gray peel product in the step 1) is Indian pumpkin gray peel 9-6.
The parental red peel line "2013-12" and the Indian Pumpkin gray peel line "9-6" specifically used in the present invention are described in "morphology, transformed and homenic Characterization of trimonoecia and subandoecious Pumpkin (Cucurbita maxima) of sunlight apple of Ethylene in Sex Expression" (Wang et al, 2019), which is available to the public at northeast university of agriculture.
Advantageous effects
The invention obtains F by hybridizing Indian pumpkin red peel ' 2013-12 ' and gray peel ' 9-61Plant, F1Selfing the plants to obtain 1627F2Segregating the population, and determining that the Indian pumpkin red peel character belongs to the dominant character controlled by a single gene through phenotypic analysis. Then take F respectively2And (3) extracting genome DNA from each young leaf of the segregation population, and screening a molecular marker closely linked with the Indian pumpkin red fruit peel character gene R by combining a SLAF-seq method. By usingThe molecular markers of the high-density map, which are finally provided, are closely linked with the Indian pumpkin red fruit peel and are respectively positioned at two sides of the gene for controlling the target character, and the genetic distance of the two markers is 64.47Kb, so that the establishment of a molecular marker-assisted breeding system of the red fruit peel is facilitated. The molecular marker of the invention can be applied and bred in practice simply, conveniently, quickly and at high flux.
The invention also provides application of the molecular marker in rapid cultivation of the red fruit peel character Indian pumpkin. By using a backcross transformation method, the red peel '2013-12' is used as a non-recurrent parent, the gray peel '9-6' is used as a recurrent parent, a backcross population is scanned by using the two molecular markers FC-01 and FC-02, and the peel color of the gray peel '9-6' is rapidly and accurately improved according to the enzyme digestion band of a PCR amplification product. The CAPS molecular markers have high stability, and the molecular markers are used for constructing a high-density genetic map and a high-density physical map, so that the establishment of a molecular marker-assisted breeding line for screening the target genes of the color of the red pericarp of the pumpkin of India is facilitated.
Drawings
FIG. 1 shows that CAPS markers FC-01 and FC-02 are closely linked to the control of the R gene of the red pericarp of pumpkins, each cross representing 2 single crossover events;
FIG. 2 shows CAPS tag FC-01P1，P2，F1，F2The enzyme digestion strip of the colony PCR product, wherein M is D2000Marker, the length of the strip is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from high to low, and P1Represents the peel line of fructus crataegi '2013-12', P2Representing the lime peel line '9-6', F1Is P1And P2First hybrid generation of (1), F2Is F1Progeny after generation selfing, F2Peel population of red fruit and F2The non-red peel populations are shown at F2Randomly selecting 10 red pericarps and 10 non-red pericarp plants from the population;
FIG. 3 shows CAPS flag FC-02P1，P2，F1，F2The enzyme digestion strip of the colony PCR product, wherein M is D2000Marker, the length of the strip is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from high to low, and P1Represents the red pericarp series '2013-12', P2Represents the gray pericarp series '9-6', F1Is P1And P2First hybrid generation of (1), F2Is F1Progeny after generation selfing, F2Peel population of red fruit and F2The non-red peel populations are shown at F2Randomly selected 10 of the red pericarp and 10 of the non-red pericarp plants in the population.
Detailed Description
The invention is further illustrated below with reference to specific embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
The red fruit peel does not represent chromatically pure red, and the red fruit peel is orange or red with color intensity between orange and red.
Example 1 screening of molecular markers closely linked to control of the red peel trait of pumpkin indicum.
1.F2Construction of the population
In the example, Indian pumpkin high-generation inbred line red fruit peel '2013-12' and gray fruit peel '9-6' are selected as parents to construct F2A population in which the parent non-red pericarp '9-6' is gray pericarp cucurbita maxima. Using the two parents to prepare the hybrid combination obtained F1Red peel, F1Generation selfing to produce F2Passage segregating population, identifying 1627F in field2The red/gray phenotype in generation individuals is verified by chi-square analysis to obtain the dominant property that the red peel property of the Indian pumpkin is under single gene control, the genetic analysis is carried out by constructing a six-generation population to find that the red peel property is dominant, and the red peel gene is named RED (R).
