CN118086574A - InDel marker detection primer for identifying potato diploid and application thereof - Google Patents
InDel marker detection primer for identifying potato diploid and application thereof Download PDFInfo
- Publication number
- CN118086574A CN118086574A CN202410494498.5A CN202410494498A CN118086574A CN 118086574 A CN118086574 A CN 118086574A CN 202410494498 A CN202410494498 A CN 202410494498A CN 118086574 A CN118086574 A CN 118086574A
- Authority
- CN
- China
- Prior art keywords
- seq
- nucleotide sequence
- primer
- potato
- set forth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 244000061456 Solanum tuberosum Species 0.000 title claims abstract description 52
- 235000002595 Solanum tuberosum Nutrition 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 239000003550 marker Substances 0.000 title claims abstract description 15
- 239000002773 nucleotide Substances 0.000 claims description 44
- 125000003729 nucleotide group Chemical group 0.000 claims description 44
- 239000012634 fragment Substances 0.000 claims description 30
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 25
- 108020004414 DNA Proteins 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 7
- 238000009396 hybridization Methods 0.000 claims description 6
- 235000012015 potatoes Nutrition 0.000 claims description 6
- 239000003068 molecular probe Substances 0.000 claims description 3
- 238000007899 nucleic acid hybridization Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 17
- 238000000684 flow cytometry Methods 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 238000012268 genome sequencing Methods 0.000 abstract description 2
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 description 23
- 208000035199 Tetraploidy Diseases 0.000 description 13
- 230000003321 amplification Effects 0.000 description 7
- 208000026487 Triploidy Diseases 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 210000003763 chloroplast Anatomy 0.000 description 3
- 239000003147 molecular marker Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000017020 Ipomoea batatas Species 0.000 description 2
- 235000002678 Ipomoea batatas Nutrition 0.000 description 2
- 235000002634 Solanum Nutrition 0.000 description 2
- 241000207763 Solanum Species 0.000 description 2
- 241000706359 Solanum phureja Species 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000554155 Andes Species 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108091060290 Chromatid Proteins 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 241000381708 Solanum x curtilobum Species 0.000 description 1
- 241000965130 Solanum x juzepczukii Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004756 chromatid Anatomy 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to an InDel marker detection primer for identifying a potato diploid and application thereof, which can be used for rapidly and accurately identifying and analyzing ploidy and genome purity of a 'green potato No. 9' reduced population, and compared with genome sequencing and flow cytometry, the primer does not need special instruments and equipment, and has lower detection cost; compared with methods such as embryo spot marking, plant morphology observation, physiological and biochemical index measurement and the like, the method has more objective and accurate detection results and good repeatability, and has important application value in the aspects of potato breeding parent selection and filial generation selection research.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an InDel marker detection primer for identifying a potato diploid and application thereof.
Background
Potatoes are a generic term for the partial species of Solanaceae (Solanacea) Solanum (Solanum) with edible tubers, the origin of the south America Andes line. Currently, potatoes have been found to have 235 varieties, of which there are 7 cultivars, including the normal cultivar and the original cultivar, 228 wild varieties. Ploidy of these species is also complex, and there are diploids (e.g. S. phureja), triploids (e.g. S. juzepczukii), tetraploids (e.g. S.turbosum), and galls (e.g. S. curtilobum), etc., and most are diploids. The cross of different ploidy materials is difficult, so that the tetraploid common cultivar S. turboosum needs to be reduced to be diploid in order to develop and utilize rich diploid resources.
