CN113881801A - Molecular marker composition with tightly linked dark green stem characters of American pumpkin and application thereof - Google Patents
Molecular marker composition with tightly linked dark green stem characters of American pumpkin and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker composition closely linked with the dark green stem character of American pumpkin and application thereof, belonging to the technical field of biology. In order to quickly and efficiently identify the dark green stem character of the American pumpkin and accelerate the breeding process of the dark green stem pumpkin. The invention discloses a molecular marker composition: (a) 1 and 2 SEQ ID NO; (b) 3 and 4, and a primer pair for amplifying the molecular markers of the group (a) and the group (b), respectively: 5 and 6, 7 and 8. The molecular marker provided by the invention has higher stability, can be simply, conveniently and quickly used for screening the types of the dark green stems in the seedling stage of the pumpkin by utilizing the molecular auxiliary selection of the dark green stems of the American pumpkin, and can quickly culture the variety improved strain of the dark green stems of the American pumpkin by a backcross transfer method.
Description
Technical Field
The invention belongs to the technical field of biomolecules, and particularly relates to a molecular marker composition with tightly linked dark green stem characters of American pumpkin and application thereof.
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.
Cucurbita maxima L, an annual vine-grown herbaceous plant of the Cucurbitaceae family Cucurbita (Cucurbitaceae), also known as zucchini, originally produced in the south of north america, and introduced from europe to china in the middle of the 19 th century, and is distributed throughout the world. 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 American pumpkin has two colors of dark green stem and light green stem, and the character separation gradually occurs along with the growth of the plant. The stem color is mainly regulated and controlled by chlorophyll, the accumulation of chlorophyll is different in the continuous growth process of the pumpkin, a dark stem and a light stem are formed, and the dark green stem color is one of important agronomic characters of the pumpkin, so that the American pumpkin with the dark green stem color character is bred by utilizing the dark green stem color character of the pumpkin, and the American pumpkin has wide application prospect and great economic value.
In the process of breeding the dark green stems of the American pumpkins by the traditional breeding method, the stems in the seedling stage are all in a light green state, so that the color of the stems can be distinguished only by waiting for the American pumpkins to grow to the flowering stage, and the problems of wide seedling selection occupation, long breeding period and the like exist. 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 closely linked with the dark green stem character of the cucurbita maxima is of great significance for understanding the molecular mechanism of the change of the stem color of the cucurbita maxima.
Disclosure of Invention
The invention aims to rapidly and efficiently identify the dark green stem character of the American pumpkin at the seedling stage of the American pumpkin and accelerate the breeding process of the dark green stem pumpkin.
The invention provides a molecular marker composition closely linked with a cucurbita maxima dark green stem character, wherein the molecular marker composition is (a) or (b) or a combination of the (a) and the (b):
(a) the nucleotide sequence shown as SEQ ID NO.1 and the nucleotide sequence shown as SEQ ID NO. 2;
(b) the nucleotide sequence shown as SEQ ID NO.3 and the nucleotide sequence shown as SEQ ID NO. 4.
Further defined, the primers for amplifying the nucleotide sequences of the group (a) are: the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO. 6.
Further defined, the primers for amplifying the nucleotide sequences of group (b) are: the nucleotide sequence shown as SEQ ID NO.7 and the nucleotide sequence shown as SEQ ID NO. 8.
The invention provides a kit for identifying the dark green stem character of American pumpkin, which is characterized by comprising the molecular marker composition.
Further defined, the kit further comprises: 10 XBuffer, dNTP, DNA template, Taq enzyme and ddH2O。
The invention discloses a method for identifying the dark green stem character of American pumpkin, which is characterized by comprising the following specific steps:
(1) extracting DNA of a sample to be detected;
(2) and (2) carrying out PCR reaction by using the DNA described in the step (1) as a template and using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6, and then carrying out PCR reaction by using the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, wherein if the amplified bands are 128nt and 331nt, the plant to be detected is homozygous, and if the amplified segments are 128nt and 137nt or 331nt and 306nt, the plant to be detected is heterozygous.
