CN113881801B - Molecular marker composition closely linked with deep green stem traits of cucurbita moschata and application of molecular marker composition - Google Patents
Molecular marker composition closely linked with deep green stem traits of cucurbita moschata and application of molecular marker composition Download PDFInfo
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Abstract
The invention discloses a molecular marker composition closely linked with the dark green stem character of cucurbita moschata and application thereof, belonging to the technical field of biology. In order to rapidly 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) SEQ ID NO 1 and SEQ ID NO 2; (b) SEQ ID NO.3 and SEQ ID NO.4, and further a primer pair for amplifying the molecular markers of the (a) set and the (b) set, respectively: SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8. The molecular marker provided by the invention has higher stability, and can be simply, conveniently and quickly used for screening the dark green stem types in the pumpkin seedling stage by utilizing the molecular auxiliary selection of the dark green stems of the cucurbita moschata, and the improved strain of the dark green stem varieties of the cucurbita moschata can be quickly cultivated by a backcross transformation method.
Description
Technical Field
The invention belongs to the technical field of biological molecules, and particularly relates to a molecular marker composition closely linked with the deep green stem character of cucurbita moschata and application thereof.
Background
Molecular marker assisted breeding is a new technology which is developed rapidly, and the direct selection of genotypes is realized by rapidly and accurately analyzing the genetic composition of individuals on a molecular level, so that molecular breeding is performed.
Pumpkin in the AmericanCucurbita maxima L.) Is of Cucurbitaceae%Cucurbitaceae) Pumpkin belonging to the genus CucurbitaCucurbita) Vining herbs, cucurbita pepo, also known as zucchini, are originally produced in the south of north america, china was introduced from europe in the middle of the 19 th century and distributed around the world. The pulp and seed can be eaten, and has the advantages of easy cultivation, high yield, and campingThe nutrition value is rich, and the like; the american pumpkin has two colors of dark green stems and light green stems, and the character separation gradually occurs along with the growth of plants. The color of the stems is mainly regulated and controlled by chlorophyll, and in the continuous growth process of the pumpkin, the accumulation of chlorophyll is different to form dark stems and light stems, and the dark green stem color is one of important agronomic characters of the pumpkin, so that the pumpkin dark green stem color character is utilized, and the pumpkin with the dark green stem color character is bred, so that the pumpkin dark green stem color character has wide application prospect and great economic value.
In the process of breeding dark green stems of the american pumpkins by the traditional breeding method, the stems in the seedling stage are in a light green state, and the color of the stems can be distinguished only after the american pumpkins grow to the flowering stage, so that the problems of wide seedling selection occupation, long breeding period and the like exist. The molecular marker closely linked with the target trait is used for marker assisted selection, which is a very effective method in pumpkin genetic breeding, can effectively widen the application of the trait and promote the quality breeding process. Therefore, the development of the molecular marker closely linked with the dark green stem character of the american pumpkin has very important significance for understanding the molecular mechanism of the color change of the american pumpkin stem.
Disclosure of Invention
The invention aims to rapidly and efficiently identify the dark green stem character of the cucurbita pepo in the seedling stage of the cucurbita pepo and accelerate the breeding process of the dark green stem cucurbita pepo.
The invention provides a molecular marker composition closely linked with the deep green stem character of cucurbita moschata, which is (a) or (b) or a combination of (a) and (b) as follows:
(a) A nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2;
(b) The nucleotide sequence shown in SEQ ID NO.3 and the nucleotide sequence shown in SEQ ID NO.4.
Further defined, 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.
Further defined, the primers for amplifying the nucleotide sequences of group (b) are: the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO. 8.
The invention provides a kit for identifying the dark green stem character of cucurbita moschata, which is characterized by comprising the molecular marker composition.
