CN112725503B - Molecular marker closely linked with hypocotyl color related gene of American pumpkin and application - Google Patents
Molecular marker closely linked with hypocotyl color related gene of American pumpkin and application Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with a gene related to the hypocotyl color of a American pumpkin and application thereof, wherein a re-sequencing and marker development technology is utilized to locate and analyze the dark green character for the first time, and two stable and reliable In-Del molecular markers G4 and G5 which are closely linked with the dark green character and have polymorphism are obtained. The molecular marker can be used for cloning and functional analysis of the CpeSC1 gene, and can be used for auxiliary selection of the molecular marker for filial generation of the hypocotyl of the American pumpkin in the seedling stage. The method is simple, rapid and accurate, can accelerate the breeding process to a certain extent, and improves the breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a molecular marker closely linked with a hypocotyl color related gene of a American pumpkin and application thereof.
Background
One of the most important links in the breeding process is selection of target traits, and the process of selecting the target traits is actually the process of selecting genotypes. However, in conventional breeding, it is generally difficult to know the genotype of the offspring, and breeders often select based on the phenotype observed with the naked eye. The disadvantage of this selection is that it is time consuming and may deviate from the genotype, resulting in inaccurate and inefficient selection. With the rapid development of molecular biology, the technology of breeding new varieties by combining molecular marker-assisted selection with conventional crossbreeding is more and more mature, the molecular marker-assisted selection has the advantages of accuracy, rapidness and no environmental interference, and loci closely linked with target character genes can be rapidly detected, so that target characters can be selected, and the breeding process is shortened.
Pumpkin is one of the vegetables which are generally cultivated in the world, wherein the cultivated area in Asia is the largest. According to FAO data, the yield of the Chinese pumpkin in 2016 is 778.94 ten thousand tons (accounting for 21.6 percent of the yield all over the world), and the cultivation area is 42.28 thousand hectares (accounting for 29.4 percent of the cultivation area all over the world). At present, China is the country with the largest pumpkin cultivation area all over the world. The Cucurbita genus includes three common cultivars, cushaw (Cucurbita moschata), Cucurbita pepo (Cucurbita pepo), and Cucurbita maxima (Cucurbita maxima), wherein Cucurbita pepo is one of the important vegetables cultivated in early spring in northern regions. With the recent publication of cucurbita genome (Sun, 2017), development of molecular markers and assisted selection breeding are also gradually expanding in cucurbita, but are slow in overall and seriously lack a large number of reliable molecular markers.
The dark hypocotyl can enhance the growth vigor of plants and effectively improve the chlorophyll content and the photosynthesis efficiency of the plants. In addition, phenotypically, seedlings containing the marker have obvious dark green color as a marker, can be used for identifying the marker characters in the seedling stage, and can be applied to the hybridization combination configuration of breeding. At present, few research reports are reported on the color positioning work of the stem or hypocotyl. Zhaliyu et al (2016) mapped the tomato green stem gene Anthocynin Absent to the 96.3kb interval of chromosome 2 and screened for slGSTAA (glutathione transferase GST) as a candidate gene. The molecular marker has the characteristics of high accuracy, simple operation, high efficiency and the like, and the close linkage marker can greatly reduce the breeding process and improve the breeding efficiency. Therefore, the method has important significance for genetic improvement of pumpkin by positioning the gene (Stem color gene, SC) of the pumpkin hypocotyl color, screening closely linked molecular markers and establishing an early auxiliary selection technical system.
Disclosure of Invention
One of the purposes of the invention is to provide two pairs of molecular markers which are closely linked with the hypocotyl color related genes of the American pumpkin.
The invention also aims to provide the application of the molecular marker which is closely linked with the hypocotyl color related gene of the American pumpkin.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses two pairs of molecular markers which are closely linked with hypocotyl color related genes of a pumpkin American, wherein the molecular markers are G4 or/and G5 and are positioned on a No. 15 chromosome of the pumpkin American.