2. Screening of haw peel-linked CAPS marker
Extraction of pumpkin genome DNA
Extraction of parent and F by CTAB method2Total DNA from leaves of the population was isolated. The method comprises the following steps: quickly grinding two leaves of true leaf in liquid nitrogen into powder, and placing into 2.0ml centrifuge tubePerforming the following steps; adding preheated 1000 mul CTAB extraction buffer solution, and carrying out water bath at 65 ℃ for 1 h; taking out, cooling to room temperature, centrifuging at 12000r/min at 4 ℃ for 17min, and sucking 800 μ l of supernatant into a new 2.0ml centrifuge tube; adding a chloroform isoamyl alcohol mixed solution with the same volume, wherein the volume ratio of chloroform to isoamyl alcohol is 24: 1, evenly mixing, and centrifuging at 12000r/min at 4 ℃ for 13 min; sucking 700 mul of supernatant into a new 2.0ml centrifuge tube; continuously adding equal volume of chloroform isoamyl alcohol, mixing uniformly, and centrifuging at 12000r/min at 4 ℃ for 13 min; taking 600 mu l of supernatant liquid to a new 1.5ml centrifuge tube, adding isopropanol with the same volume, gently mixing uniformly, and storing for 1h in a refrigerator at the temperature of-20 ℃; centrifuging at 12000r/min at 4 ℃ for 10 min; pouring out the supernatant, washing the precipitate twice with 70% ethanol by volume fraction, drying, and adding 40 μ lddH2Dissolving O, adding 10 mu g/ml RNase to remove RNA, and carrying out water bath at 37 ℃ for 30 min; the concentration of the resulting DNA was estimated by electrophoresis on a 0.8% agarose gel using 50 ng/. mu.l of lambda DNA as a standard and stored at-20 ℃ until use.
Acquisition of molecular markers
According to F2And randomly selecting 100 individual plants of the red pericarp and the ash pericarp according to the field survey results of the generation groups, constructing an SLAF library, developing 8406 polymorphic SLAF labels, and then positioning the R gene of the red pericarp of the pumpkin to a No. 14 chromosome end association region by utilizing SNP-index marker association analysis.
SNP results obtained by the genome re-sequencing of the pumpkin of Indian squash are analyzed, and CAPS marker primers are designed. Using a haw peel parent, a lime peel parent and F1DNA is taken as a template, and an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6 (an amplification molecular marker FC-01) are respectively used; the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8 (an amplification molecular marker FC-02) are subjected to PCR amplification, and then the PCR product is subjected to restriction enzyme digestion, wherein the primers are synthesized by the Huada gene. The PCR amplification and restriction endonuclease system and procedure were as follows:
TABLE 1 PCR reaction System
The PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 5 min; 35 cycles; final extension at 72 deg.C for 5 min; finally, the mixture is stored at 4 ℃.
Enzyme digestion system: mu.l of the PCR product obtained above, 3.8. mu.l of ddH2O, 10 XBuffer 1. mu.l, 0.2. mu.l of restriction endonuclease; the reaction conditions of the enzyme digestion test are as follows: the enzyme cutting temperature is 37 ℃, and the enzyme cutting reaction time is 3 h. Wherein, the molecular marker FC-01 amplification product is subjected to enzyme digestion by using an AluI restriction endonuclease (the enzyme cutting site of the AluI is AGCT), and the molecular marker FC-02 amplification product is subjected to enzyme digestion by using an AseI restriction endonuclease (the enzyme cutting site of the AseI is ATTAAT). The cleavage products were detected by electrophoresis using 2% agarose gel. Molecular markers FC-01 and FC-02 in the rind, non-rind parent and F1With polymorphisms in between and clearly banded markers.
③ CAPS molecular marker scanning F2Segregating populations
Scanning two parents and the F by using the two CAPS primer pairs (the primer sequences of the molecular marker FC-01 are shown as SEQ ID NO.5 and SEQ ID NO.6, and the primer sequences of the molecular marker FC-02 are shown as SEQ ID NO.7 and SEQ ID NO.8, both synthesized by Beijing university.) developed in the way2And searching for the differential individuals of the marker type and the character phenotype in the population, and obtaining the cross-over individuals of the target section.