Further defined, the kit further comprises: 10X Buffer, dNTP, DNA template, taq enzyme and ddH 2 O。
The invention relates to a method for identifying dark green stem characters of cucurbita moschata, which is characterized by comprising the following specific steps:
(1) Extracting DNA of a sample to be detected;
(2) And (3) taking the DNA in the step (1) as a template, carrying out PCR reaction by using a nucleotide sequence shown in SEQ ID NO.5 and a nucleotide sequence shown in SEQ ID NO.6, and carrying out PCR reaction by using a nucleotide sequence shown in SEQ ID NO.7 and a 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 fragments 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 pumpkins, which comprises the following specific steps:
1) Hybridization is carried out on the female parent and male parent by taking the American pumpkin dark green stem strain 19 as female parent and the pumpkin light green stem strain 113 as male parent, thus obtaining F 1 Backcrossing the offspring and the male parent to obtain offspring BC 1 F 1 BC is scanned by 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, respectively 1 F 1 Selecting plants with 128nt and 137nt nucleotide fragments after scanning by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and 331nt and 306nt nucleotide fragments after scanning by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, and backcrossing with the male parent to obtain offspring BC 2 F 1 ；
2) Respectively utilizing rightsThe nucleotide sequence shown in SEQ ID No.5 and the nucleotide sequence shown in SEQ ID No.6 as described in claim 1, and the nucleotide sequence shown in SEQ ID No.7 and the nucleotide sequence shown in SEQ ID No.8, to scan BC 2 F 1 Selecting plants with 128nt and 137nt nucleotide fragments after scanning by the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 and 331nt and 306nt nucleotide fragments after scanning by the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8, and then backcrossing with the male parent to obtain the backcrossed third-generation BC 3 F 1 ；
3) BC is scanned using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 with the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 3 F 1 Selecting plants with 128nt and 137nt nucleotide fragments after scanning by the nucleotide sequence shown in a molecular marker SEQ ID NO.5 and the nucleotide sequence shown in a SEQ ID NO.6 and 331nt and 306nt nucleotide fragments after scanning by the nucleotide sequence shown in a SEQ ID NO.7 and the nucleotide sequence shown in a SEQ ID NO.8 from a population, carrying out selfing, and marking the offspring as BC 3 F 2 ；
4) BC is scanned using the nucleotide sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6 with the nucleotide sequence shown in SEQ ID NO.7 and the nucleotide sequence shown in SEQ ID NO.8 3 F 2 And selecting a plant which has 128nt 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 has 331nt 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, namely the improved dark green stem inbred line.
The molecular marker composition or the kit is applied to the identification of the dark green stem character of the homozygous american pumpkin.
The molecular marker composition or the kit is applied to breeding of the american pumpkins with dark green stems or light green stems.
The beneficial effects are that: the invention is obtained by hybridization of dark green stems '19' and light green stems '113' of the american pumpkinsF 1 Plants, F 1 Plant selfing to obtain 1054 plants F 2 Segregating the populations, and determining that the dark green stem trait of the cucurbita moschata belongs to the dominant trait of single gene control through phenotypic analysis. Respectively taking F 2 Extracting genome DNA from tender leaves of each plant of the isolated population, and screening dark green stem character genes of cucurbita moschata by combining BSA-seq methodDClosely linked molecular markers. And finally providing two molecular markers closely linked with the dark green stems of the cucurbita moschata by using the molecular markers of the BAS-seq resequencing, wherein the two molecular markers are respectively located at two sides of a control target trait gene, the genetic distance of the two markers is 61.58 Kb, and the establishment of a molecular marker assisted breeding system of the dark green stems is facilitated. The molecular marker provided by the invention has the accuracy of distinguishing the dark green stems from the light green stems of over 99%, and can be simply, conveniently and rapidly applied to high-throughput application and breeding practice.
The invention also provides application of the molecular marker in rapid cultivation of dark green stem property of the american pumpkin. By using a backcross transformation method, the dark green stem '19' is used as a non-recurrent parent, the light green stem '113' is used as a recurrent parent, and the nucleotide sequence shown in the SEQ ID NO.1 of the two molecular markers is utilized, and the sequence is matched with the pumpkin dark green stem character geneDLinkage with the nucleotide sequence shown in SEQ ID NO.2, the RA-01 primer (SEQ ID NO.5 and SEQ ID NO. 6) and the RA-02 primer (SEQ ID NO.7 and SEQ ID NO. 8) scan the backcross population, and rapidly and accurately improve the stem color of the light-colored stem '113' according to the PCR amplification product. Indel molecular markers have high stability, and the construction of high-density genetic maps and physical maps by using the molecular markers can be helpful for establishing molecular marker assisted breeding lines for screening the pumpkin dark green stem target genes.