Amplifying a molecular marker primer pair closely linked with the hypocotyl color related gene of the American pumpkin, wherein the sequence of the primer pair corresponding to the molecular marker G4 is as follows:
G4-F：CCCTCGAGTTGTTCTTCTGC
G4-R：ATTGTTCCCAGCTCAAACCA；
the sequence of the primer pair corresponding to the molecular marker G5 is as follows:
G5-F：TCGTCTCCATAAGGAAATAG
G5-R：TTCAAGCAAACTACCATAAA。
the invention also discloses application of the molecular marker primer pair in assisted breeding of the American pumpkin. The molecular marker can be used for molecular marker assisted breeding in the future, and DNA of leaves is extracted in the seedling stage, and whether the molecular marker exists or not is detected, so that the dark green hypocotyl character of the American pumpkin variety is screened. The detection may be a method of PCR detection, and specifically, the above-mentioned primer pair of the molecular marker of the present invention may be used, and the detection may also be performed by a sequencing method.
In addition, the molecular marker can also be used for positioning genes related to the hypocotyl color of the American pumpkin. These applications can be carried out in accordance with conventional methods.
The invention also discloses application of the molecular marker in identification of the seedling-stage characters of the American pumpkin, in particular application in screening and identification of the hypocotyl color of the American pumpkin, wherein the hypocotyl color of the American pumpkin mainly comprises light green and dark green, and the specific steps for identification of the hypocotyl color character are as follows:
(1) using DNA of a test germplasm as a template for PCR amplification and using markers G4 or/and G5 as primers;
(2) reaction systems for PCR amplification, e.g.The following: the PCR amplification system is 10.0. mu.L and comprises ddH2O5.0 mu L, Taq enzyme 3.0 mu L, template DNA 1.0 mu L, positive and negative primers 0.5 mu L respectively;
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, 24 cycles, extension at 72 deg.C for 7min (to complete extension of product), and storage at 4 deg.C;
(3) and (3) detecting the PCR product by polyacrylamide gel electrophoresis: taking 1ul of PCR product, using a pipette to punch a 96-hole gel, using 2000/750 fragment size as Marker, and 220v voltage, and running for about 1.2-2h (set according to fragment size); with 0.1% AgNO3And (3) carrying out a decolorization reaction, then carrying out a color development reaction by using NaOH and formaldehyde, washing the strip by using clear water, airing the strip, and judging the color of the hypocotyl of the American pumpkin according to the strip result.
If a single band of 220bp or double bands of 220bp and 227bp can be amplified, the color of the hypocotyl of the American pumpkin is dark green; if the single band of 227bp can be amplified, the hypocotyl of the American pumpkin is light green; or/and amplifying the American pumpkin material genome DNA serving as a template by utilizing a primer pair G5-F, G5-R, wherein the color of the hypocotyl of the American pumpkin is dark green if a 165bp single band or both the 165bp and 160bp bands can be amplified, and the color of the hypocotyl of the American pumpkin is light green if only the 160bp single band can be amplified;
wherein, the base sequence of the 220bp strip is shown as SEQ ID NO.1 in the sequence table; a 227bp strip, the base sequence of which is shown as SEQ ID NO.3 in the sequence table; the base sequence of the 165bp strip is shown as SEQ ID NO.2 in the sequence table, and the base sequence of the 160bp strip is shown as SEQ ID NO.4 in the sequence table.
The invention has the following advantages:
(1) the invention obtains two molecular markers G4/G5 which are closely linked with the hypocotyl color related gene of the pumpkin, the genetic distance between the marker G4 and the gene is 11.9cm, and the genetic distance between the marker G5 and the gene is 2.5cm, and by using the result, the cloning of the hypocotyl color related gene CpeSC1 of the pumpkin can be finally realized, thereby laying a foundation for the research of a molecular mechanism formed by the hypocotyl color of the pumpkin.
(2) Identification of the accuracy of the trait by markers G4 and G5, F2The match degree of the genotype and the phenotype in the 210 strains of the population is 100 percent, which is beneficial to realizing accurate breeding.
(3) The screening of the linkage markers of the following hypocotyl color (light green/dark green) characters is beneficial to the molecular marker-assisted selective breeding, and the method is simple and feasible, is beneficial to improving the efficiency and saving the cost.