The scanning in the invention is to amplify the corresponding molecular marker by using the primer, then carry out enzyme digestion on the molecular marker, and judge the color character of the peel according to the enzyme digestion result.
The PCR system, PCR program and restriction enzyme system used in the scanning process are the same as the second step. And (3) carrying out electrophoresis detection on the enzyme digestion product through 2% agarose gel, and observing an imaging result through an agarose gel imaging system.
The results of the partial scan of the molecular marker FC-01 are shown in FIG. 2, and the plant with the fruit peel all presents bands of 44nt, 722nt or 44nt, 396nt, 326nt and 722nt, wherein the 44nt fragment is too small to be shown in the agarose gel image, so the size of the restriction enzyme cutting band shown in FIG. 2 is 396nt and 326 nt. The ashpericarp plants exhibited bands of 44nt, 396nt and 326nt, with the 44nt fragment being too small to be visible on the agarose gel image, so the size of the cleavage bands shown in FIG. 2 were 396nt and 326 nt. The results of the scanning of the FC-02 part of the molecular marker are shown in FIG. 3, wherein the pericarp plants all show bands of 841nt or 420nt, 421nt and 841nt, and the pericarp plants show bands of 420nt and 421 nt.
1627F strains marked by the above 2 molecules2The separated population is verified, the result shows that 2 molecular markers, FC-01 and FC-02 can better distinguish the red peel from non-red peel plants, the 2 molecular markers are tightly linked with the red peel gene R and respectively located on two sides of the R gene, the genetic distance of the two markers is 64.47Kb (figure 1), wherein, the sequence shown by SEQ ID NO.1 in the molecular marker FC-01 is linked with the red peel gene R of the pumpkin of India pumpkin and can be cut into 44nt and 722nt nucleotide fragments by the restriction enzyme AluI enzyme, the sequence shown by SEQ ID NO.2 is linked with the gray peel gene R and can be cut into 44nt, 396nt and 326nt nucleotide fragments by the restriction enzyme AluI enzyme; the sequence shown in SEQ ID NO.3 in the molecular marker FC-02 is linked with the Indian pumpkin red peel gene R and cannot be cut by restriction enzyme AseI; the sequence shown in SEQ ID NO.4 is linked with the ash peel gene r and can be cut into nucleotide fragments of 420nt and 421nt by a restriction enzyme AseI enzyme.
There were 14 and 10 single crossover events for marker FC-1, marker FC-2 and R gene, each closely linked to the target segment. The CAPS molecular markers have high stability, and the molecular markers are used for constructing a high-density genetic map and a high-density physical map, so that the establishment of a molecular marker-assisted breeding line for screening the target genes of the color of the red pericarp of the pumpkin of India is facilitated.
3. Sequencing of polymorphic fragments
Taking genome DNA of a red fruit peel parent '2013-12' and a lime fruit peel parent '9-6' as templates, and respectively taking an upstream primer shown by SEQ ID NO.5 and a downstream primer shown by SEQ ID NO.6 (an amplification molecular marker FC-01); the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8 (an amplification molecular marker FC-02) are subjected to PCR amplification to perform PCR amplification. The total PCR reaction was 20. mu.l: 10 XBuffer 2. mu.l, dNTP 2. mu.l, DNA template 1.2. mu.l, upper and lower primers 0.8. mu.l, Taq enzyme 0.2. mu.l, ddH2O13. mu.l. The PCR reaction conditions are as follows: 95 deg.CPre-denaturation for 5min, denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 1min, 35 cycles; final extension at 72 deg.C for 5 min; finally, the mixture is stored at 4 ℃.
