CN115976251B - InDel marker for cassava genetic diversity analysis and application thereof - Google Patents
InDel marker for cassava genetic diversity analysis and application thereof Download PDFInfo
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Abstract
The invention provides a primer group based on InDel marker development of a cassava whole genome and application thereof. Based on the result of 22 parts of cassava germplasm resources genome-wide resequencing analysis, the InDel locus is excavated to develop a molecular marker, the effectiveness of 64 pairs of primers is verified, and 20 pairs of primers have polymorphism, and the polymorphism rate is 31.25%. The genetic diversity of 72 cassava germplasm resources is revealed based on the result of InDel marker cluster analysis with optimal amplification effect of 18, the gene diversity index of each marker is between 0.21 and 0.50, and the shannon diversity index is between 0.36 and 0.70. The results of the cluster analysis showed that 72 parts of cassava material were divided into 2 clusters at a genetic similarity coefficient of 0.62. The research result lays a certain foundation for the subsequent development of cassava breeding work.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an InDel marker for cassava genetic diversity analysis and application thereof.
Background
Cassava (Manihot esculenta Crantz) belongs to the family Euphorbiaceae, is one of the sixth important crops in the world, and is parallel to sweet potato and potato as the third potato crop in the world. The original Brazil is originally from America, and is introduced and popularized and planted in more than nearly one hundred countries in Africa and Asia. The Guangdong province of China is introduced for the first time in the 20 th century, and the current is mainly distributed in the southeast coastal areas of China, such as Guangxi, hainan and other provinces. In addition, there are also plants in Fujian, jiangxi, hunan and Guizhou, etc. The starch-rich food is widely applied to industrial raw material production, and can be used as a source of calories required by human bodies, so that the starch-rich food becomes a staple food for nearly 10 hundred million people in the world. As important raw materials for key grain crops and industries, the material has important roles in industries such as international livestock, chemical industry, food, environmental protection and the like.
The introduction of cassava into China has been recently two hundred years old, and at present, researchers in China have screened a series of main plant varieties from introduced germplasm. Because of complex genetic background and various germplasm resources, the collection and preservation of germplasm materials and the development progress of breeding work in China are slower. However, whether the breeding work breaks through or not depends on the degree of genetic diversity of resources among varieties, so that analysis of the genetic diversity among populations is of great significance to the breeding work of the species. The prior report shows that the molecular marker has the advantages of stable heredity, uneasy influence of environment and materials, etc. The method is currently proved to be a valuable tool for germplasm resource analysis and identification evaluation, and has the advantages of stable heredity, difficult influence of environment and materials, and the like. Genetic diversity analysis of cassava germplasm has been performed by a learner using markers such as SRAP (Sequence-related amplified polymorphism), SSR (Simple Sequence Repeats), AFLP (Amplified Fragment Length Polymorphism), RAPD (random amplified polymorphic DNA), etc. InDel marker (insertion-deletion) refers to individual variation caused by insertion and deletion of nucleotide fragments among populations due to different individual allele point sequences, namely, comparison of homologous sequences, and then, gap is found. Compared with SNP, inDel is quicker to apply, and can be rapidly detected by utilizing an electrophoresis technology platform, so that the requirements on facilities and methods are simpler. And students compare the SSR amplification result with the SSR amplification result, and the stability and the separation effect of the SSR amplification result are superior to those of SSR. Besides, the InDel marker has the advantages of low development cost, wide distribution in genome and the like, so that the InDel marker is gradually becoming a popular research in the field of crops and is largely developed and utilized, such as capsicum, rape, tomato, upland cotton, cabbage mustard and the like. There are few research reports currently applied to cassava germplasm resource related analysis using InDel markers.
The research utilizes the whole genome resequencing result of 22 parts of cassava germplasm resources to analyze, digs and develops InDel marks suitable for electrophoresis apparatus detection, and selects 5 parts of cassava germplasm resources to carry out molecular mark validity identification. Finally, 18 pairs of InDel markers with polymorphism and clear strips are screened out to analyze the genetic diversity and population structure of 72 parts of cassava germplasm resources, and a certain foundation is laid for the subsequent development of cassava breeding work.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a primer group based on InDel marker development of a cassava whole genome and application thereof.