The commonly used potato ploidy identification method in the current scientific research comprises the following steps of (1) identifying the plant morphology: compared with the material with lower ploidy, the leaf of the high-ploidy material is thicker, the stalk is thicker, the flowers and fruits are bigger, etc. However, the effect of controlling each trait gene is not clear at present, and the method can only be used as an auxiliary means for ploidy identification in many exceptional cases. (2) stomatal guard cell chloroplast count: the number of chloroplasts in the stomatal guard cells of a species is substantially constant and generally increases with increasing ploidy, so that ploidy of a species can be roughly judged by the number of chloroplasts in the stomatal guard cells. (3) flow cytometry: the DNA content of the cells can be directly measured by using a flow cytometer, thereby identifying the ploidy of the plant. However, this method cannot determine aneuploidy in organisms. (4) chromosome count: the ploidy of organisms is related to the number of chromosomes in cells, and the selection of cells with vigorous cell division tissue in the later stages of division period for chromosome counting is the most intuitive and accurate ploidy identification method. However, the method has low working efficiency and high requirements on experimental skills of personnel. (5) molecular marker identification: the molecular marker is a special DNA sequence in genome, and the difference of the DNA sequences can be used as auxiliary evidence to judge the gene (genome) source and ploidy of plants. (6) Whole genome sequencing: the whole genome sequencing is carried out on the experimental materials, splicing and assembling are carried out, ploidy judgment is carried out according to the assembled chromosome sequence, but the cost is high, and the skill requirement on operators is very high.
Zheng Yingzhuai et al have studied the identification of potato tri-, tetraploids and diploids with SSR markers in the potato "Cooperation 88" reduced population. The study performed genetic relationship analysis on two, three, and four tetraploids in the "collaboration 88" reduced-power population with 7 pairs of SSR primers. According to the detection result, the ploidy false detection rate of the 7 pairs of primer pairs of the reduced population is 12.0%. The 7 pairs of primers are used for detecting the reduced population of the green potato No. 9, and the result confusion is found to be unavailable for ploidy detection of the parent population and the reduced population. Therefore, it is necessary to find a molecular marker primer suitable for the "green potato No. 9" reduced population, which is very important for rapidly and accurately identifying potato ploidy, and InDel marker is the first choice for developing molecular markers in the invention because of the convenience of detection and interpretation.
Disclosure of Invention
In order to overcome the defects of the technology, the invention provides an InDel marker detection primer for identifying the diploid of potatoes and application thereof, which can be used for rapidly identifying the ploidy of a high-yield potato variety 'green potato No. 9' and derivative materials thereof.
In order to achieve the above purpose, the present invention is realized by the following scheme:
InDel scan analysis was performed on the "green potato No. 9" genomic sequence (PRJCA 006096 and PRJCA 006099) downloaded from the China national center for biological information (http:// bigd.big.ac.cn /). And (3) extending the two wings of the obtained InDel fragment by 100-200 bp respectively, and designing primers to obtain corresponding detection primers. The PCR amplification is carried out by using detection primers in the reduced-power population of the green potato No. 9, the electrophoresis detection is carried out on amplified products, and screening is carried out according to electrophoresis results, and the inventor determines 6 pairs of primers which can be used for combining and distinguishing the ploidy of the population (the screening flow is shown in figure 1), so that the method can be used for rapidly and accurately identifying and analyzing the ploidy and the genome purity of the reduced-power population of the green potato No. 9.
Accordingly, in a first aspect the present invention provides an InDel marker detection primer for use in the identification of potato diploids comprising a primer pair as follows:
primer pair QSINDEL-4: comprises nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 and a reverse primer;
Primer pair QSINDEL-7: comprises nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 and a reverse primer;
Primer pair QSINDEL-8: comprises nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6, a forward primer and a reverse primer;
primer pair QSINDEL-9: comprises nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8 and a reverse primer;
primer pair QSINDEL-43: comprises nucleotide sequences shown in SEQ ID NO:9 and SEQ ID NO:10 and a reverse primer;
Primer pair QSINDEL-77: comprises nucleotide sequences shown in SEQ ID NO:11 and SEQ ID NO:12 and a reverse primer.
The 6 pairs of primers are used for ploidy detection of parent and reduced population of potato tetraploid variety 'green potato No. 9', and the accuracy is up to 98.3%.