Adding 5 μ l of BioTeke SYBR Green I nucleic acid dye into 20 μ l of PCR amplification product, standing at room temperature for 10min to combine the dye with DNA, separating by 2% Agarose Gel electrophoresis, cutting down the target fragment under an ultraviolet lamp, and respectively recovering and purifying the DNA fragment by using TaKaRaMiniBEST Agarose Gel DNA Extraction Kit, wherein the specific steps refer to Kit instructions and product number 9762. The purified fragments were sequenced by Chida Gene Co., Ltd, and the sequencing results were analyzed by DNAMAN software. The fragment size of the fragment amplified by using the red fruit peel '2013-12' as a template and using the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 is 766nt, the amplification sequence is shown in SEQ ID NO.1, and the fragment comprises a restriction enzyme AluI restriction site. The size of a fragment amplified by taking the ash peel parent '9-6' as a template and utilizing an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6 is 766nt, an amplification sequence is shown in SEQ ID NO.2, and the fragment comprises two restriction enzyme AluI restriction sites. The size of an amplified fragment is 841nt by taking the red fruit peel '2013-12' as a template and utilizing an upstream primer shown by SEQ ID NO.7 and a downstream primer shown by SEQ ID NO.8, the amplified sequence is shown in SEQ ID NO.3, and the fragment does not contain a restriction enzyme AseI restriction site. The size of an amplified fragment is 841nt by taking the ash peel parent '9-6' as a template and utilizing an upstream primer shown by SEQ ID NO.7 and a downstream primer shown by SEQ ID NO.8, the amplified sequence is shown in SEQ ID NO.4, and the amplified fragment contains a restriction endonuclease AseI restriction site.
Example 2 Breeding method of red peel pumpkin ayurvedic.
In this embodiment, a backcross transformation method is used, wherein a haw peel '2013-12' is used as a non-recurrent parent, a lime peel '9-6' is used as a recurrent parent, and the color of the peel of the superior lime peel strain '9-6' "9-6" is directionally improved, and the specific operation steps are as follows:
(1) hybridizing the red peel '2013-12' and the gray peel '9-6', and obtaining F1Backcrossing the generations with the lime peel '9-6' to obtain the offspring BC1F1Two molecular markers of the invention, FC-01 and FC-02 scansBC1F1Population, i.e. at BC1F1Taking DNA of each plant in the population as a template, and respectively utilizing an upstream primer shown by SEQ ID NO.5 and a downstream primer shown by SEQ ID NO. 6; and carrying out PCR amplification on an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO.8, and then carrying out restriction enzyme digestion on an amplification product. The PCR reaction system, PCR reaction conditions and enzyme digestion system were the same as described in EXAMPLE 1. Selecting plants which are scanned by the molecular marker FC-01 and have nucleotide fragments of 44nt, 396nt, 326nt and 722nt and nucleotide fragments of 420nt, 421nt and 841nt and are scanned by the molecular marker FC-02, and backcrossing the plants with the ash pericarp '9-6' to obtain the offspring BC2F1。
(2) Scanning of BC Using molecular markers FC-01 and FC-02 of the invention2F1Continuously selecting plants which are marked with QC-01 and have nucleotide fragments of 44nt, 396nt, 326nt and 722nt and nucleotide fragments of 420nt, 421nt and 841nt after being scanned by molecular marker FC-02, and backcrossing with the gray pericarp '9-6' to obtain the backcross third generation BC3F1。
(3) Scanning of BC Using molecular markers FC-01 and FC-02 of the invention3F1Continuously selecting plants which are scanned by the molecular marker FC-01 and have nucleotide fragments of 44nt, 396nt, 326nt and 722nt and scanned by the molecular marker FC-02 and have nucleotide fragments of 420nt, 421nt and 841nt, selfing to obtain progeny BC3F2。