The first aspect of the invention provides a primer set based on InDel marker development of cassava whole genome, which comprises 18 pairs of InDel primers with nucleotide sequences shown in SEQ ID NO. 1-36.
Preferably, the primer set further comprises 2 pairs of InDel primers with nucleotide sequences shown in SEQ ID NO. 37-40.
In a second aspect of the invention there is provided a kit comprising a primer set according to the first aspect of the invention.
Preferably, the kit further comprises reagents required for performing a PCR reaction and/or electrophoresis.
In a third aspect, the invention provides the use of a primer set according to the first aspect of the invention or a kit according to the second aspect of the invention in the analysis of genetic diversity of cassava.
In a fourth aspect, the invention provides a method for analyzing genetic diversity of cassava, which is carried out by using the primer set described in the first aspect or the kit described in the second aspect.
Preferably, the analysis method comprises the steps of:
(1) Extracting genomic DNA of cassava to be analyzed;
(2) Using the DNA to be detected in the step (1) as a template, and performing PCR amplification by using the primer set according to the first aspect of the invention or the kit according to the second aspect of the invention;
(3) And carrying out genetic diversity analysis on the cassava to be analyzed according to the amplified genomic DNA polymorphism strips.
Genetic diversity analysis includes, but is not limited to, analysis of genetic parameters such as effective allele factors, gene diversity index, aroma diversity index, and the like, construction of UPMGA cluster dendrograms, and the like.
In a fifth aspect, the invention provides the use of a primer set according to the first aspect of the invention or a kit according to the second aspect of the invention for differentiating the germplasm of cassava.
Based on the result of 22 parts of cassava germplasm resources genome-wide resequencing analysis, the InDel locus is excavated to develop a molecular marker, the effectiveness of 64 pairs of primers is verified, and 20 pairs of primers have polymorphism, and the polymorphism rate is 31.25%. The genetic diversity of 72 cassava germplasm resources is revealed based on the result of InDel marker cluster analysis with optimal amplification effect of 18, the gene diversity index of each marker is between 0.21 and 0.50, and the shannon diversity index is between 0.36 and 0.70. The results of the cluster analysis showed that 72 parts of cassava material were divided into 2 clusters at a genetic similarity coefficient of 0.62. The research result lays a certain foundation for the subsequent development of cassava breeding work.
Drawings
FIGS. 1 to 18 are respectively electrophoresis charts of amplification products of 18 pairs of primers shown in Table 3in 72 parts of cassava material.
FIG. 19 is an electrophoretogram of the amplification product of primer pair IDC49 in 72 parts of cassava material.
FIG. 20 is a UPMGA cluster dendrogram of 72 cassava diversity.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1 cassava Material and field test
22 parts of genetically stable cassava material (22 parts of cassava material basic information are shown in Table 1) were selected for whole genome resequencing. 5 parts of cassava germplasm material were randomly selected for InDel marker validity identification, and 72 parts of cassava material (72 parts of cassava germplasm resource basic information are shown in Table 4) were used for cluster analysis. All the materials are planted in a cassava germplasm resource garden of the national institute of tropical crop variety resource research of the national academy of tropical agricultural sciences in 2021 and 3 months.