Further, the nucleotide sequence of the primer is (a), (b) or (c), so that the same detection purpose is achieved;
(a) Nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 12;
(b) A nucleotide sequence which hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12 under stringent conditions;
(c) A nucleotide sequence which has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12 and codes.
In some specific embodiments, the invention provides that the nucleotide sequence of the InDel marker detection primer set has 80% identity to the sequence shown in SEQ ID NO. 1-SEQ ID NO. 12; preferably 85% identical, more preferably 90% identical, more preferably 95% identical, and most preferably 99% identical.
Illustratively, as used herein, "stringent conditions" refers to conditions under which a probe will hybridize to its target sequence to a detectable extent that hybridizes to other sequences (e.g., at least 2-fold background). Stringent conditions are sequence-dependent and will be different from one environment to another. By controlling the stringency of hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified. Alternatively, stringent conditions may be adjusted to allow for some sequence mismatches so that a lower degree of similarity is detected. These nucleotide sequences which hybridize under stringent conditions can be used, for example, for the expression of variant proteins of SEQ ID NO.1 or can be used as primers, probes, exogenous donor sequences, guide RNAs, antisense RNAs, shRNAs and siRNAs.
In a second aspect, the invention provides the use of an InDel marker detection primer set in the ploidy identification of distant filial generation of potato.
Further, the potato is "green potato No. 9".
Further, determining whether the DNA fragments shown in (a) - (n) are included in the sample to be tested;
(a) As set forth in SEQ ID NO:13, a nucleotide sequence shown in seq id no;
(b) As set forth in SEQ ID NO:14, a nucleotide sequence shown in seq id no;
(c) As set forth in SEQ ID NO:15, a nucleotide sequence shown in seq id no;
(d) As set forth in SEQ ID NO:16, a nucleotide sequence shown in seq id no;
(e) As set forth in SEQ ID NO:17, a nucleotide sequence shown in seq id no;
(f) As set forth in SEQ ID NO:18, and a nucleotide sequence shown in seq id no.
In some specific examples, 6 pairs of primers (the nucleotide sequences of which are shown as SEQ ID NO. 1-SEQ ID NO. 12) can be co-amplified in the female parent to obtain 6 DNA fragments (the nucleotide sequences of which are shown as SEQ ID NO. 13-SEQ ID NO. 18), and the primers can be used for identifying ploidy of distant filial generation according to the existence of the DNA fragments.
In a third aspect, the invention provides the use of an InDel marker detection primer set in the identification of the genomic purity of progeny of distant hybridization of potatoes.
Further, the potato is "green potato No. 9".
Further, determining whether the DNA fragment shown in (a) - (i) is included in the sample to be tested;
(a) As set forth in SEQ ID NO:19, a nucleotide sequence shown in seq id no;
(b) As set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;
(c) As set forth in SEQ ID NO:21, a nucleotide sequence shown in seq id no;
(d) As set forth in SEQ ID NO:22, a nucleotide sequence shown in seq id no;
(e) As set forth in SEQ ID NO: 23.
In some embodiments, 6 pairs of primers (having nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 12) co-amplify 5 DNA fragments (having nucleotide sequences shown in SEQ ID NO. 19-SEQ ID NO. 22) in the male parent IVP101, and based on the presence or absence of these DNA fragments, it can be deduced whether there is a male parent gene introgression in the doubling material, thereby allowing for the identification of distant hybrid progeny genome purity.
Further, the application comprises the steps of nucleic acid hybridization detection, molecular marking, gene chip preparation, molecular probe preparation and detection kit preparation.
Compared with the prior art, the invention has the following beneficial effects:
(1) The amplification result of 6 pairs of InDel marked primers is used for detecting and judging the ploidy of the 'green potato No. 9' reduced population, compared with genome sequencing and flow cytometry, special instruments and equipment are not needed, and the detection cost is lower;
(2) Compared with methods such as embryo spot marking, plant morphology observation, physiological and biochemical index measurement and the like, the method has the advantages of more objective and accurate detection result and good repeatability.