(4) Scanning of BC Using molecular markers FC-01 and FC-02 of the invention3F2And selecting plants which are scanned by the molecular marker FC-01 and have 44nt and 722nt nucleotide fragments and scanned by the molecular marker FC-02 and have 841nt nucleotide fragments, namely the improved red pericarp inbred line. The inbred lines were established and the pericarp color of the fruits was investigated from the day of pollination to the 45d maturity stage. The color of the pericarp at the mature period is red, which indicates that the improved inbred line has the red pericarp character, and other agronomic characters are similar to '9-6'. The molecular markers FC-01 and FC-02 of the invention can be used for rapidly and accurately improving the pericarp of the pumpkin indicum.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Nucleotide sequence listing
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<211> 841
<212> DNA
<213> molecular marker FC-02
<400> 3
tttattcaaa gcggtgcggg acttttattg gagattattt aggctcctcc ccttagacac 60
gatttcctta acctatgcta taacaacgta agctcaccgc tcgcttttat attttcaata 120
tataatattc tttttttttt ttccaaggta atgatgatca catggaaggc aatccattgg 180
cagatgaaaa gaagaaaaaa catgagaaag agaaaaaaaa aggcctttag gaaaatatgt 240
aagacagaaa caggaacaac ctggcatttc caaacaacat aatccaaaca aaaaaatcca 300
taaaagaaga aaattgtcga aaatagatga aaagaaagca gaggacattc attcattaca 360
gccatcatca tcctaataac agtacagcac aaaaaccaaa ctaaatcaga catgtcctaa 420
ttagtcaaga ttaagaaaaa tgtgtgaatc taacgatgga aagttggtat taaaaccttc 480
cctaattttc actgttgtgg tggtggtggt ggtggtggtg tggtggtggt gatagtcaaa 540
tctcaaatgg taatttggta agaacaatca gaaagcctag atcatgttca tatcagatca 600
atgtggtcag caaggaggat tatctgatgt tttttttggg taagcaacta attagattag 660
cccgggagcg atatattgat gtcggtgggc gaatttctcg tctcctgcaa ggaagaacgc 720
acggcagcat tggcacccca attgtctctc tggaaagccg aaggagcaaa actggccggt 780
tttagggacc cgggatttgg ttgaccgtta ccgtttaaac tgtcgtctgt atcggcattg 840
g 841
<210> 4
<211> 841
<212> DNA
<213> molecular marker FC-02
<400> 4
tttattcaaa gcggtgcggg acttttattg gagattattt aggctcctcc ccttagacac 60
gatttcctta acctatgcta taacaacgta agctcaccgc tcgcttttat attttcaata 120
tataatattc tttttttttt ttccaaggta atgatgatca catggaaggc aatccattgg 180
cagatgaaaa gaagaaaaaa catgagaaag agaaaaaaaa aggcctttag gaaaatatgt 240
aagacagaaa caggaacaac ctggcatttc caaacaacat aatccaaaca aaaaaatcca 300
taaaagaaga aaattgtcga aaatagatga aaagaaagca gaggacattc attcattaca 360
gccatcatca tcctaataac agtacagcac aaaaaccaaa ctaaatcaga catgtcctaa 420
ttaatcaaga ttaagaaaaa tgtgtgaatc taacgatgga aagttggtat taaaaccttc 480
cctaattttc actgttgtgg tggtggtggt ggtggtggtg tggtggtggt gatagtcaaa 540
tctcaaatgg taatttggta agaacaatca gaaagcctag atcatgttca tatcagatca 600
atgtggtcag caaggaggat tatctgatgt tttttttggg taagcaacta attagattag 660
cccgggagcg atatattgat gtcggtgggc gaatttctcg tctcctgcaa ggaagaacgc 720
acggcagcat tggcacccca attgtctctc tggaaagccg aaggagcaaa actggccggt 780
tttagggacc cgggatttgg ttgaccgtta ccgtttaaac tgtcgtctgt atcggcattg 840
g 841
<210> 5
<211> 20
<212> DNA
<213> molecular marker FC-01 upstream primer
<400> 5
caacggctat gaatcactgc 20
<210> 6
<211> 22
<212> DNA
<213> molecular marker FC-01 downstream primer
<400> 6
tcaattagaa taccaaagcc tg 22
<210> 7
<211> 18
<212> DNA
<213> molecular marker FC-02 upstream primer
<400> 7
tttattcaaa gcggtgcg 18
<210> 8
<211> 18
<212> DNA
<213> molecular marker FC-02 downstream primer
<400> 8
ccaatgccga tacagacg 18
Claims (8)
1. The molecular marker closely linked with the property of controlling the red fruit peel of the pumpkin indicum is characterized by comprising FC-01 and FC-02, wherein the FC-01 molecular marker consists of a DNA fragment shown in SEQ ID NO.1 and a DNA fragment shown in SEQ ID NO. 2; the molecular marker FC-02 consists of a DNA fragment shown by SEQ ID NO.3 and a DNA fragment shown by SEQ ID NO. 4.