TABLE 1 basic information of cassava germplasm resources 22 parts
| Numbering device | Material name | Germplasm type | Source | Numbering device | Material name | Germplasm type | Source |
| C4 | SC201 | Breeding variety | China | C37 | GR024-8 | Breeding variety | China |
| C6 | Yunnan No. 7 | Local variety | China | C40 | CMR37-14-9 | Introduced strain | Thailand (Thailand) |
| C9 | Swiss W4 | Introduced strain | Swiss (Swiss) | C46 | COL739 | Introduced strain | Columbia |
| C12 | ZM8625 | Strain of strain | China | C47 | Swiss P11 | Introduced strain | Swiss (Swiss) |
| C15 | CM4040-1 | Introduced strain | Columbia | C49 | Indonesia seed | Introduced strain | Indonesia ink |
| C17 | R5 | Introduced strain | Thailand (Thailand) | C57 | Swiss Q10 | Introduced strain | Swiss (Swiss) |
| C22 | KM98-6 | Introduced strain | Vietnam (Vietnam) | C58 | COL2626 | Introduced strain | Columbia |
| C27 | Swiss H19 | Introduced strain | Swiss (Swiss) | C60 | R80 | Introduced strain | Thailand (Thailand) |
| C31 | Brazil No. 7 | Introduced strain | Brazil | C69 | Gui Hui heat No. 5 | Breeding variety | China |
| C35 | SC6 | Breeding variety | China | C71 | SC12 | Breeding variety | China |
2 Whole genome high throughput sequencing and InDel site mining
And (3) placing young leaves of the cassava material in liquid nitrogen, extracting genome total DNA by using a CTAB method, detecting the concentration, purity and integrity of the genome total DNA, and respectively using qualified samples for subsequent library construction and sequencing. The above work is done by Beijing Baimaike Biotech Co. And (3) carrying out quality evaluation on the obtained raw read, filtering to obtain clear Reads, comparing the clear Reads with a reference genome sequence by using BWA, and carrying out InDel detection and annotation based on the comparison result.
And filtering the raw Data obtained by sequencing 22 parts of cassava germplasm resources through Illumina to obtain Clean Data of 671.57G. Q20 is more than 85% between clear data of each sample, and GC content is between 36.9% and 45.04%. After comparing reads to the reference genome, the contrast ratio was between 84.93% and 98.60%. In conclusion, the sequencing quality, GC content and data amount of 22 parts of cassava germplasm resources are qualified, and the method is applicable to development of InDel marks. After alignment with the cassava reference genome sequence, 1737846 indes were detected in a total of 22 cassava germplasm resources whole genome, of which the total of indes located in the coding region was 15499.
3InDel primer design and marking effectiveness identification
Screening InDel loci identified by sequencing results, wherein the screening conditions are as follows: the number of the base numbers of the Insertion/deletion is 13bp or more and 5bp or more, and the sequencing depth is more than 10. Based on the position of the InDel locus on the reference genome (Mescunta_v7.0), 250bp each on the upstream and downstream of the InDel locus was taken and primer design was performed using Primer5.0. The design ranges of the upstream primer and the downstream primer are respectively 1-225bp and 270-501 bp, the primer length is 20-25 bp, and the annealing temperature is 47-65 ℃. Random selection was performed based on the InDel markers detected across the genome, and a total of 64 InDel sites were finally selected (Table 2).
64 pairs of InDel primers are used for marking effectiveness identification, and 5 parts of cassava germplasm DNA are randomly selected as templates for PCR amplification. PCR reaction System (20 ul): 3ul of DNA template (50-100 ng/ml), 10ul of 2X Rapid Tap Master Mix (Vazyme), 0.3ul of upstream primer (20 ng/ul), 0.3ul of downstream primer (20 ng/ul) and 6.4ul of ddH 2O. Amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 15s, annealing at 52-60℃for 15s, extension at 72℃for 15s, 32 cycles total; extension at 72℃for 5min,4℃hold. The amplified products are subjected to 3% agarose gel electrophoresis for detection, then the gel effect system is used for photographing and recording the gel running fruits, and the types of the stripes are counted. In the detection result, 62 pairs of primers amplify target bands, 2 pairs of primers do not amplify the bands, and the effective rate reaches 96.88%. The agarose gel electrophoresis detection result is counted to find that 20 pairs of primers in 64 pairs of primers have polymorphism in 5 parts of cassava, and the polymorphism rate is 31.25%.
