CN117385085A - SNP (Single nucleotide polymorphism) marker associated with castor bean and application thereof - Google Patents
SNP (Single nucleotide polymorphism) marker associated with castor bean and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular markers, and provides an SNP marker associated with castor bean and application thereof. The SNP molecular marker related to the castor bean thorn provided by the invention is as follows: one or more of mk2189, mk5539, mk5692 and mk 5693. The invention obtains the character that the castor thorn is controlled by a pair of incomplete dominant genes by analyzing the genetic rule of the thorn character of a group of genetic groups of the castor, wherein thorns are dominant, and no thorns are recessive. Carrying out QTL positioning on the castor bean thorn character through linkage analysis of the character surface type value and the molecular marker, screening SNP molecular markers related to the castor bean thorn character in a QTL interval through gene annotation, and providing a theoretical basis for auxiliary breeding of the castor bean marker.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SNP marker associated with castor bean and application thereof.
Background
Castor (ricius communication L.) is an important industrial oil crop native to the southeast and Africa eastern parts of India, mediterranean basins. India, brazil, china, russia, thailand philippines are the most important castor-planting countries. Castor oil is rich in a particular hydroxy fatty acid and is therefore used as a feedstock for many different industrial products. The thorn trait of castor is one of the main agronomic traits. Common castor capsules are classified into thorn-type and thornless type. However, in actual field breeding, the castor capsule traits are classified into two types of comparison systems, namely thorn-free and thorn-free. For example, small amounts of thorns, medium amounts of thorns, and dense thorns are also common in thorn bearing breeding materials. At present, genetic and molecular mechanisms of the castor bean thorn character are still unclear, and research on markers and genes related to the castor bean thorn character, which are obtained based on spectrogram positioning, is not yet seen.
Disclosure of Invention
The invention aims to provide an SNP marker associated with castor bean thorn and application thereof, wherein the SNP marker is used for carrying out QTL positioning on castor bean thorn traits through linkage analysis of trait phenotype values and molecular markers based on constructed high-density SNP genetic map, and screening SNP molecular markers associated with the castor bean thorn traits in a QTL interval through gene annotation, so that theoretical basis is provided for auxiliary breeding of the castor bean markers.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SNP molecular marker associated with castor bean, which is characterized in that: one or more of mk2189, mk5539, mk5692 and mk 5693;
the scafold position of mk2189 is 28470, the physical position is 118469, the reference genome base type is C, the reference base type of the detection sample is T, and the gene is 28470.t000014;
the scafold position of mk5539 is 29010, the physical position is 74050, the reference genome base type is T, the reference base type of a detection sample is A, and the gene is 29010.t000006;
the scafold position of mk5692 is 29491, the physical position is 6385, the reference genome base type is C, the reference base type of the detection sample is G, and the gene is 29491.t000001;
the scafold position of mk5693 is 29491, the physical position is 14928, the reference genome base type is G, the reference base type of the detection sample is A, and the gene is 29491.t000004 or 29491.t000005.
The invention also provides application of the SNP molecular marker related to the castor bean thorn in identifying the castor bean thorn.
Preferably, the castor varieties are SL1 and HCH1.
The invention provides an SNP marker associated with castor bean and application thereof. The SNP molecular marker related to the castor bean thorn provided by the invention is as follows: one or more of mk2189, mk5539, mk5692 and mk 5693. The invention obtains the character that the castor thorn is controlled by a pair of incomplete dominant genes by analyzing the genetic rule of the thorn character of a group of genetic groups of the castor, wherein thorns are dominant, and no thorns are recessive. Carrying out QTL positioning on the castor bean thorn character through linkage analysis of the character surface type value and the molecular marker, screening SNP molecular markers related to the castor bean thorn character in a QTL interval through gene annotation, and providing a theoretical basis for auxiliary breeding of the castor bean marker.
Drawings
FIG. 1 shows a parent and its partial offspring single capsule wherein A is the female parent HCH1, B is the male parent SL1, C is the partial F 2 Replacing capsules;
FIG. 2 is a graph showing a linkage group Marker profile, wherein the X-axis is chromosome number, the Y-axis is genetic distance (cM), the left end of the horizontal line is Marker genetic distance, and the right end of the horizontal line is Marker name;
FIG. 3 shows the results of QTL localization for the thorn trait, wherein the ordinate represents the LOD value and the abscissa represents the linkage group number.