The invention provides a method for breeding dark green stem American pumpkin, which comprises the following specific steps:
1) hybridizing the female parent and the male parent by taking the deep green stem line 19 of the American pumpkin as the female parent and the light green stem line 113 of the American pumpkin as the male parent to obtain F1Backcrossing the generations with the male parent to obtain offspring BC1F1The nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6, the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 are used to scan BC respectively1F1A population, selecting plants which are scanned by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and have 128nt and 137nt nucleotide fragments and scanned by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 and have 331nt and 306nt nucleotide fragments, and backcrossing the plants with the male parent to obtain a progeny BC2F1；
2) Scanning BC using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 according to claim 1, respectively2F1A population, selecting plants which have 128nt and 137nt nucleotide fragments after being scanned by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and have 331nt and 306nt nucleotide fragments after being scanned by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, and then carrying out backcross with the male parent to obtain the backcross third generation BC3F1；
3) The nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6, and the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 are used for scanning BC3F1Selecting plants which have 128nt and 137nt nucleotide fragments after being scanned by the nucleotide sequence shown in the molecular marker SEQ ID NO.5 and the nucleotide sequence shown in the SEQ ID NO.6 and have 331nt and 306nt nucleotide fragments after being scanned by the nucleotide sequence shown in the SEQ ID NO.7 and the nucleotide sequence shown in the SEQ ID NO.8, selfing, and marking the progeny as BC3F2；
4) Scanning BC with the nucleotide sequence shown in SEQ ID NO.5 and 6 and the nucleotide sequence shown in SEQ ID NO.7 and 83F2The nucleotide sequence shown by SEQ ID NO.5 and the nucleotide sequence shown by SEQ ID NO.6 are selected to haveA plant with a 128nt nucleotide fragment and 331nt nucleotide fragment after being scanned by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 is the improved dark green stem inbred line.
The application of the molecular marker composition or the kit in identifying the dark green stem character of the homozygous pumpkin americana.
The application of the molecular marker composition or the kit in breeding of the cucurbita moschata with the characteristics of dark green stems or light green stems.
Has the advantages that: the invention uses the hybridization of the deep green stem '19' and the light green stem '113' of the American pumpkin to obtain F1Plant, F1Obtaining 1054F through plant selfing2Separating the population, and determining that the dark green stem character of the American pumpkin belongs to the dominant character controlled by a single gene through phenotypic analysis. Then take F respectively2And (3) extracting genome DNA from each tender leaf of the segregation population, and screening a molecular marker closely linked with the character gene D of the dark green stem of the American pumpkin by combining a BSA-seq method. By utilizing the BAS-seq re-sequencing molecular markers, two finally provided molecular markers which are closely linked with the dark green stems of the American pumpkin are respectively located on two sides of the gene for controlling the target characters, the genetic distance of the two markers is 61.58Kb, and the establishment of a molecular marker assisted breeding system of the dark green stems is facilitated. The molecular marker of the invention has the accuracy of distinguishing the dark green stem from the light green stem of more than 99 percent respectively, and can be applied and bred in a simple, convenient and high-throughput manner.
The invention also provides application of the molecular marker in fast cultivation of the dark green stem character pumpkin. By using a backcross transformation method, dark green stem '19' is used as a non-recurrent parent, light green stem '113' is used as a recurrent parent, two molecular markers of the invention are used for nucleotide sequences shown in SEQ ID NO 1, the sequences are linked with the dark green stem character gene D of the American pumpkin and scan a backcross population with the nucleotide sequence shown in SEQ ID NO 2, RA-01 primers (SEQ ID NO:5 and SEQ ID NO:6) and RA-02 primers (SEQ ID NO:7 and SEQ ID NO:8), and the stem color of the light-colored stem '113' is rapidly and accurately improved according to PCR amplification products. The Indel 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 molecular marker-assisted breeding line for screening the deep green stem target genes of the American pumpkin can be established.