Drawings
FIG. 1 shows Indel markers RA-01 and RA-02 and control of dark green stem genes of American pumpkinDClosely linked, each cross represents 2 single exchange events;
FIG. 2 is an Indel marker RA-01P 1 ，P 2 ，F1，F 2 The restriction enzyme strip of the colony PCR product, wherein M is D2000Marker, the strip length is respectively 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom, P1 represents a dark green stem strain '19',P 2 represents a light green stem line '113', F1 is P 1 And P 2 First generation of hybridization of F 2 Is F 1 Filial generation of selfing offspring, F 2 Dark green stem population, F 2 The pale green stem populations are shown at F respectively 2 10 dark green stems and 10 light green stem plants randomly selected from the population;
FIG. 3 is an Indel marker RA-02P 1 ，P 2 ，F 1 ，F 2 The enzyme cutting band of colony PCR product, wherein M is D2000Marker, the band length is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom, P1 represents dark green stem strain '19', P 2 Represents a pale green stem line '113', F 1 Is P 1 And P 2 First generation of hybridization of F 2 Is F 1 Filial generation of selfing offspring, F 2 Dark green stem population, F 2 The pale green stem populations are shown at F respectively 2 10 dark green stems and 10 light green stem plants randomly selected from the population.
Detailed Description
The invention is further described below in connection with specific embodiments. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Standard for dark green stems: the chlorophyll content of the plant stem epidermis is more than 6 mg/L;
standard for pale green stems: the chlorophyll content of the plant stem epidermis is less than 1 mg/L;
the stems mentioned in the present invention refer to the basal stem segments of the flowering period of the cucurbita moschata.
Nucleotide sequence shown in SEQ ID NO.1 (the sequence and the pumpkin dark green stem character gene)DLinkage) with a nucleotide sequence shown in SEQ ID NO.2 (the sequence is linked with a pumpkin light green stem character genedLinkage) is an RA-01 molecular marker;
nucleotide sequence shown in SEQ ID NO.3 (the sequence and the pumpkin dark green stem character gene)DLinkage) with a nucleotide sequence shown in SEQ ID NO.4 (the sequence is linked with a pumpkin light green stem character genedLinkage) is an RA-02 molecular marker.
Example 1 screening of molecular markers closely linked to the dark green stem trait of Anchorage pumpkin
1.F 2 Construction of populations
In the example, dark green stems '19' and light green stems '113' of the high-generation selfing lines of the American pumpkin are selected as parents to construct F 2 A population. Hybrid combinations are formulated using these two parents to obtain F 1 The generation is dark green stem, F 1 Generation of F by selfing 2 Generation segregating population, identifying 1054F in the field 2 The dark/light green stem phenotype in the generation individuals is verified by chi-square analysis to obtain the dark green stem character of the american pumpkin as the dominant character controlled by a single gene, genetic analysis is carried out by constructing a six-generation population to find the dark green stem character as dominant, and the dark green stem gene is named asDOR（D）。
2. Screening of dark green stem linked Indel markers
(1) Extraction of pumpkin genomic DNA
Parent and F are extracted by CTAB method 2 The total DNA of the leaves of the population was isolated. The method comprises the following steps: taking two leaves which are unfolded and then quickly grinding the leaves into powder in liquid nitrogen, and placing the powder into a 2.0ml centrifuge tube; adding 1000 μl of preheated CTAB extraction buffer, and water-bathing at 65deg.C for 1 hr; taking out, cooling to room temperature, centrifuging at 12000r/min and 4 ℃ for 17min, and sucking 800 μl of supernatant into a new 2.0ml centrifuge tube; adding an equal volume of chloroform isoamyl alcohol mixed solution, wherein the volume ratio of chloroform to isoamyl alcohol is 24:1, after mixing evenly, centrifuging at the temperature of 12000r/min and 4 ℃ for 13min; aspirate 700 μl supernatant into a new 2.0ml centrifuge tube; continuously adding equal volume chloroform isoamyl alcohol, uniformly mixing, and centrifuging at the temperature of 12000r/min and 4 ℃ for 13min; adding 600 μl supernatant into a new 1.5ml centrifuge tube, adding equal volume of isopropanol, gently mixing, and storing in a refrigerator at-20deg.C for 1 hr; centrifuging at 12000r/min and 4 ℃ for 10min; pouring out supernatant, washing the precipitate with 70% ethanol twice, drying, and adding 40 μlddH 2 After O dissolution, 10. Mu.g/ml RNase was added to remove RNA, and the mixture was subjected to a water bath at 37℃for 30min; the concentration of the obtained DNA was estimated by 0.8% agarose gel electrophoresis using 50 ng/. Mu.l lambda.DNA as a standard and stored at-20℃for use.