Detailed Description
The invention provides an SNP molecular marker associated with castor bean, which is characterized in that: one or more of mk2189, mk5539, mk5692 and mk 5693;
the scafold position of mk2189 is 28470, the physical position is 118469, the reference genome base type is C, the reference base type of the detection sample is T, and the gene is 28470.t000014;
the scafold position of mk5539 is 29010, the physical position is 74050, the reference genome base type is T, the reference base type of a detection sample is A, and the gene is 29010.t000006;
the scafold position of mk5692 is 29491, the physical position is 6385, the reference genome base type is C, the reference base type of the detection sample is G, and the gene is 29491.t000001;
the scafold position of mk5693 is 29491, the physical position is 14928, the reference genome base type is G, the reference base type of the detection sample is A, and the gene is 29491.t000004 or 29491.t000005.
The invention also provides application of the SNP molecular marker related to the castor bean thorn in identifying the castor bean thorn.
In the present invention, the varieties of castor are preferably SL1 and HCH1.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Experimental materials
In 2018, a homozygous dense thorn type amphoteric strain SL1 male parent and a thorn-free mosaic type female line (female ear mosaic type distribution of very little male flowers) HCH1 female parent (figures 1, A and B) are selected, and the quadrature F is obtained by hybridization 1 Reverse cross F 1 .2019 planting parent and orthorhombic F 1 Reverse cross F 1 . Orthogonal F 1 Self-copulation harvest F 2 Seeds (FIG. 1, C) and backcrossed with parental controls to harvest BC, respectively 1 P 1 ，BC 1 P 2 . Parent and offspring group are planted in 2020. Experimental materials are provided by the institute of industrial crops at Shanxi university, and the planting of materials of each generation is completed. The plant spacing of the material planting rows is 1m multiplied by 1m, and the test land is required to be uniform in fertility and flat in topography.
2. Investigation of the Ricinus communis thorn character
And (3) after the plant maturity period, investigating the number of the non-thorn, less thorn, medium thorn and dense thorn plants of castor capsules in each generation population, and counting the medium thorn and the less thorn as intermediate types. By usingExcel2007 performs data statistics and SPSS22.0 performs chi-square test.
3. Construction of high-Density genetic map
And carrying out polymorphic marker development among parents based on the genotype detection result of the castor parents according to the whole genome sequencing data of the two parents and 150 offspring thereof in the earlier stage. Abnormal base inspection, integrity filtration and partial separation marker filtration are carried out on the offspring markers after typing. The markers of each linkage group are sequenced by using Joinmap4.1 for each linkage group, and finally a high-density SNP marker map is obtained. Linkage maps were drawn with the perl SVG module.
4. Castor fruit thorn character QTL positioning analysis
Based on the genetic map result, 150F are combined with castor 2 And (3) carrying out QTL analysis on the thorn trait of the castor by using CIM mapping method of MapQTL and WinQTL software.
5. Screening for molecular markers and candidate genes
The castor genome database (Ricinus communis V0.1.0.1) was downloaded from the PYTOZOME V13 database (http:// phytozome. Jgi. Doe. Gov/pz /), and the relevant molecular markers and candidate genes within the QTL localization interval were screened by gene annotation information.
6. Genetic analysis
By forward and backward crossing F to parents 1 The investigation of the thorn trait of (2) shows that whether the number of thorns of the orthogonal or backcross progeny is between two parents, belongs to the intermediate type, and indicates that the trait is controlled by a nuclear gene, and the allele is incompletely dominant. F (F) 2 The progressive significance of the generation separation ratio is greater than 0.05, which indicates F 2 Generation without thorn: middle thorn: the dense thorn separation ratio is not significantly different from the theoretical value of 1:2:1, and accords with the separation of a pair of gene allele controlsSeparation ratio, and has thorns: no thorn is 3:1, the thorns are dominant, and the no thorns are recessive. This conclusion was verified in the backcross population at the same time. Thorn orthogonal F 1 The filial generation of the female parent is thorn-shaped, and the filial generation of the female parent is thorn-free and has a separation ratio of 1:1. Investigation and chi-square test analysis of different genetic generation fruit thorn traits (table 1) led to the result that the fruit thorn traits are controlled by a pair of incompletely dominant alleles, with thorns as dominant and no thorns as recessive.