Drawings
FIG. 1 is a flow chart of InDel marker detection primer screening;
FIG. 2 is a ploidy detection graph of "sweet potato No. 9" hybridization-induced reduced-magnification population material;
FIG. 3 is a graph of agarose gel electrophoresis detection of InDel labeled primers;
FIG. 4 is a graph of a cluster analysis of the detection results of InDel molecular markers of the "sweet potato No. 9" hybridization-induced fold-reduction population.
Detailed Description
The present invention will be described in detail with reference to specific embodiments thereof, so that those skilled in the art can better understand the technical solutions of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Based on the invention, in order to develop diploid resources of tetraploid good potato variety 'green potato No. 9', the 'green potato No. 9' is subjected to de-multiplication, and de-multiplication groups comprising diploid, triploid and tetraploid materials are obtained. After the inventor analyzes the genome of the green potato No. 9, an InDel marker primer suitable for the reduced population of the green potato No. 9 is developed, and the InDel marker primer can be used for better judging the ploidy of the reduced population material and estimating the flow direction of the parent gene.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1
The potato tetraploid variety 'green potato No. 9' is subjected to a reduction treatment, part of leaves of the obtained material are cut off, and after liquid nitrogen quick freezing, the leaves are stored at-80 ℃ and detected by a flow cytometry to determine the ploidy of the material (see fig. 2), and as shown in fig. 2, a: green potato No. 9 (Q9) (tetraploid, female parent); b: IVP101 (diploid, male parent); c: diploid progeny; d: triploid progeny; e: tetraploid progeny.
Example 2
Performing InDel scanning analysis on a known 'green potato No. 9' genome, and performing primer design on each extension 100-200 bp of two wings of the obtained InDel fragment, wherein fragments with lower GC% content at two ends of the fragment and incapability of designing amplification primers are removed; then, selecting fragments of the amplified fragment specific for a certain chromosome and different on different chromatids of the same chromosome; comparing the expected amplified fragment with the published diploid potato Phureja DM 1-3 516R 44 v8.1 genome, carrying out ePCR on the genome by using a primer, and reserving fragments and primers which can distinguish a parent; the primer reserved in screening is subjected to PCR detection verification in a reduced population of 'green potato No. 9', and an amplified product is subjected to electrophoresis detection, and screening is carried out according to an electrophoresis result, so that 6 pairs of InDel mark detection primers which can be used for reduced population ploidy screening are determined to be primer pairs QSINDEL-4 (the nucleotide sequences of which are shown as SEQ ID NO:1 and SEQ ID NO: 2), primer pairs QSINDEL-7 (the nucleotide sequences of which are shown as SEQ ID NO:3 and SEQ ID NO: 4), primer pairs QSINDEL-8 (the nucleotide sequences of which are shown as SEQ ID NO:5 and SEQ ID NO: 6), primer pairs QSINDEL-9 (the nucleotide sequences of which are shown as SEQ ID NO:7 and SEQ ID NO: 8), primer pairs QSINDEL-43 (the nucleotide sequences of which are shown as SEQ ID NO:9 and SEQ ID NO: 10) and primer pairs QSINDEL-77 (the nucleotide sequences of which are shown as SEQ ID NO:11 and SEQ ID NO: 12) respectively.