2. The molecular marker of claim 1, wherein the DNA fragment shown in SEQ ID No.1 of the molecular marker FC-01 is linked to the pumpkin red peel trait gene R, and the DNA fragment shown in SEQ ID No.2 is linked to the pumpkin non-red peel trait gene R.
3. The molecular marker of claim 1, wherein the DNA fragment shown in SEQ ID No.3 of the molecular marker FC-02 is linked to the pumpkin red peel trait gene R, and the DNA fragment shown in SEQ ID No.4 is linked to the pumpkin non-red peel trait gene R.
4. The primer for amplifying the molecular marker of claim 1, wherein the primer sequence for amplifying the molecular marker FC-01 is shown as SEQ ID No.5 and SEQ ID No. 6; the primer sequence of the amplification molecular marker FC-02 is shown as SEQ ID NO.7 and SEQ ID NO. 8.
5. The use of the molecular marker of any one of claims 1-3 in the breeding of pumpkin with the peel trait.
6. The application of the primer of claim 4 in pumpkin breeding with the red fruit peel trait.
7. A method for breeding pumpkin with a haw peel character comprises the following steps:
1) taking the Indian pumpkin red peel line 2013-12 as a female parent and the pumpkin gray peel line as a male parent, hybridizing the female parent and the male parent to obtain F1Backcrossing the generations with the male parent to obtain offspring BC1F1Scanning BC with the molecular marker FC-01 and the molecular marker FC-02 according to claim 1, respectively1F1Selecting plants which are scanned by the molecular marker FC-01 and have nucleotide fragments of 44nt, 396nt, 326nt and 722nt and scanned by the molecular marker FC-02 and have nucleotide fragments of 420nt, 421nt and 841nt, backcrossing the plants with the male parent to obtain offspring BC2F1；
2) Scanning BC Using molecular marker FC-01 and molecular marker FC-02, respectively2F1Selecting plants which have nucleotide fragments of 44nt, 396nt, 326nt and 722nt after being scanned by a molecular marker FC-01 and have nucleotide fragments of 420nt, 421nt and 841nt after being scanned by a molecular marker FC-02, and backcrossing the plants with the male parent to obtain the backcross third generation BC3F1；
3) Scanning of BC Using molecular marker FC-01 and molecular marker FC-023F1Selecting plants with 44nt, 396nt, 326nt and 722nt nucleotide fragments after being scanned by the molecular marker FC-01 and 420nt, 421nt and 841nt nucleotide fragments after being scanned by the molecular marker FC-02 for selfing, and marking the progeny as BC3F2；
4) Scanning of BC Using molecular markers FC-01 and FC-023F2Selecting the plant which has 44nt and 722nt nucleotide segments after being scanned by the molecular marker FC-01 and has 841nt nucleotide segments after being scanned by the molecular marker FC-02, namely the improved red pericarp inbred line.
8. The method of claim 7, wherein the pumpkin gray peel product is ayurvedic gray peel 9-6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114015796A (en) * | 2021-10-29 | 2022-02-08 | 东北农业大学 | Molecular marker closely linked with control of light white peel of American pumpkin, primer and application |
| CN116497152A (en) * | 2023-06-19 | 2023-07-28 | 东北农业大学 | Molecular marker closely linked with gene Cmamg for controlling gray peel color of Indian pumpkin and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114015796A (en) * | 2021-10-29 | 2022-02-08 | 东北农业大学 | Molecular marker closely linked with control of light white peel of American pumpkin, primer and application |
| CN114015796B (en) * | 2021-10-29 | 2023-09-26 | 东北农业大学 | Molecular marker closely linked with control of white light peel of cucurbita moschata, primer and application |
| CN116497152A (en) * | 2023-06-19 | 2023-07-28 | 东北农业大学 | Molecular marker closely linked with gene Cmamg for controlling gray peel color of Indian pumpkin and application thereof |
| CN116497152B (en) * | 2023-06-19 | 2023-09-29 | 东北农业大学 | Molecular marker closely linked with gene Cmamg for controlling gray peel color of Indian pumpkin and application thereof |
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