Drawings
FIG. 1 shows Indel markers RA-01 and RA-02 closely linked to the gene D controlling the dark green stem of Cucurbita maxima, each cross representing 2 single crossover events;
FIG. 2 shows the Indel marker RA-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 and 100bp from high to low respectively, and P1 represents the deep green stem strain '19', P2Represents a light green stem line '113', F1 is P1And P2First hybrid generation of (1), F2Is F1Progeny after generation selfing, F2Dark green stem population and F2The light green stem population is shown at F2Randomly selecting 10 dark green stems and 10 light green stem plants from the population;
FIG. 3 shows the Indel marker RA-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 and 100bp from high to low respectively, and P1 represents the deep green stem strain '19', P2Represents the light green stem line '113', F1Is P1And P2First hybrid generation of (1), F2Is F1Progeny after generation selfing, F2Dark green stem population and F2The light green stem population is shown at F2Randomly selected 10 dark green stems and 10 light green stem plants from 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.
Standard for dark green stems: the chlorophyll content of the plant stem epidermis is more than 6 mg/L;
standard for light green stems: the chlorophyll content of the plant stem epidermis is less than 1 mg/L;
the stem mentioned in the invention refers to the stem segment at the base of the flowering period of the American pumpkin.
The nucleotide sequence shown in SEQ ID NO.1 (the sequence is linked with the deep green stem character gene D of the American pumpkin) and the nucleotide sequence shown in SEQ ID NO.2 (the sequence is linked with the light green stem character gene D of the American pumpkin) are RA-01 molecular markers;
the nucleotide sequence shown in SEQ ID NO.3 (the sequence is linked with the deep green stem character gene D of the American pumpkin) and the nucleotide sequence shown in SEQ ID NO.4 (the sequence is linked with the light green stem character gene D of the American pumpkin) are RA-02 molecular markers.
Example 1 screening of molecular markers closely linked to the dark green stem trait of Cucurbita maxima
1.F2Construction of the population
In the example, the American pumpkin high-generation inbred line dark green stem '19' and light green stem '113' are selected as parents to construct F2And (4) a group. Using the two parents to prepare the hybrid combination obtained F1The generation is dark green stem, F1Generation selfing to produce F2Passage segregating population, identifying 1054F in field2The phenotype of the dark/light green stem in the generation individual is verified by chi-square analysis, the character of the dark green stem of the American pumpkin is a dominant character controlled by a single gene, the character of the dark green stem is found to be dominant by constructing a six-generation population for genetic analysis, and the gene of the dark green stem is named as DOR (D).
2. Screening of dark green stem-linked Indel markers
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 with true leaves unfolded in liquid nitrogen into powder, and placing the powder in a 2.0ml centrifuge tube; 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 30 dark green stems and light green stem single plants according to the field survey result of the generation group, constructing a dark green stem pool and a light green stem pool, carrying out BSA-seq, and then positioning the pumpkin dark green stem D gene to a No. 15 chromosome correlation area by utilizing InDel-index marker correlation analysis.
Example 2 method for identifying dark green stem pumpkin
1. Analyzing Indel results obtained by resequencing of the American pumpkin genome and designing Indel labeled primers. Using dark green stem parent, light green stem 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 RA-01) are respectively used; the upstream primer shown in SEQ ID NO.7 and the downstream primer (amplified molecular marker RA-02) shown in SEQ ID NO.8 are subjected to PCR amplification, and the primers are synthesized by Huada gene. The PCR amplification system was programmed 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 ℃.
Indel molecular marker scanning F2Segregating populations
Scanning two parents and the above F using the two pairs of Indel primers developed above2The population searches for the differential individuals of the marker type and the character phenotype to obtain the cross with the target sectionAnd (5) changing the single plant.