(4) The molecular marker disclosed by the invention has the characteristics of convenience in detection, stable amplified product and high specificity, and can be simply, conveniently, quickly and high-flux applied to American pumpkin breeding practice and variety identification.
Drawings
FIG. 1 is a distribution of InDel-index correlation values on chromosomes;
in the figure, the abscissa is the chromosome name, the colored points represent the calculated InDel-index (or. DELTA. InDel-index) values, and the black lines represent the fitted InDel-index (or. DELTA. InDel-index) values. The upper graph is a distribution graph of InDel-index values of the recessive mixed pool; the middle graph is a distribution graph of InDel-index values of the dominant mixed pool; the lower graph is a distribution plot of Δ InDel-index values, where the red lines represent the 99 percentile threshold lines;
FIG. 2 is a genetic linkage map prepared by JoinMap4.0;
FIG. 3 shows the molecular marker G4 in parent F1And F2Validation in the population;
in the figure, lane 6 is marker, 2000 bp; lane 1 is recessive at P1(V05B0318), recessive at P2(V05B0372), dominant at "1", lane 2, and F at lane 31Read as "3" dominant, and 47 strains F in the other lanes2Population, read gel references P1, P2, F1Incoming read band, no band or no definite read to "4";
FIG. 4 shows the molecular marker G5 in parent F1And F2Validation in the population;
in the figure, lane 1 is marker, 750 bp; lane 2 is recessive at P1(V05B0318) and dominant at "1", lane 3 at P2(V05B0372) and F at lane 41Read as "3" dominant, and 47 strains F in the other lanes2Population, read gel referenceBands were read at P1, P2, F1, with no bands or an indeterminate reading of "4".
Detailed Description
The present invention will be described in detail below with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The materials and reagents selected for use in the present invention can be purchased from the market without specific reference.
In the following examples, 1500F strains were constructed using a light green hypocotyl pumpkin "V05B 0318" as the female parent and a dark green hypocotyl pumpkin "V05B 0372" as the male parent2Isolating the population; primary mapping by Cluster isolation (BSA, 30 strains), development of candidate Interval markers in combination with parental sequencing, and sequencing with F2The population was verified for co-segregation of markers. The markers G4 and G5 which are closely linked with the hypocotyl of the dark green are screened, and the markers can be used for screening the hypocotyl color of the American pumpkin and identifying the seedling stage. The method comprises the following specific steps:
description of the materials
The American pumpkin materials V05B0318 and V05B0372 can be obtained by a germplasm bank (a national crop germplasm resource platform-a vegetable germplasm resource sub-platform) or a key laboratory of fruit melon biology in Henan province.
The color of hypocotyl of the two materials is identified in the field, and the result shows that V05B0372 is dark green, V05B0318 is light green, and F1Is dark green.
Construction of genetically isolated population
Parent V05B0318 of the American pumpkin material in 2019 spring "V05B0372 ", hybridization to obtain F1(V05B0318×V05B0372)，F1Selfing to obtain F2Generation, then, 210 strains F2Phenotypic identification and genetic analysis of the segregating population revealed that V05B0372 was dark green, V05B0318 was light green, and F1Phenotype matched with parental V05B0372, and F2The separation ratio of light green to dark green was about 1: 3 (Table 1). The above shows that: dark green versus light green is a dominant trait controlled by a single gene, which was designated CpeSC 1.
TABLE 1 pumpkin F of P1(V05B0318) x P2(V05B0372)2Analysis of genetic rules of colony hypocotyl color
Second, American pumpkin genetic map construction and primary positioning of hypocotyl color
1. Extraction of American pumpkin genome DNA
Extraction of American pumpkin parent and F by CTAB method2Group genome DNA, wherein the extracted individual plant DNA is used for constructing and analyzing a genetic map;
2. construction and analysis of genetic maps
Genome re-sequencing of Illumina HiSeq was performed using 30 dark green rods and 30 light green rods, and candidate genes were located in the 2.1Mb 15 interval by the BSA-index marker analysis method, and 139 genes were contained, and the distribution of InDel-index correlation values on chromosomes is shown in FIG. 1.
