CN115896327A - InDel marker closely linked with leaf of Lance mustard and application thereof - Google Patents
InDel marker closely linked with leaf of Lance mustard and application thereof Download PDFInfo
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- CN115896327A CN115896327A CN202211184195.0A CN202211184195A CN115896327A CN 115896327 A CN115896327 A CN 115896327A CN 202211184195 A CN202211184195 A CN 202211184195A CN 115896327 A CN115896327 A CN 115896327A
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- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 title description 2
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- 238000000034 method Methods 0.000 claims abstract description 8
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Abstract
The invention belongs to the technical field of plant breeding, and particularly relates to an InDel marker closely linked with leaf of Lance mushroom and application thereof. The InDel marker comprises a sequence shown as SEQ ID NO:1 and SEQ ID NO:2, and (b) a primer set shown in (2). The InDel marker is based on PCR amplification, has clear amplification band, high polymorphism and high resolution, and can accurately identify whether the cabbage mustard contains mushroom leaves; and the method is simple, convenient and easy to implement, strong in operability, economical and practical.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to an InDel marker closely linked with leaf of Lance mushroom and application thereof.
Background
Cabbage mustard (Brassica oleracea var. Chinensis Lei) is a Brassica vegetable in cruciferae, and has high nutritional value and good market prospect. The cabbage mustard has low genetic diversity, but has various morphological traits and obvious genetic differences, the mushroom leaves are malformed leaves born on the leaf veins of the cabbage mustard, and the current researches on the leaf traits of the cabbage mustard are few. The mushroom leaf, also called mushroom flower, is a metamorphosis lobule similar to mushroom growing on the leaf vein of cabbage mustard, and is a special botanical trait. At present, the growing mushroom leaves are found only on the cabbage mustard leaves of a small number of farmhouses such as small mushrooms, mushroom mosses, short-foot mushrooms, mushroom bouquet mustards, old-variety mushrooms and the like, and the mushroom leaf varieties of the cabbage mustard belong to the cabbage mustard.
With the increasing maturity and perfection of molecular marker technology, the InDel marker plays an important role in the aspects of germplasm resource analysis, molecular marker-assisted breeding, population analysis, gene localization, genetic map construction and the like, but the research on the development and utilization of molecular markers related to characteristic shapes of leaves of the Lance mushroom and the like is relatively weak.
Disclosure of Invention
Aiming at the problems, the invention aims to provide an InDel marker which is closely linked with leaves of the kalium bicolor, is based on PCR amplification, has clear amplification band, high polymorphism and high resolution, and can accurately identify whether the kalium bicolor contains the mushroom leaves; and the method is simple, convenient and easy to implement, strong in operability, economical and practical.
In order to achieve the purpose, the following technical scheme can be adopted:
the invention provides an InDel marker closely linked with leaves of Lancet mustards, which comprises a sequence shown as SEQ ID NO:1 and SEQ ID NO:2, and (b) a primer set shown in (2).
The invention further provides application of the InDel marker which is tightly linked with the leaves of the kale mustard in breeding of the kale mustard.
In another aspect, the invention provides an application of the InDel marker which is closely linked with leaves of the kalium sinense in purity identification of the kalium sinense hybrid.
The invention further provides an application of the InDel marker which is tightly linked with the leaf of the kalium bicolor in the construction of a kalium bicolor genetic linkage map.
In another aspect, the present invention provides a method for identifying leaf traits of Echinacea purpurea, comprising: the DNA of the cabbage mustard to be identified is used as a DNA template, and the InDel marker which is closely linked with the leaf of the cabbage mustard is used for PCR amplification.
Further, the method for identifying the leaf traits of the Echinacea purpurea may comprise the following steps: extracting DNA of the cabbage mustard to be identified as a DNA template, carrying out PCR amplification by using the InDel marker which is tightly linked with the leaf of the cabbage mustard to obtain an amplification product, carrying out gel electrophoresis on the amplification product, and when the banding pattern is consistent with that of a parent with mushroom leaves, expressing the plant as the mushroom leaves; when the banding pattern is consistent with the parent strain without the mushroom leaves, the plant shows no mushroom leaves; when the banding pattern is heterozygous, the banding pattern is controlled by another independently inherited gene, and when the gene is heterozygous, the plant shows the mushroom leaves.
The beneficial effects of the invention at least comprise: the InDel marker which is tightly linked with the leaves of the kalium bicolor provided by the invention is based on PCR amplification, has clear amplification band, high polymorphism and high resolution, and can accurately identify whether the kalium bicolor contains the leaves; and the method is simple, convenient and easy to implement, strong in operability, economical and practical.
Drawings
FIG. 1 shows the presence or absence of a mushroom leaf phenotype in cabbage mustard;
FIG. 2 shows agarose gel electrophoresis detection of a portion of a DNA sample, in lane 1: DL2000 DNA Marker, lanes 2-13: randomly extracting partial DNA samples;
FIG. 3 is a genetic linkage map of a mushroom leaf locus on chromosome 4;
FIG. 4 is a partial gel electrophoresis image labeled InDel-64, wherein A represents a PCR banding pattern consistent with the banding pattern of the mushroom-leaf-containing parent No. 55, B represents a PCR banding pattern consistent with the banding pattern of the mushroom-leaf-free parent No. 52, and H represents a heterozygous banding pattern, i.e., the banding pattern of both the mushroom-leaf-containing parent and the mushroom-leaf-free parent;
FIG. 5 is a schematic diagram of reasoning about the genetic pattern of the mushroom leaf trait.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context has a significantly different meaning, the singular forms of expressions include the plural forms of expressions. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to refer to the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the circumstances.
For a better understanding of the present invention, the following further illustrates the contents of the present invention with reference to specific examples, but the contents of the present invention are not limited to the following examples.
In the following examples, the high-generation selfing lines of cabbage mustard for testing are all from southern China agricultural university, and the No. 52 selfing line is a mushroom leaf material and is bred from old seed incense; no. 55 inbred line is a mushroom-free leaf material. Taking cabbage mustard No. 52 as a female parent and cabbage mustard No. 55 as a male parent, hybridizing to obtain F1 generation plants, and selfing the F1 to construct an F2 generation segregation population with or without mushroom leaves.
In the following examples, primers were synthesized by Oncorhynchus Biotechnology Ltd.
Example 1
(1) InDel labeled primer
Taking No. 52 cabbage mustard and No. 55 cabbage mustard as parent pools, after character investigation, selecting 30 plants of each of mushroom leaf-containing and mushroom leaf-free cabbage mustard in F2 generation segregation populations, and taking tender leaves with the diameter of 0.5cm from each plant by using a puncher to form two extreme pools of mushroom leaf-containing and mushroom leaf-free; entrusted Wuhan Feisha gene information limited company with the mixed pool sample to carry out DNA extraction and genome re-sequencing;
taking genome at the chromosome level of the arabidopsis thaliana as a reference genome, analyzing by a QTL-seqr package based on an R language, respectively counting the proportion of Reads of mutant parent genotypes of the extreme mixed pool and the reference genome in the same base site to the total Reads, taking 1Mb as a window and 10Kb as a step length, and calculating the average value of SNP-index of each window to reflect SNP-index of two extreme mixed pools of filial generation; delta (SNP-index) is the difference between SNP-indices of two extreme pools; performing 1000 times of replacement tests, and selecting 95% and 99% confidence levels as thresholds to obtain the distribution of the delta (SNP-index) of the two mixed pools on the chromosome; the alpha value detected by Grubbs was set to 0.001, resulting in the distribution of G' value in the genome. Determining a region above a threshold line in the Δ (SNP-index) >95% confidence level and Grubbs detection as a potential QTL candidate region;
the screened InDel sites are positioned on the middle flowering cabbage mustard reference genome, 200bp sequences of the upstream and downstream of the InDel sites are taken, primers (shown in the following table 1) are designed by using Primer Premier 5 software, and PCR products contain the InDel sites and have the size of 150bp-400b.
TABLE 1 information on InDel-labeled primers
(2) Primer validation
In order to research the genetic characteristics of leaf traits of the kalium bicolor, no. 52 kalium bicolor (shown as a in figure 1) with mushroom leaves is taken as a female parent, no. 55 kalium bicolor (shown as b in figure 1) without mushroom leaves is taken as a male parent, and F obtained by hybridization is used 1 All the generation plants show mushroom leaves, which indicates that the characteristics of the mushroom leaves are controlled by dominant genes; mixing the above materials to obtain a mixture F 1 Selfing the cabbage mustard to produce F 2 Separating the population; 404 Strain F 2 The generation individual plants comprise 264 strains of the leaf mustard and no leaf mushroomBrassica juncea 140 strain, using Microsoft Excel 2019 software on F 2 The character of leaf of Chinese cabbage mustard in the colony is X 2 Goodness-of-fit testing found the separation ratio to follow 10:6, P value of 0.237>0.05; thus, the existence of the leaf of the Lancet mustards is probably controlled by two pairs of independently inherited major genes.
For the two parents No. 52 and No. 55 and F 2 Two mixed pool genomes with mushroom leaves and without mushroom leaves are sequenced, and the sequencing depth is 30 times; based on a BGI (MGI-seq 2000) high-throughput sequencing platform, 98.92Gb data are generated jointly, the sequencing sequences (Reads) obtained after filtration are 638614928, and the effective rate is more than 96.64%; the original data showed that Q20 was above 96.5% and Q30 was above 89.8%, the GC content of the genome was normal, 36%, 35.8% and 36.8%, respectively (see Table 2), with 99.65% of the high quality Reads compared to the medium flower cabbage reference genome; the data volume of all samples is enough, the quality of sequencing data is qualified, the comparison efficiency with the reference genome of the Chinese flowering cabbage is high, and the method can be used for subsequent variation detection and character gene positioning.
TABLE 2 summary of retest data
1) Determination of candidate intervals
Analysis of the delta (SNP-index) and G' value of the mixed pool with and without mushroom leaves was carried out with the genome of Brassica napobrassica as reference, at F 2 4,663,771 SNP loci are detected in the extreme mixed pool of the colony, and 3593233 SNP loci are obtained after filtration; according to a delta (SNP-index) distribution diagram, two obvious peaks are found to appear on chromosome 4 by taking a 95% confidence level as a screening threshold, and are respectively positioned at 60217093bp-61017093bp and 65817093bp-67617093 bp; according to the distribution diagram of the G' value, only chromosome 4 above the threshold line is found to have a peak at 58243405bp-67606738bp, the maximum value of the G' value is 43.1097, the position is 67078568bp, and the delta (SNP-index) value of the position is 0.777; according to the results of the Δ (SNP-index) analysis and Grubbs detection, the intersection of the regions associated with the two analysis methods was taken as a candidate region which was 60217093bp-67606738bp on chromosome 4 and had a total length of about 7.4Mb.
2) InDel marker-based mushroom leafgenesis related gene locus positioning
Will be 404F 2 Detecting the absorbance of 406 DNA mother solutions of a single plant and 2 parents by using an ultramicro ultraviolet spectrophotometer, and finding that the absorbance ratio (A260/A280) of nucleic acid and protein of the DNA mother solutions is more than 1.8, and the absorbance ratio (A260/A230) of nucleic acid and carbohydrate of the DNA mother solutions is more than 2.0; randomly extracting the detection result of the DNA sample (see table 3), which indicates that the extracted DNA mother liquor is purer and the contents of protein, polyphenols, salts, organic solvents and other impurities are less; and (3) performing agarose gel electrophoresis on the extracted DNA, wherein the electrophoresis detection result is shown in figure 2, and the DNA presents a clear and complete bright band, which indicates that the genomic DNA mother solution has good quality and can be used for subsequent tests.
TABLE 3 absorbance of partial DNA samples
| DNA mother liquor numbering | 5 | 17 | 53 | 60 | 108 | 117 |
| Concentration (μ g/. Mu.g) | 654.9 | 524.4 | 1233.5 | 1141.1 | 1516.1 | 1281.4 |
| A260/A280 | 2.18 | 2.16 | 2.14 | 2.14 | 2.16 | 2.15 |
| A260/A230 | 2.15 | 2.09 | 2.11 | 2.13 | 2.20 | 2.18 |
3) Construction of linkage map and QTL analysis
According to the result of BSA analysis, the gene related to leaf development of the kale mushroom is located in a region with the physical position of 60217093bp-67606738bp on the No. 4 chromosome; with ICIMapping software, at 404F 2 DNA of the individual and two parents as templates, using 10 pairs of InDel markers for F in the candidate regions 2 Constructing a linkage map of the population; the total genetic distance between the markers was 41.89cM, the QTL was located between the markers InDel-64 and InDel-69, the genetic distance was 2.44cM, the physical distance was 255kb, the LOD value was 44.2259 at the maximum, the contribution rate of the QTL was 43.19% (see FIG. 3), calculated as 95%Confidence level as the screening threshold, two distinct peaks were found to occur on chromosome 4, at 60.21Mb-61.01Mb and 65.8Mb-67.61Mb, respectively.
4) Phenotypic and banding analysis
The phenotype of the plant marked by the InDel is analyzed, and the primer InDel-64 is found to be closely linked with the characteristics of the mushroom leaves, and the banding patterns of 101F with the mushroom leaf parent are consistent 2 Among the individual plants, 98 individual plants with mushroom leaves exist; in 95 strains of F with the same banding pattern as the non-mushroom leaf parent 2 Among the individual plants, 82 plants exist in the single plant without the mushroom leaf; f in heterozygous ribbon type 203 2 In the single plant, 151 plants with mushroom leaves and 52 plants without mushroom leaves are provided, and the proportion is about 3:1 (table 4);
TABLE 4 data statistics of banding patterns and plant phenotypes of the marker InDel-64
| Tape type | A | H | B |
| Leaf of Pleurotus Ostreatus | 98 | 151 | 13 |
| Without |
3 | 52 | 82 |
Analyzing the banding pattern of the InDel marker, and when the banding pattern of an amplification product marked with InDel-64 is consistent with that of a parent with mushroom leaves, showing that the plant presents with mushroom leaves as shown in figure 4; when the banding pattern is consistent with the parent without mushroom leaves, the plant shows without mushroom leaves; when the banding pattern is heterozygous, the banding pattern is controlled by another independently inherited gene, and when the gene is heterozygous, the plant shows the mushroom leaves; the reasoning of the inheritance pattern of the mushroom leaf trait is shown in FIG. 5, which shows that the gene linked to InDel-64 has an epistatic effect, while another gene has a dominant effect.
Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.
Claims (6)
1. An InDel marker closely linked to a leaf of Echinacea purpurea, comprising a sequence shown in SEQ ID NO:1 and SEQ ID NO:2 under the control of a control.
2. The use of the InDel marker tightly linked to leaves of Echinacea purpurea according to claim 1 in breeding of Echinacea purpurea.
3. The use of the InDel marker of claim 1, which is tightly linked to leaves of Echinacea purpurea, for purity determination of an Echinacea purpurea hybrid.
4. The use of the InDel marker tightly linked to leaves of Echinacea purpurea according to claim 1 in the construction of genetic linkage maps of Echinacea purpurea.
5. A method for identifying leaf traits of Echinacea purpurea comprises the following steps: the DNA of the cabbage mustard to be identified is used as a DNA template, and the InDel marker which is closely linked with the leaf of the cabbage mustard and is described in claim 1 is used for PCR amplification.
6. The method for identifying the leaf trait of Echinacea purpurea according to claim 5, comprising: extracting DNA of the cabbage mustard to be identified as a DNA template, carrying out PCR amplification on the InDel marker which is in close linkage with the leaf of the cabbage mustard as claimed in claim 1 to obtain an amplification product, carrying out gel electrophoresis on the amplification product, and when the band type is consistent with that of a parent with mushroom leaf, showing that a plant shows the mushroom leaf; when the banding pattern is consistent with the parent strain without the mushroom leaves, the plant shows no mushroom leaves; when the banding pattern is heterozygous, it is controlled by another independently inherited gene, and when the gene is heterozygous, the plant shows mushroom leaves.
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2022
- 2022-09-27 CN CN202211184195.0A patent/CN115896327A/en active Pending
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