The scanning in the invention refers to the judgment of the stem color character according to the molecular marker amplification result by using the corresponding molecular marker amplified by the primer. The PCR system and PCR program used in the scanning process are the same as those described in step 1 of this example. And (3) carrying out electrophoresis detection on the PCR product through polyacrylamide gel, and observing an imaging result through a polyacrylamide gel imaging system.
As a result: the scanning result of the molecular marker RA-01 part is shown in figure 2, and the dark green stem plants show 128nt bands or 128nt and 137nt bands. The light green stem plants exhibited a band of 137 nt. The results of the partial scan of the molecular marker RA-02 are shown in FIG. 3, and the dark green stem plants all show a band of 331nt or 331nt and 306nt, while the light green stem plants show a band of 331 nt.
The 2 molecular markers are 1054 strain F2The isolated population is verified, and the result shows that 2 molecular markers, namely RA-01 and RA-02, can better distinguish dark green stem plants from light green stem plants, are tightly linked with a dark green stem gene D and are respectively located on two sides of the D gene, and the genetic distance between the two markers is 61.58Kb (shown in figure 1), wherein the sequence shown by SEQ ID NO.1 in the molecular marker RA-01 is linked with the dark green stem gene D of the American pumpkin, and the sequence shown by SEQ ID NO.2 is linked with the light green stem gene D; the sequence shown by SEQ ID NO.3 in the molecular marker RA-02 is linked with the deep green stem gene D of the American pumpkin, and the sequence shown by SEQ ID NO.4 is linked with the light green stem gene D.
There were 7 and 2 single crossover events for marker RA-1, marker RA-2 and D genes, each closely linked to the target segment. The Indel 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 molecular marker-assisted breeding line for screening the deep green stem target genes of the American pumpkin can be established.
3. Sequencing of polymorphic fragments
Taking genome DNA of a dark-color stem parent '19' and a light-color stem parent '113' as templates, and respectively taking an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6 (an amplified molecular marker RA-01); the upstream primer shown in SEQ ID NO.7 and the downstream primer (amplification molecular marker RA-02) shown in SEQ ID NO.8 are subjected to PCR amplificationPCR amplification was performed. 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: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ 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 TaKaRa miniBEST 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. Wherein the size of a fragment amplified by using the deep green stem '19' 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 128nt, and the amplified sequence is shown in SEQ ID NO. 1. The fragment size amplified by using the light green stem parent '113' 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 137nt, and the amplified sequence is shown in SEQ ID NO. 2. The size of the amplified fragment is 331nt using the deep green stem '19' as a template and the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8, and the amplified sequence is shown in SEQ ID NO. 3. The size of the amplified fragment is 306nt by using the light green stem parent '113' as a template and using the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8, and the amplified sequence is shown in SEQ ID NO. 4.
American pumpkin with characteristic of identifying green stem
And extracting DNA of true leaves of the American pumpkin to be detected, and performing PCR to identify whether the green stem character of the plant is homozygous at the seedling stage.
The formula for calculating the accuracy is as follows: the plant is characterized by the genotype/plant phenotype identified by the molecular marker.
The size of the amplified fragment of the homozygous dark green stem plant is 128nt by using the upstream primer shown by SEQ ID NO.5 and the downstream primer shown by SEQ ID NO.6, and the size of the amplified fragment is 331nt by using the upstream primer shown by SEQ ID NO.7 and the downstream primer shown by SEQ ID NO.8, so that the accuracy is 99.23%.
The sizes of the fragments of the heterozygous dark green stem plants amplified by the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 are 128nt and 137nt, and the accuracy is 99.26%.
The heterozygous dark green stem plant utilizes the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8, the amplified fragments are 331nt and 306nt in size, and the accuracy is 99.44%.