Strain 210F2The group is subjected to polyacrylamide gel electrophoresis for genotyping, and the genotype of the group is compared with a field statistical phenotype, analyzed, counted and converted to prepare a genetic map. Markers such as G1, G2, G3, G4, G5, G6 and the like are selected for map construction and analysis, and the CpeSC1 gene is preliminarily positioned by using JoinMap4.0 software. The results show that: the CpeSC1 gene was located between markers G4 and G5 (fig. 2), the genetic distance for markers G4 and CpeSC1 gene was 11.9em, and the genetic distance for markers G5 and CpeSC1 gene was 2.5 em.
Third, design of primer for close linkage marker with CpeSC1 gene
According to the position of the marker, the nucleotide sequence of the upstream and downstream 150bp is taken as a template, the sequence is extracted into primer5, an In-Del primer is designed, and the amplification lengths of molecular markers G1, G2, G3, G4, G5 and G6 are respectively 194bp, 219bp, 208bp, 220bp, 165bp and 151 bp.
The primer sequence of marker G1 is: f: GGCAGAAACATAATCTGGAGGA
R：AGGTCGGGGAGAAGAACAAAAC
The primer sequence of marker G2 is: f: TATCTGTCAACAGACGGGGAGT
R：GGTCCTGTAGACATTAGGTGCT
The primer sequence of marker G3 is: f: GCCCCTATAAAGCTAACA
R：ATGCAAATACAACTCAAACT
The primer sequence of marker G4 is: f: CCCTCGAGTTGTTCTTCTGC
R：ATTGTTCCCAGCTCAAACCA
The primer sequence of marker G5 is: f: TCGTCTCCATAAGGAAATAG
R：TTCAAGCAAACTACCATAAA
The primer sequence of marker G6 is: f: TCGACGGATTCAAGGTTTTC
R：ACAGGGTCAATTCACGAACC
Among them, G4, G5 are closest to CpeSC1 gene genetic distance, so G4 and G5 are selected as optimal molecular markers, the molecular marker G4 closely linked with CpeSC1 gene amplifies the sequence shown in SEQ ID NO.1, the length is 220bp, wherein the marker G4 amplifies a single band of 227bp (shown in SEQ ID NO. 3) in a light green single strain, and amplifies a single band of 220bp or double bands of 220bp and 227bp in a dark green single strain. Referring to FIG. 3, Lane 1 shows that the reading of P1(V05B0318) is "1" recessive, Lane 2 shows that the reading of P2(V05B0372) is "2" dominant, and Lane 3 shows that the reading of F1 is "3" dominant; the parent P1(V05B0318) has an insertion of 7bp relative to P2(V05B0372) resulting in a difference in the bands of the parents.
The molecular marker G5 closely linked to the CpeSC1 gene amplified a sequence as shown in SEQ ID NO.2 and had a length of 165 bp. Wherein the marker G5 can amplify a single band of 160bp (shown in SEQ ID NO. 4) in a light green single strain, and can amplify a single band of 165bp or double bands of 160bp and 165bp in a dark green single strain. Referring to FIG. 4, Lane 2 shows that the reading of P1(V05B0318) is "1" recessive, Lane 3 shows that the reading of P2(V05B0372) is "2" dominant, and Lane 4 shows that the reading of F1 is "3" dominant; the deletion of 5bp of parent P1(V05B0318) relative to P2(V05B0372) resulted in a difference in the bands of the parents.
The molecular markers have high stability and good repeatability, and play a key role in the construction of the American pumpkin hypocotyl color related gene molecular marker assisted breeding system.