(2) Molecular marker acquisition
According to F 2 Randomly selecting 30 dark green stems and light green stems single plants from the generation population field investigation result, constructing a dark green stem pool and a light green stem pool for BSA-seq, and then carrying out association analysis on pumpkin dark green stems by utilizing InDel-index markersDThe gene maps to chromosome 15-associated region.
Example 2 method of identifying dark green-stem cucurbita moschata
1. Indel results from the sequencing of the pumpkin genome were analyzed and Indel marker primers were designed. With dark green stem parent, light green stem parent and F 1 The DNA is used as a template, and an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6 (amplified molecular marker RA-01) are respectively used; the PCR amplification was performed with the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8 (amplification molecular marker RA-02), which were synthesized from the Huada gene. The PCR amplification system was as follows:
TABLE 1 PCR reaction System
The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 95℃for 30s, annealing at 60℃for 30s, and extension at 72℃for 5 min;35 cycles; final extension at 72℃for 5 min; finally, the mixture is preserved at the temperature of 4 ℃.
Indel molecular marker Scan F 2 Segregating populations
Scanning two parents and F above using the two pairs of Indel primers developed above 2 And (3) the population, searching for a differential single plant of the marker type and the character phenotype, and obtaining an exchange single plant with the target section.
The scanning in the invention refers to the process of amplifying corresponding molecular markers by using the primers and judging stem color properties according to the molecular marker amplification result. The PCR system and the PCR program adopted in the scanning process are the same as those in the step 1 of the embodiment. The PCR products are detected by electrophoresis through polyacrylamide gel, and imaging results are observed through a polyacrylamide gel imaging system.
Results: the partial scan of the molecular marker RA-01 is shown in FIG. 2, and the dark green stem plants each show a band of 128nt or 128nt and 137 nt. The light green stem plants exhibited a band of 137 nt. The partial scanning result of the molecular marker RA-02 is shown in FIG. 3, dark green stem plants all show bands of 331nt or 331nt and 306nt, and light green stem plants show bands of 306 nt.
The above 2 molecular markers pass through 1054 strain F 2 The result of verification of the segregating population shows that 2 molecular markers, RA-01 and RA-02, can better distinguish dark green stem plants from light green stem plants, and are both similar to dark green stem genesDClosely interlocked and respectively sit onDThe two sides of the gene are 61.58 Kb (figure 1) of the genetic distance of the two markers, wherein the sequence shown by SEQ ID NO.1 in the molecular marker RA-01 shows the dark green stem gene of the American pumpkinDLinkage, sequence shown in SEQ ID NO.2 and light green stem genedInterlocking; SEQ ID NO.3 sequence in molecular marker RA-02 and deep green stem gene of American pumpkinDLinkage, SEQ ID NO.4 shows sequence and light green stem genedAnd (3) interlocking.
Tag RA-1, tag RA-2DThere are 7 and 2 single crossover events for each gene, each closely linked to the target segment. Indel molecular markers have high stability, and the construction of high-density genetic maps and physical maps by using the molecular markers can be helpful for establishing molecular marker assisted breeding lines for screening the pumpkin dark green stem target genes.
3. Polymorphic fragment sequencing
Taking genome DNA of a dark stem parent '19' and a light 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 (amplified molecular marker RA-01); the upstream primer shown in SEQ ID No.7 and the downstream primer shown in SEQ ID No.8 (amplification molecular marker RA-02) were subjected to PCR amplification for PCR amplification. The total PCR reaction system was 20. Mu.l: 10 XBuffer 2 mu l, dNTP 2 mu l, DNA template 1.2 mu l, upper and lower primers respectively 0.8 mu l, taq enzyme 0.2 mu l, ddH 2 O13 μl. The PCR reaction conditions were: pre-denaturation at 95℃for 5 min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 1 min,35 cycles; final extension at 72℃for 5 min; finally, the mixture is preserved at the temperature of 4 ℃.