Example 3 Breeding method of homozygous dark green stem cucurbita moschata
In this embodiment, a backcross transformation method is used to directionally improve the stem color property of the superior line '113' of light green stems by using the dark green stem '19' as a non-recurrent parent and the light green stem '113' as a recurrent parent, and the specific operation steps are as follows:
(1) the dark green stem '19' and light green stem '113' were crossed to give F1Backcrossing the generations with a light green stem '113' to obtain progeny BC1F1BC was scanned with two molecular markers RA-01 and RA-02 of the present invention1F1Population, 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 the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8 are subjected to PCR amplification. The PCR reaction system and the PCR reaction conditions were the same as those described in example 1. Selecting plants with 128nt and 137nt nucleotide fragments after being scanned by the molecular marker RA-01 and 331nt and 306nt nucleotide fragments after being scanned by the molecular marker RA-02, and backcrossing the plants with a light green stem '113' to obtain progeny BC2F1。
(2) BC scanning by using molecular markers RA-01 and RA-02 of the invention2F1Continuously selecting plants marked with RA-01 and having 128nt and 137nt nucleotide fragments and scanned by molecular marker RA-02 and having 331nt and 306nt nucleotide fragments, and backcrossing with light green stem '113' to obtain backcross third generation BC3F1。
(3) BC scanning by using molecular markers RA-01 and RA-02 of the invention3F1Continuing to select the molecular marker RA-01After being scanned, the plant with 128nt and 137nt nucleotide segments and 331nt and 306nt nucleotide segments after being scanned by the molecular marker RA-02 is selfed to obtain the offspring BC3F2。
(4) BC scanning by using molecular markers RA-01 and RA-02 of the invention3F2And selecting plants which have 128nt nucleotide fragments after being scanned by the molecular marker RA-01 and 331nt nucleotide fragments after being scanned by the molecular marker RA-02 as the population, namely the improved dark green stem inbred line. The inbred lines were established and investigated for stem color from the day of pollination to the 45d maturity stage. The chlorophyll content of the stem and leaf in the mature period is 7.8mg/L, and the stem and leaf meets the dark green stem standard, which shows that the improved inbred line has the dark green stem character, and other agronomic characters are similar to '113'. The molecular markers RA-01 and RA-02 of the invention can be used for rapidly and accurately improving the stem color of the American pumpkin.
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.
SEQUENCE LISTING
<110> northeast university of agriculture
<120> American pumpkin dark green stem character tightly-linked molecular marker composition and application
<160> 8
<170> PatentIn version 3.5
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<400> 4
atagtcataa ataactcgtg atagaaatat ttagctaaat tacaaaaata cctttaaatt 60
ttatgtttta tttcaaacat ttttaaaggt tatacattaa gtaattgaaa tatttggcta 120
aattacaaaa atacccttaa cctttatgtt ttgtttcaaa cgttttaaaa agttatacct 180
ttacttttct taaacatttc aaaactatct ggcgagaaat tttagaatgt caaatcaaaa 240
ctaaatctga gaaagtttaa acgtatgaca attttaattt tcgagtttta ccaccacatc 300
cttcta 306
<210> 5
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 5
agtgccacct gtttctcc 18
<210> 6
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 6
tcccaccatc aatcaataa 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 7
atagtcataa ataactcgtg 20
<210> 8
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 8
tagaaggatg tggtggta 18
Claims (9)
1. A molecular marker composition closely linked with the dark green stem character of the American pumpkin is characterized in that the molecular marker composition is (a) or (b) or a combination of the (a) and the (b):
(a) 1, linked with the dark green stem character gene D of the American pumpkin; 2, the nucleotide sequence is linked with the character gene d of the light green stem of the American pumpkin;
(b) 3, the sequence is linked with the character gene D of the dark green stem of the American pumpkin; and the nucleotide sequence shown in SEQ ID NO.4 is linked with the character gene d of the light green stem of the American pumpkin.
2. The molecular marker composition according to claim 1, wherein the primers for amplifying the nucleotide sequences of group (a) are: the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO. 6.