Detection of markers in segregating populations of parents and progeny
With parent strains of "V05B 0318", "V05B 0372", F1And F2The DNA of (1) was used as a template, and PCR amplification was carried out using G4 and G5 as markers, respectively, and the results of the experiments are shown in FIGS. 3 and 4, respectively. And further, to obtain markers co-segregating for phenotype and genotype, increasing marker accuracy, we used markers G4 and G5 for F2The colony is detected by PCR amplification and polyacrylamide gel electrophoresis, and the result shows that: the CpeSC1 gene is located between two molecular markers, G4 and G5.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Henan university of agriculture
<120> molecular marker closely linked with hypocotyl color related gene of American pumpkin and application
<130> 2021
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 220
<212> DNA
<213> Cucurbita pepo
<400> 1
ccctcgagtt gttcttctgc ttcagttatt tgtttcgaca tatttcgatc gtgggcaatt 60
tccaacattt ggtatcatgt acgcccacat ccaacgacgg taatcgtcaa caagcaggaa 120
aatacttatt accagccatc gggtgatcgg gccacacaaa tctgcatgta ccagttgcag 180
tgatcgctca acatggaaac tggtttgagc tgggaacaat 220
<210> 2
<211> 165
<212> DNA
<213> Cucurbita pepo
<400> 2
tcgtctccat aaggaaatag atttgattaa atatcgaggt tcgttccttt tccccttcgt 60
attataaagt tatagtactc agtgtagctc cattatgtgt tcttttggaa cacttcttgt 120
atatgttgtg tatgagattc caaaatttat ggtagtttgc ttgaa 165
<210> 3
<211> 227
<212> DNA
<213> Cucurbita pepo
<400> 3
ccctcgagtt gttcttctgc ttcagttatt tgtttcgaca tatttcgatc gtgggcaatt 60
tccaacattt ggtatcatgt acgcccacat ccaacgacgg taatcgtcaa caagcaggaa 120
aatacttatt accagccatc gatggtgggg tgatcgggcc acacaaatct gcatgtacca 180
gttgcagtga tcgctcaaca tggaaactgg tttgagctgg gaacaat 227
<210> 4
<211> 160
<212> DNA
<213> Cucurbita pepo
<400> 4
tcgtctccat aaggaaatag atttgattaa atatcgaggt tcgttccttt tccccttcgt 60
attataaagt tatagtactc agtgtagctc cattgtgttc ttttggaaca cttgtatatg 120
ttgtgtatga gattccaaaa tttatggtag tttgcttgaa 160
Claims (4)
1. The molecular marker primer pair closely linked with the hypocotyl color related gene of the pumpkin america is characterized in that the primer pair has the sequence as follows:
G4-F：CCCTCGAGTTGTTCTTCTGC
G4-R：ATTGTTCCCAGCTCAAACCA；
or
G5-F：TCGTCTCCATAAGGAAATAG
G5-R：TTCAAGCAAACTACCATAAA。
2. The use of the pair of molecular tagged primers of claim 1 in assisted breeding of cucurbita sativa molecular tags.
3. The application of the molecular marker primer pair of claim 1 in the localization of hypocotyl color-related genes of the cucurbita maxima.
4. The application of the molecular marker primer pair in screening and identifying the hypocotyl color of the American pumpkin in claim 1 is characterized by comprising the following specific identification steps:
amplifying by using a primer pair G4-F, G4-R and using the detected genome DNA of the American pumpkin material as a template, wherein if a single band of 220bp or double bands of 220bp and 227bp can be amplified, the hypocotyl of the American pumpkin is dark green; if the single band of 227bp can be amplified, the hypocotyl of the American pumpkin is light green; or/and amplifying the American pumpkin material genome DNA serving as a template by utilizing a primer pair G5-F, G5-R, wherein the color of the hypocotyl of the American pumpkin is dark green if a 165bp single band or a 165bp and 160bp double band can be amplified, and the color of the hypocotyl of the American pumpkin is light green if only a 160bp single band can be amplified;
wherein, the base sequence of the 220bp strip is shown as SEQ ID NO.1 in the sequence table; a 227bp strip, the base sequence of which is shown as SEQ ID NO.3 in the sequence table; the base sequence of the 165bp strip is shown as SEQ ID NO.2 in the sequence table, and the base sequence of the 160bp strip is shown as SEQ ID NO.4 in the sequence table.
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| CN111979350A (en) * | 2020-09-29 | 2020-11-24 | 北京市农林科学院 | Method for identifying authenticity of cucurbita pepo variety |
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