TABLE 2 64 InDel tag information
Application of 4InDel primer in identifying specific germplasm resource
And (3) carrying out group gene analysis on 72 parts of germplasm resource materials by using 20 pairs of primer pairs with polymorphism, wherein the polymorphism strips are amplified only at 184bp and 198bp in IDC46 amplification strips, and the polymorphism strips are amplified at 184bp and 170bp in two parts of cassava materials. Based on the analysis of the band amplification results, IDC46 was found to directly identify specific cassava materials, brazil 14 (C38) and COL1395 (C70). After two specific germplasm are identified, germplasm distinction can be carried out by combining other polymorphic Indel primer electrophoresis bands, as shown in the amplification result of IDC06 in Brazil No. 14, the InDel primer only amplifies the band at 152bp, and in COL1395, the band is amplified at 152bp and 173 bp. In addition to this, IDC03, IDC06, IDC07, IDC17, IDC21, IDC28, IDC43, IDC50, IDC51, IDC53, IDC56, IDC49 can be used for two subsequent authentications of the cassava material (table 3). Therefore, the IDC46 is utilized for germplasm identification, and the statistics of the band amplification result combined with the primers can directly identify and distinguish the cassava material Brazil No. 14 from COL 1395.
If a plurality of specific InDel primers capable of identifying specific cassava materials are selected and combined, an effective tool can be provided for cassava germplasm resource identification work. The findings also fully illustrate that the InDel mark is expected to be used for constructing the cassava fingerprint and the molecular identity card.
TABLE 3 amplification results of other polymorphic Indel primers for Brazil No. 14 and COL1395
5 cassava germplasm resource genetic diversity analysis
And selecting InDel marks with clear bands and polymorphism, and carrying out PCR amplification and agarose gel electrophoresis detection by taking 72 parts of cassava germplasm resource genome DNA as a template, wherein the method is the same as the method. Finally, shooting and counting the amplification result. Finally, 18 pairs of primers with optimal amplification effect (table 2) are selected to carry out PCR amplification on 72 parts of cassava varieties. Through PCR amplification detection, the 18 pairs of primers can amplify target bands, and the types of the amplified bands of the 18 pairs of primers are counted, so that the band counting method comprises the following steps: the alleles were marked with a band "1", no band "0" and the deletions "9". After the strip matrix is generated, primers Nei and Shannon are calculated by using Popgene32, and 72 parts of cassava germplasm resources are subjected to cluster analysis by using a UPGMA algorithm in NTSYS-pc. The results are shown in tables 4-5 and FIG. 4.
As can be seen from Table 4, in 72 cassava germplasm, the effective allele factor variation range is 1.2712-1.9862, the IDC53 locus amplified alleles are the least, the IDC15 locus amplified alleles are the most, the gene diversity index variation range is 0.2134-0.4965, the aromatic diversity index variation range is 0.3698-0.6897, the effective allele factor, the gene diversity index and the aromatic diversity index show similar variation trend, the maximum value is at the IDC15 locus, and the minimum value is at the IDC53 locus.
As can be seen from Table 5 and FIG. 4, cluster analysis of 72 parts of cassava germplasm resources using 18 InDel markers with better polymorphisms revealed that at a genetic similarity coefficient of 0.62, 72 parts of cassava were divided into 2 populations. The maximum group is a 1# group, the germplasm sources and types of the maximum group are wide, local varieties and breeding varieties from China are mostly divided into 1# sub-groups, the rest 1# sub-group materials have no geographic distribution rule, and 2# sub-group materials are mainly from Columbia and China. As shown by the clustering analysis result, the minimum genetic similarity coefficient of 72 parts of cassava germplasm resource groups is 0.61, and 7 parts of cassava materials have 100% of genetic similarity coefficient, which indicates that the genetic basis of the cassava germplasm is relatively narrow and similar to the previous research result. According to the research results of scholars at home and abroad, the existing cassava has lower genetic variation level among germplasm, very narrow genetic basis and difficult satisfaction of the cassava breeding work requirement.
TABLE 4 genetic diversity of Indel primers in 72 parts of cassava germplasm
TABLE 5 basic information of 72 parts of cassava germplasm resources
| Numbering device | Material name | Germplasm type | Source | Subpopulations | Numbering device | Material name | Germplasm type | Source | Subpopulations |
| C1 | No leaf stalk | Wild variety | 1# | C37 | GR024-8 | Breeding variety | China | 1# | |
| C2 | KM94 | Introduced strain | Vietnam (Vietnam) | 1# | C38 | Brazil No. 14 | Introduced strain | Brazil | 1# |
| C3 | SC14 | Breeding variety | China | 1# | C39 | CM965-3 | Introduced strain | Columbia | 1# |
| C4 | SC201 | Breeding variety | China | 1# | C40 | CMR37-14-9 | Introduced strain | Thailand (Thailand) | 1# |
| C5 | Yunnan No. 8 | Local variety | China | 1# | C41 | Swiss T7 | Introduced strain | Swiss (Swiss) | 1# |
| C6 | Yunnan No. 7 | Local variety | China | 1# | C42 | ZM9242 | Strain of strain | China | 1# |
| C7 | SC7 | Breeding variety | China | 1# | C43 | ZM99250 | Strain of strain | China | 1# |
| C8 | Yunnan No. 2 | Local variety | China | 1# | C44 | COL629-4 | Introduced strain | Columbia | 1# |
| C9 | Swiss W4 | Introduced strain | Swiss (Swiss) | 1# | C45 | CM4031-2 | Introduced strain | Columbia | 1# |
| C10 | ZM93236 | Strain of strain | China | 1# | C46 | COL739 | Introduced strain | Columbia | 1# |
| C11 | SC205 | Breeding variety | China | 1# | C47 | Swiss P11 | Introduced strain | Swiss (Swiss) | 1# |
| C12 | ZM8625 | Strain of strain | China | 1# | C48 | Brazil No. 15 | Introduced strain | Brazil | 1# |
| C13 | Swiss R9 | Introduced strain | Swiss (Swiss) | 1# | C49 | Indonesia seed | Introduced strain | Indonesia ink | 1# |
| C14 | ZM99247 | Strain of strain | China | 1# | C50 | Swiss S8 | Introduced strain | Swiss (Swiss) | 1# |
| C15 | CM4040-1 | Introduced strain | Columbia | 1# | C51 | COL244 | Introduced strain | Columbia | 1# |
| C16 | SC8002 | Breeding variety | China | 1# | C52 | R90 | Introduced strain | Thailand (Thailand) | 1# |
| C17 | R5 | Introduced strain | Thailand (Thailand) | 1# | C53 | Indonesia fine leaf | Introduced strain | Indonesia ink | 1# |
| C18 | R72 | Introduced strain | Thailand (Thailand) | 1# | C54 | CM7595 | Introduced strain | Columbia | 1# |
| C19 | COL198 | Introduced strain | Columbia | 1# | C55 | R7 | Introduced strain | Thailand (Thailand) | 1# |
| C20 | Seed of mucilage | Introduced strain | Indonesia ink | 1# | C56 | CM1568-2 | Introduced strain | Columbia | 1# |
| C21 | R60 | Introduced strain | Thailand (Thailand) | 1# | C57 | Swiss Q10 | Introduced strain | Swiss (Swiss) | 1# |
| C22 | KM98-6 | Introduced strain | Vietnam (Vietnam) | 1# | C58 | COL2626 | Introduced strain | Columbia | 1# |
| C23 | CM3993-9 | Introduced strain | Columbia | 1# | C59 | KM98-7 | Introduced strain | Vietnam (Vietnam) | 1# |
| C24 | Swiss N13 | Introduced strain | Swiss (Swiss) | 1# | C60 | R80 | Introduced strain | Thailand (Thailand) | 1# |
| C25 | GR911 | Breeding variety | China | 1# | C61 | GR024-3 | Breeding variety | China | 1# |
| C26 | Brazil No. 14 | Introduced strain | Brazil | 1# | C62 | COL1061 | Introduced strain | Columbia | 1# |
| C27 | Swiss H19 | Introduced strain | Swiss (Swiss) | 1# | C63 | Swiss U6 | Introduced strain | Swiss (Swiss) | 1# |
| C28 | Brazil No. 9 | Introduced strain | Brazil | 1# | C64 | CM483-2 | Introduced strain | Columbia | 1# |
| C29 | Swiss B25 | Introduced strain | Swiss (Swiss) | 1# | C65 | COL777 | Introduced strain | Columbia | 1# |
| C30 | CMR36-40-9 | Introduced strain | Thailand (Thailand) | 1# | C66 | SC11 | Breeding variety | China | 2# |
| C31 | Brazil No. 7 | Introduced strain | Brazil | 1# | C67 | SC8 | Breeding variety | China | 2# |
| C32 | COL1049-1 | Introduced strain | Columbia | 1# | C68 | CM901 | Introduced strain | Columbia | 2# |
| C33 | ZM7901 | Strain of strain | China | 1# | C69 | Gui Hui heat No. 5 | Breeding variety | China | 2# |
| C34 | ZM8229 | Strain of strain | China | 1# | C70 | COL1395 | Introduced strain | Columbia | 2# |
| C35 | SC6 | Breeding variety | China | 1# | C71 | SC12 | Breeding variety | China | 2# |
| C36 | SC13 | Breeding variety | China | 1# | C72 | CM3327-4 | Introduced strain | Columbia | 2# |
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (8)
1. The primer group based on the InDel marker development of the cassava whole genome is characterized by comprising 18 pairs of InDel primers with nucleotide sequences shown in SEQ ID NO. 1-36.
2. The primer set of claim 1, further comprising 2 pairs of InDel primers having nucleotide sequences as set forth in SEQ ID nos. 37-40.
3. A kit comprising the primer set of claim 1 or 2.
4. A kit according to claim 3, further comprising reagents required for performing a PCR reaction and/or electrophoresis.
5. Use of the primer set of claim 1 or 2 or the kit of claim 3 or 4in analysis of genetic diversity of cassava.
6. A method for analyzing genetic diversity of cassava, which is characterized in that the method is carried out by using the primer set as claimed in claim 1 or 2 or the kit as claimed in claim 3 or 4.
7. The method of analysis according to claim 6, comprising the steps of:
(1) Extracting genomic DNA of cassava to be analyzed;
(2) Using the DNA to be detected in the step (1) as a template, and performing PCR amplification by using the primer set as claimed in claim 1 or 2 or the kit as claimed in claim 3 or 4;
(3) And carrying out genetic diversity analysis on the cassava to be analyzed according to the amplified genomic DNA polymorphism strips.
8. Use of a primer set according to claim 1 or 2 or a kit according to claim 3 or 4 for differentiating the germplasm of cassava.
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| CN106434971A (en) * | 2016-11-17 | 2017-02-22 | 中国热带农业科学院热带作物品种资源研究所 | PCR (polymerase chain reaction) primers, method and kit for analyzing genetic diversity in gramineous plants |
| KR101961653B1 (en) * | 2017-11-08 | 2019-03-26 | 대한민국 | SNP molecular marker for selecting cultivars of sweet potatoes and uses thereof |
| CN114622035A (en) * | 2022-04-29 | 2022-06-14 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato whole genome high-throughput specific InDel molecular marker primer group and application thereof |
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| CN106434971A (en) * | 2016-11-17 | 2017-02-22 | 中国热带农业科学院热带作物品种资源研究所 | PCR (polymerase chain reaction) primers, method and kit for analyzing genetic diversity in gramineous plants |
| KR101961653B1 (en) * | 2017-11-08 | 2019-03-26 | 대한민국 | SNP molecular marker for selecting cultivars of sweet potatoes and uses thereof |
| CN114622035A (en) * | 2022-04-29 | 2022-06-14 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato whole genome high-throughput specific InDel molecular marker primer group and application thereof |
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