3. The molecular marker composition according to claim 1, wherein the primers for amplifying the nucleotide sequences of group (b) are: the nucleotide sequence shown as SEQ ID NO.7 and the nucleotide sequence shown as SEQ ID NO. 8.
4. A kit for identifying the dark green stem character of cucurbita maxima, wherein the kit comprises the molecular marker composition of any one of claims 1 to 3.
5. The kit of claim 4, further comprising: 10 XBuffer, dNTP, DNA template, Taq enzyme and ddH2O。
6. A method for identifying the dark green stem character of American pumpkin is characterized by comprising the following specific steps:
(1) extracting DNA of a sample to be detected;
(2) and (2) carrying out PCR reaction by using the DNA described in the step (1) as a template and using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6, and then carrying out PCR reaction by using the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, wherein if the amplified bands are 128nt and 331nt, the plant to be detected is a dark green stem homozygous plant, and if the amplified fragments are 128nt and 137nt, or 331nt and 306nt, the plant to be detected is a dark green stem heterozygous plant.
7. A method for breeding dark green stem American pumpkin is characterized by comprising the following specific steps:
1) hybridizing the female parent and the male parent by taking the deep green stem line 19 of the American pumpkin as the female parent and the light green stem line 113 of the American pumpkin as the male parent to obtain F1Backcrossing the generations with the male parent to obtain offspring BC1F1Respectively using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and the nucleotide sequence shown in SEQ ID NO.7Nucleotide sequence shown and nucleotide sequence scan BC shown in SEQ ID NO.81F1A population, selecting plants which are scanned by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and have 128nt and 137nt nucleotide fragments and scanned by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 and have 331nt and 306nt nucleotide fragments, and backcrossing the plants with the male parent to obtain a progeny BC2F1；
2) Scanning BC using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 according to claim 1, respectively2F1A population, selecting plants which have 128nt and 137nt nucleotide fragments after being scanned by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and have 331nt and 306nt nucleotide fragments after being scanned by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, and then carrying out backcross with the male parent to obtain the backcross third generation BC3F1；
3) The nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6, and the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 are used for scanning BC3F1Selecting plants which have 128nt and 137nt nucleotide fragments after being scanned by the nucleotide sequence shown in the molecular marker SEQ ID NO.5 and the nucleotide sequence shown in the SEQ ID NO.6 and have 331nt and 306nt nucleotide fragments after being scanned by the nucleotide sequence shown in the SEQ ID NO.7 and the nucleotide sequence shown in the SEQ ID NO.8, selfing, and marking the progeny as BC3F2；
4) Scanning BC with the nucleotide sequence shown in SEQ ID NO.5 and 6 and the nucleotide sequence shown in SEQ ID NO.7 and 83F2Selecting a nucleotide sequence shown as SEQ ID NO.5 and a nucleotide sequence shown as SEQ ID NO.6 which have 128nt nucleotide fragments after scanning, and a nucleotide sequence shown as SEQ ID NO.7 and a nucleotide sequence shown as SEQ ID NO.8 which have 331nt nucleotide fragments after scanningThe acid-fragment plant is the improved dark green stem inbred line.
8. Use of the molecular marker composition of any one of claims 1 to 3 or the kit of claim 4 or 5 for identifying the dark green stem trait of homozygous cucurbita moschata.
9. Use of the molecular marker composition of any one of claims 1 to 3 or the kit of claim 4 or 5 for breeding cucurbita moschata with a dark green stem or light green stem trait.
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| CN116219058A (en) * | 2023-03-07 | 2023-06-06 | 河南农业大学 | Molecular marker closely linked with major QTL of seed grain length of cucurbita moschata and application |
| CN116497144A (en) * | 2023-03-07 | 2023-07-28 | 河南农业大学 | Molecular marker closely linked with more or less related genes of side branches of cucurbita moschata and application thereof |
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