Example 3
Extracting total DNA of 'green potato No. 9', IVP101 and leaves respectively by an improved CTAB method, carrying out PCR (polymerase chain reaction) amplification detection verification on 6 pairs of designed InDel marker detection primers in two parents and reduced populations identified as two, three and four-fold by flow cytometry, wherein an amplification system and an amplification program are shown in tables 1 and 2:
TABLE 1 amplification System
TABLE 2 amplification procedure
The amplified products were electrophoretically detected with 2% agarose (see FIG. 3), and 6 pairs of primers were found to be useful for distinguishing the parent and the parent. In fig. 3, M is Maker, Q is green potato No. 9, I is IVP101, and C is a negative control; as can be seen from FIG. 3, in the female parent green potato No. 9, 6 pairs of designed primers can amplify 6 fragments altogether, wherein the primer pair QSINDEL-4 can amplify 1 target fragment QSINDEL-4 (the nucleotide sequence of which is shown as SEQ ID NO: 13) with the length of 280 bp respectively; primer pair QSINDEL-7 can amplify 1 item target fragment QSINDEL-7-1 (the nucleotide sequence of which is shown as SEQ ID NO: 14) with the length of 186 bp; primer pair QSINDEL-8 can amplify 1 item target fragment QSINDEL-8-1 (the nucleotide sequence of which is shown as SEQ ID NO: 15) with length of 110 bp; primer pair QSINDEL-9 can amplify 1 item target fragment QSINDEL-9-1 (the nucleotide sequence of which is shown as SEQ ID NO: 16) with length of 255 bp; primer pair QSINDEL-43 can amplify 1 item target fragment QSINDEL-43-1 (the nucleotide sequence of which is shown as SEQ ID NO: 17) with length of 215 bp; primer pair QSINDEL-77 can amplify 1 item target fragment QSINDEL-77-1 (the nucleotide sequence of which is shown as SEQ ID NO: 18) with length of 244 bp; and, each amplified fragment was subjected to clone sequencing, and the sequence was identical to that of the corresponding fragment in the published genome. Based on the presence or absence of these DNA fragments, the method can be used to identify ploidy of distant filial offspring.
In addition, it is found that in the male parent IVP101, 5 fragments can be amplified by 6 pairs of designed primers, wherein 1 target fragment IVP 101-QSINDEL-4 (the nucleotide sequence of which is shown as SEQ ID NO: 19) can be amplified by the primer pair QSINDEL-4, and the lengths of the fragments are 238 and bp respectively; primer pair QSINDEL-7 can amplify 1 target segment IVP 101-QSINDEL-7 (the nucleotide sequence of which is shown as SEQ ID NO: 20) with length of 155 bp; primer pair QSINDEL-8 can amplify 1 target segment IVP 101-QSINDEL-8 (its nucleotide sequence is shown in SEQ ID NO: 21) with length of 193 bp; primer pair QSINDEL-9 can amplify 1 target segment IVP 101-QSINDEL-9 (the nucleotide sequence of which is shown as SEQ ID NO: 22) with length of 214 bp; primer pair QSINDEL-43 can amplify 1 item target segment IVP 101-QSINDEL-43 (its nucleotide sequence is shown as SEQ ID NO: 23) with length of 248 bp; it can be deduced from the presence or absence of these fragments whether there is introgression of the male parent gene in the reduced material.
As a result of material genome source evaluation and ploidy analysis according to the PCR detection result, it was found that 44.2% of the genome of the diploid material in the reduced-power population was derived from the female parent, the remaining 55.8% of the diploid contained male parent bands, and the genomes of the triploid and tetraploid materials contained parent bands.
The detection results of 6 pairs of InDel labeled primers in 120 parts of "green potato No. 9" reduced population materials (marked as 1 with a band and 0 without a corresponding band) are labeled, and the overall result is subjected to clustering analysis by a UPGMA Method (Unweighted Pair-Group Method WITH ARITHMETIC MEANS) (see FIG. 4). As shown in fig. 4, the blue ground trace portion represents Q9 (female parent), the blue-green ground trace portion represents IVP101 (male parent), the green ground trace portion represents tetraploid, the red ground trace portion represents triploid, and the yellow ground trace portion represents diploid; as a result, only one part of triploid material and one part of tetraploid material were classified into diploid material, and the clustering results of other materials were consistent with the detection results of flow cytometry.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. An InDel marker detection primer for identifying a potato diploid is characterized by comprising a primer pair shown as follows:
primer pair QSINDEL-4: comprises nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 and a reverse primer;
Primer pair QSINDEL-7: comprises nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 and a reverse primer;
Primer pair QSINDEL-8: comprises nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6, a forward primer and a reverse primer;
primer pair QSINDEL-9: comprises nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8 and a reverse primer;
primer pair QSINDEL-43: comprises nucleotide sequences shown in SEQ ID NO:9 and SEQ ID NO:10 and a reverse primer;
Primer pair QSINDEL-77: comprises nucleotide sequences shown in SEQ ID NO:11 and SEQ ID NO:12 and a reverse primer.