Table 1 Each generation of separation ratio and chi-square test
Note that a.0 units (.0%) have a desired frequency of less than 5.
7. High density genetic map
By comparison of sequencing data and population SNP marker development, the markers of each linkage group were ranked using Joinmap4.1 for each linkage group (linkage groups were ranked using a maximum likelihood algorithm, and the genetic distances were calculated by the Kosambi function), giving a total of 10 linkage groups (Table 2, FIG. 2), a total genetic distance of 3355.03cM, a total number of upper markers of 5713, and an average genetic distance between markers of 0.59cM.
TABLE 2 genetic linkage group information statistics
Qtl positioning results
The LOD value threshold for the phenotype was determined using PT (persistence test 1000 times) in MapQTL. Selecting LOD threshold value of the experiment, selecting LOD value corresponding to each linkage group at 95% confidence (namely selecting LOD of the second column corresponding to the sixth column at 0.95 as LOD threshold value of the linkage group; selecting value which is larger than 0.95 and is closest to 0.95 when the sixth column is not 0.95); at the genome-wide level, LOD at 95% confidence Interval (i.e., the value of the Interval column) is selected as the phenotypic LOD threshold; and finally determining the LOD threshold value of the castor capsule thorn character positioning to be 3.9. The threshold results of the fruit thorn trait phenotype LOD values are shown in Table 3.
And carrying out QTL quantitative analysis on the thorn traits by using a CIM (common information model) mapping method of WinQTL software, and selecting a QTL section with an LOD value of not less than 3.9. Finally, a major QTL cluster is obtained in linkage group 9 (as shown in figure 3). The QTL interval 138.31cM-164.91cM, the genetic distance 26.60cM (shown in Table 4), explains that the phenotype variation rate is 5% -90%.
TABLE 3 QTL replacement test results
| Group | Interval | Count | Cum.count | Rel.count | Rel.cum.count |
| 1 | 2.8 | 6 | 952 | 0.006 | 0.952 |
| 2 | 2.7 | 10 | 953 | 0.01 | 0.953 |
| 3 | 2.8 | 10 | 951 | 0.01 | 0.951 |
| 4 | 2.8 | 5 | 952 | 0.005 | 0.952 |
| 5 | 2.7 | 7 | 951 | 0.007 | 0.951 |
| 6 | 2.1 | 12 | 953 | 0.012 | 0.953 |
| 7 | 2.7 | 4 | 953 | 0.004 | 0.953 |
| 8 | 2.2 | 17 | 957 | 0.017 | 0.957 |
| 9 | 2.6 | 5 | 952 | 0.005 | 0.952 |
| 10 | 2.3 | 11 | 957 | 0.011 | 0.957 |
| GW | 3.9 | 10 | 956 | 0.01 | 0.956 |
Note that: group, linkage Group number; GW represents the entire genome; interval, LOD Interval; count, substitution test 1000 times, the number of times that the LOD interval value appears; cum count, substitution check 1000 times, < = LOD occurrences; count, count/permutation test total number of times (e.g., 1000); totally, count/permutation test total number.
TABLE 4 QTL analysis results
Note that: trait; CT fruit thorn english Capsule chord abbreviation; chromasomome linkage group numbering; positon/cM genetic distance, in cM; LOD value of the QTL; additive effects; domino Dominant effect RIL or DH population Dominant effect value is 0; r is R 2 The proportion of QTL interpreted phenotypic variation (fractional representation, if 0.63, indicates an interpreted phenotypic variation rate of 63%); confidence intervals of "lod1_l" and "lod1_r" 99%; confidence intervals of "lod2_l" and "lod2_r" 95%.
9. Closely related SNP markers and candidate genes
17 SNP markers in the QTL interval 138.31cM-164.91cM are subjected to gene annotation analysis, and 4 SNP markers associated with the castor bean thorn trait are obtained through screening (table 5), wherein 2 variants occur in the coding region of the gene and 2 variants occur in 1Kb upstream and downstream of the gene. The coding region of a gene and a marker mutated within 1Kb upstream and downstream are generally considered as molecular markers that may be associated with a trait. According to the annotation information of the reference genes, candidate genes corresponding to mk2189, mk5539 and mk5692 markers all encode conserved putative proteins, and the functions of the genes are unknown. The upstream gene of mk5693 encodes a protein containing a pentapeptide repeat, and the downstream gene of mk5693 encodes a serine/threonine protein kinase. These 5 genes are presumed to be 28470.t000014, 29010.t000006, 29491.t000001, 29491.t000004, 29491.t000005. Is a gene related to the character of the castor bean.