20. Mu.l of BioTeke SYBR Green I nucleic acid dye was added to the PCR amplified product, the mixture was allowed to stand at room temperature for 10 minutes to bind the dye to DNA, 2% agarose gel electrophoresis was performed to separate the target fragments, the target fragments were excised under an ultraviolet lamp, and purified DNA fragments were recovered by TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, respectively, with reference to kit instructions, product No. 9762. The purified fragments were sequenced by Huada gene limited and the sequencing results were analyzed by DNAMAN software. Wherein the fragment size amplified by using the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 with dark green stem '19' as a template is 128nt, and the amplified sequence is shown in SEQ ID NO.1. The light green stem parent 113' is used as a template, the size of a fragment amplified by using an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO.6 is 137nt, and the amplified sequence is shown as SEQ ID NO.2. The dark green stem '19' is used as a template, an upstream primer shown as SEQ ID NO.7 and a downstream primer shown as SEQ ID NO.8 are used, the amplified fragment size is 331nt, and the amplified sequence is shown as SEQ ID NO.3. The light green stem parent 113' is used as a template, an upstream primer shown as SEQ ID NO.7 and a downstream primer shown as SEQ ID NO.8 are used, the amplified fragment size is 306nt, and the amplified sequence is shown as SEQ ID NO.4.
Pumpkin with green stem character identification
Extracting DNA of the true leaves of the cucurbita pepo to be detected, and carrying out PCR to identify whether the green stem characters of the plants in the seedling stage are homozygous.
The formula for calculating the accuracy is: genotype/plant phenotype of plants identified by molecular markers.
The size of the amplified fragment of the homozygous dark green stem plant is 128nt by using the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6, and then the amplified fragment size is 331nt by using the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8, so that the accuracy is 99.23%.
The amplified fragment sizes of the heterozygous dark green stem plants by using 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 uses the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8, the amplified fragment sizes are 331nt and 306nt, and the accuracy is 99.44%.
Example 3 Breeding method of homozygous dark green-stem American pumpkin
In the embodiment, a backcross transformation method is utilized, dark green stems '19' are used as non-recurrent parents, light green stems '113' are used as recurrent parents, and stem color characters of a light green stem excellent strain '113' are directionally improved, and the specific operation steps are as follows:
(1) Hybridization of dark green stem '19' and light green stem '113' to obtain F 1 Backcrossing the generation and the light green stem 113' to obtain the offspring BC 1 F 1 BC scanning with two molecular markers RA-01 and RA-02 of the present invention 1 F 1 Populations, i.e. in BC 1 F 1 The DNA of each plant in the population is used as a template, and an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO.6 are respectively utilized; and the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO. 8. The PCR reaction system and the PCR reaction conditions were the same as those described in (2) of example 1. Selecting plants with 128nt and 137nt nucleotide fragments after being scanned by the molecular marker RA-01 and with 331nt and 306nt nucleotide fragments after being scanned by the molecular marker RA-02, and carrying out backcross with the light green stem '113' to obtain offspring BC 2 F 1 。
(2) BC scanning with molecular markers RA-01 and RA-02 of the present invention 2 F 1 The colony, continue to select the plant with 128nt and 137nt nucleotide fragments marked RA-01 and 331nt and 306nt nucleotide fragments scanned by molecular marker RA-02, then backcross with the light green stem '113' to obtain the backcross third generation BC 3 F 1 。
(3) BC scanning with molecular markers RA-01 and RA-02 of the present invention 3 F 1 Continuously selecting plants with 128nt and 137nt nucleotide fragments after being scanned by the molecular marker RA-01 and with 331nt and 306nt nucleotide fragments after being scanned by the molecular marker RA-02, and selfing to obtain offspring BC 3 F 2 。
(4) BC scanning with molecular markers RA-01 and RA-02 of the present invention 3 F 2 Selecting plants with 128nt nucleotide fragments after scanning by the molecular marker RA-01 and 331nt nucleotide fragments after scanning by the molecular marker RA-02 as populationsGood dark green stem inbred line. The inbred line was colonized and stem colour was investigated during the maturation period 45d from the day of pollination. The chlorophyll content of the stem in the mature period is 7.8mg/L, accords with the dark green stem standard, and shows that the improved inbred line has the dark green stem character and other agronomic characters are similar to those of '113'. The molecular markers RA-01 and RA-02 of the invention can be used for rapidly and accurately improving the color of the pumpkin stems.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
SEQUENCE LISTING
<110> northeast agricultural university
<120> molecular marker composition closely linked with deep green stem character of cucurbita moschata and application thereof
<160> 8
<170> PatentIn version 3.5
<210> 1
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agtgccacct gtttctccca tgctgtgttt tcctattgat gtgagcggtt attgtatcat 60
tattatcatt attatttttt ttgcatttgg tagattcagt tgatacattt tattgattga 120
tggtggga 128
<210> 2
<211> 137
<212> DNA
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<400> 2
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attgattgat ggtggga 137
<210> 3
<211> 331
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<400> 3
atagtcataa ataactcgtg atagaaatat ttagctaaat tacaaaaata cctttaaatt 60
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aattacaaaa atacccttaa cctttatgtt ttgtttcaaa cgttttaaaa agttatacct 180
ttacttttct taaacatttc aaaactatct ggcgagaaat ttttagaatg tcaaatcaaa 240
actaaatcag aatgtcaaat caaaactaaa tctgagaaag tttaaacgta tgacaatttt 300
aattttcgag ttttaccacc acatccttct a 331
<210> 4
<211> 306
<212> DNA
<213> Synthesis
<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> Synthesis
<400> 5
agtgccacct gtttctcc 18
<210> 6
<211> 19
<212> DNA
<213> Synthesis
<400> 6
tcccaccatc aatcaataa 19
<210> 7
<211> 20
<212> DNA
<213> Synthesis
<400> 7
atagtcataa ataactcgtg 20
<210> 8
<211> 18
<212> DNA
<213> Synthesis
<400> 8
tagaaggatg tggtggta 18
Claims (3)
1. A method for identifying dark green stem traits of cucurbita moschata, which is characterized by comprising the following specific steps:
(1) Extracting DNA of a sample to be detected;
(2) Taking the DNA in the step (1) as a template, carrying out PCR reaction by using a nucleotide sequence shown in SEQ ID NO.5 and a nucleotide sequence shown in SEQ ID NO.6, and then carrying out PCR reaction by using a nucleotide sequence shown in SEQ ID NO.7 and a nucleotide sequence shown in SEQ ID NO.8, wherein if amplified bands are 128nt and 331nt respectively, the plant to be detected is a dark green stem homozygous plant, and if amplified fragments are 128nt and 137nt, 331nt and 306nt respectively, the plant to be detected is a dark green stem heterozygous plant; if the amplified bands are 137nt and 306nt respectively, the plants to be detected are light green stem plants.
2. The application of the method as claimed in claim 1 in identifying dark green stem traits of homozygous cucurbita moschata, wherein DNA of a sample to be detected is used as a template, a nucleotide sequence shown in SEQ ID NO.5 and a nucleotide sequence shown in SEQ ID NO.6 are used for carrying out PCR reaction, a nucleotide sequence shown in SEQ ID NO.7 and a nucleotide sequence shown in SEQ ID NO.8 are used for carrying out PCR reaction, if amplified bands are respectively 128nt and 331nt, the plant to be detected is a dark green stem homozygous plant, and if amplified fragments are respectively 128nt and 137nt, 331nt and 306nt, the plant to be detected is a dark green stem heterozygous plant.
3. The application of the method as claimed in claim 1 in breeding dark green stems or light green stems, characterized in that DNA of a sample to be detected is used as a template, a nucleotide sequence shown in SEQ ID NO.5 and a nucleotide sequence shown in SEQ ID NO.6 are used for carrying out PCR reaction, a nucleotide sequence shown in SEQ ID NO.7 and a nucleotide sequence shown in SEQ ID NO.8 are used for carrying out PCR reaction, if amplified bands are respectively 128nt and 331nt, the plant to be detected is a dark green stem homozygous plant, and if amplified fragments are respectively 128nt and 137nt, 331nt and 306nt, the plant to be detected is a dark green stem heterozygous plant; if the amplified bands are 137nt and 306nt respectively, the plants to be detected are light green stem plants.
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