2. The primer according to claim 1, wherein: the nucleotide sequence of the primer is (a), (b) or (c);
(a) Nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 12;
(b) A nucleotide sequence which hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12 under stringent conditions;
(c) A nucleotide sequence which has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12 and codes.
3. Use of a primer set according to claim 1 or 2 for ploidy identification of distant hybridization progeny of potatoes.
4. Use according to claim 3, characterized in that: determining whether the sample to be tested comprises the DNA fragments shown in (a) - (f);
(a) As set forth in SEQ ID NO:13, a nucleotide sequence shown in seq id no;
(b) As set forth in SEQ ID NO:14, a nucleotide sequence shown in seq id no;
(c) As set forth in SEQ ID NO:15, a nucleotide sequence shown in seq id no;
(d) As set forth in SEQ ID NO:16, a nucleotide sequence shown in seq id no;
(e) As set forth in SEQ ID NO:17, a nucleotide sequence shown in seq id no;
(f) As set forth in SEQ ID NO:18, and a nucleotide sequence shown in seq id no.
5. Use of a primer set according to claim 1 or 2 for the identification of the genomic purity of progeny of distant hybridization of potatoes.
6. Use according to claim 5, characterized in that: determining whether the sample to be tested comprises the DNA fragments shown in (a) - (i);
(a) As set forth in SEQ ID NO:19, a nucleotide sequence shown in seq id no;
(b) As set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;
(c) As set forth in SEQ ID NO:21, a nucleotide sequence shown in seq id no;
(d) As set forth in SEQ ID NO:22, a nucleotide sequence shown in seq id no;
(e) As set forth in SEQ ID NO: 23.
7. Use according to claim 3 or 4, characterized in that: the potato is 'green potato No. 9'.
8. Use according to claim 5 or 6, characterized in that: the potato is 'green potato No. 9'.
9. Use according to claim 3 or 4, characterized in that: the application comprises the steps of nucleic acid hybridization detection, molecular marking, gene chip preparation, molecular probe preparation and detection kit preparation.
10. Use according to claim 5 or 6, characterized in that: the application comprises the steps of nucleic acid hybridization detection, molecular marking, gene chip preparation, molecular probe preparation and detection kit preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410494498.5A CN118086574A (en) | 2024-04-24 | 2024-04-24 | InDel marker detection primer for identifying potato diploid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410494498.5A CN118086574A (en) | 2024-04-24 | 2024-04-24 | InDel marker detection primer for identifying potato diploid and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118086574A true CN118086574A (en) | 2024-05-28 |
Family
ID=91150930
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410494498.5A Pending CN118086574A (en) | 2024-04-24 | 2024-04-24 | InDel marker detection primer for identifying potato diploid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118086574A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803094A (en) * | 2016-05-06 | 2016-07-27 | 中国农业科学院蔬菜花卉研究所 | SCAR mark for appraisal or auxiliary appraisal of maturity of potatoes and application of SCAR mark |
| CN110894539A (en) * | 2019-12-04 | 2020-03-20 | 云南师范大学 | Method for identifying self-compatibility of diploid potatoes |
-
2024
- 2024-04-24 CN CN202410494498.5A patent/CN118086574A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803094A (en) * | 2016-05-06 | 2016-07-27 | 中国农业科学院蔬菜花卉研究所 | SCAR mark for appraisal or auxiliary appraisal of maturity of potatoes and application of SCAR mark |