TABLE 5 positional information of SNP markers in coding regions of localization intervals
The genetic breeding research of castor is weak, long time is needed and accuracy is lacking when selecting plants with ideal characters, particularly polygenic control characters, and the improvement process of castor varieties is slow. Molecular marker assisted breeding (MAS) methods can effectively solve these problems. Genetic maps and Quantitative Trait Loci (QTLs) are important tools for molecular marker assisted breeding (MAS).
Researchers consider that thorny varieties are prone to burst. Research results of Anjanik in India show that the castor capsule has thorns as dominant and no thorns as recessive; the Philippine Aranez study indicated that the incompletely dominant gene controlled capsule piercing, consistent with the results of this study. In 2014 Huang Feng orchid, a RAPD molecular marker technology is used for screening differential fragments of thorn-containing and thorn-free materials to obtain a 112bp DNA sequence possibly related to the thorn character of castor bean. The marker density and the resolution of the high-density SNP marker genetic map are far higher than those of the traditional SSR, ISSR, RAPD markers. At present, no research on markers and genes related to the castor bean thorn character obtained based on spectrogram positioning is seen. SNP mutation sites and corresponding genes obtained by the research result are all newly discovered.
The research obtains the character that the castor thorn is controlled by a pair of incomplete dominant genes through genetic rule analysis of the thorn character of a group of genetic groups of the castor in more than one generation. The castor fruit thorn character is positioned in the No. 9 linkage group based on the constructed high-density genetic map for the first time, and the QTL interval is 138.31cM-164.91cM (genetic distance is 26.60 cM). And 4 SNP markers mk2189, mk5539, mk5692 and mk5693 closely related to the castor bean thorn trait were selected, and 5 candidate genes 28470.t000014, 29010.t000006, 29491.t000001, 29491.t000004 and 29491.t000005 related to the castor bean thorn shape were selected.
From the above examples, the present invention provides a SNP marker associated with Ricinus communis and its application. The SNP molecular marker related to the castor bean thorn provided by the invention is as follows: one or more of mk2189, mk5539, mk5692 and mk 5693. The invention obtains the character that the castor thorn is controlled by a pair of incomplete dominant genes by analyzing the genetic rule of the thorn character of a group of genetic groups of the castor, wherein thorns are dominant, and no thorns are recessive. Carrying out QTL positioning on the castor bean thorn character through linkage analysis of the character surface type value and the molecular marker, screening SNP molecular markers related to the castor bean thorn character in a QTL interval through gene annotation, and providing a theoretical basis for auxiliary breeding of the castor bean marker.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. A SNP molecular marker associated with castor bean, wherein the SNP molecular marker is: one or more of mk2189, mk5539, mk5692 and mk 5693;
the scafold position of mk2189 is 28470, the physical position is 118469, the reference genome base type is C, the reference base type of the detection sample is T, and the gene is 28470.t000014;
the scafold position of mk5539 is 29010, the physical position is 74050, the reference genome base type is T, the reference base type of a detection sample is A, and the gene is 29010.t000006;
the scafold position of mk5692 is 29491, the physical position is 6385, the reference genome base type is C, the reference base type of the detection sample is G, and the gene is 29491.t000001;
the scafold position of mk5693 is 29491, the physical position is 14928, the reference genome base type is G, the reference base type of the detection sample is A, and the gene is 29491.t000004 or 29491.t000005.
2. Use of the SNP molecular marker associated with ricinus communis of claim 1 for identifying ricinus communis.
3. The use according to claim 2, characterized in that the varieties of castor are SL1 and HCH1.
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| CN113430299A (en) * | 2021-08-13 | 2021-09-24 | 山西农业大学经济作物研究所 | SNP molecular marker associated with castor stem color and application thereof |
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| 黄凤兰;赵永;彭木;张智勇;陈晓凤;包春光;邹千稳;吴春桃;: "与蓖麻果刺性状连锁的RAPD标记", 华北农学报, no. 01, 28 February 2014 (2014-02-28), pages 83 - 88 * |
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