| CN110894539A (en) * | 2019-12-04 | 2020-03-20 | 云南师范大学 | Method for identifying self-compatibility of diploid potatoes |
Non-Patent Citations (4)
| Title |
|---|
| FANG WANG等: "The autotetraploid potato genome provides insights into highly heterozygous species", 《PLANT BIOTECHNOL J》, vol. 20, no. 10, 1 August 2022 (2022-08-01), pages 1996 - 2005 * |
| 王鹏;李芳弟;郭天顺;窦俊焕;颉炜清;罗照霞;齐小东;杨晨;赵中梁;宋怡;吕汰;: "马铃薯种质资源遗传关系分析及指纹图谱构建", 农业生物技术学报, no. 05, 25 May 2020 (2020-05-25) * |
| 郑英转: "诱导四倍体马铃薯优良品种合作88降倍及其机制研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 8, 15 August 2021 (2021-08-15), pages 047 - 184 * |
| 高健军: "马铃薯品种的孤雌生殖诱导研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 01, 15 January 2015 (2015-01-15), pages 047 - 213 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Demeke et al. | Identification of potato cultivars and clonal variants by random amplified polymorphic DNA analysis | |
| Yamaki et al. | Development of INDEL markers to discriminate all genome types rapidly in the genus Oryza | |
| US20220298549A1 (en) | Method for identifying whether a diploid potato is self-compatible | |
| CN109609686B (en) | Molecular marker for early sex identification of actinidia arguta seedlings and application of molecular marker | |
| CN110724758B (en) | Method for identifying purity of Jingnongke 728 corn hybrid based on SNP marker | |
| CN110872633B (en) | Method for identifying purity of Jingke 968 corn hybrid based on SNP marker | |
| CN110777216B (en) | Method for identifying purity of Jingke waxy 2000 corn hybrid based on SNP marker | |
| CN110241245A (en) | Detect KASP primer and its application of cucumber bacterial angular leaf spot gene | |
| CN112176091B (en) | CAPS molecular marker closely linked with eggplant sepal color character gene and preparation method thereof | |
| CN113249509B (en) | Identification primer and identification method for interspecific hybrid progeny of populus jaborandi and populus microphylla | |
| CN118086574A (en) | InDel marker detection primer for identifying potato diploid and application thereof | |
| CN114231603B (en) | Primer, reagent, identification method and kit for identifying paeonia boita | |
| CN113278723B (en) | Composition for analyzing genetic diversity of Chinese cabbage genome segment or genetic diversity introduced in synthetic mustard and application | |
| CN106399495B (en) | SNP marker closely linked with soybean short stalk character and application thereof | |
| CN115109864A (en) | SSR molecular marker E201 for identifying Chinese pumpkin 'Zhongchuanu No. 1' hybrid, and primer, kit and method thereof | |
| CN109136392B (en) | Genetic diversity identification method and reagent for multi-generation meiotic gynogenesis megalobrama amblycephala | |
| CN107012253B (en) | Method for identifying female parent of cultivated oat | |
| CN112251530B (en) | SSR (simple sequence repeat) marker associated with ramie fiber fineness and application thereof | |
| US20210363600A1 (en) | Primer groups for detecting hybrid rice backbone parent and application thereof | |
| CN116926230B (en) | Molecular marker related to cotton fiber length and application thereof | |
| CN117305511B (en) | KASP primer group related to wheat grain width and application thereof | |
| CN115927734B (en) | KASP molecular marker for identifying hardness of pear fruit, KASP primer and application thereof | |
| CN113817839B (en) | Qidamu cashmere goat cashmere flexibility related molecular marker and application thereof | |
| CN108060258B (en) | Primer group, kit and method for pure heterozygous identification of cotton MON15985 transformant | |
| CN118166155A (en) | SNP molecular marker related to pumpkin pulp thickness, amplification primer set and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |