CN113718038A - Molecular marker assisted breeding primer group for fatty acid content of echinus intermedius and application - Google Patents

Molecular marker assisted breeding primer group for fatty acid content of echinus intermedius and application Download PDF

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CN113718038A
CN113718038A CN202110846104.4A CN202110846104A CN113718038A CN 113718038 A CN113718038 A CN 113718038A CN 202110846104 A CN202110846104 A CN 202110846104A CN 113718038 A CN113718038 A CN 113718038A
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fatty acid
acid content
intermedius
echinus
molecular marker
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CN113718038B (en
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丁君
常亚青
王姮
左然涛
赵文飞
刘晓雨
张伟杰
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Dalian Ocean University
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Abstract

The invention relates to a molecular marker assisted breeding primer group for fatty acid content of echinus intermedia and application thereof, belongs to the field of molecular breeding, and provides a combination of 4 primer pairs, wherein echinus which simultaneously satisfies A007359 marker genotype as CC, A012370 marker genotype as CC, A012381 marker genotype as TT and A012386 marker genotype AA is selected as a parent, so that individuals with high fatty acid content in gonads can be bred.

Description

Molecular marker assisted breeding primer group for fatty acid content of echinus intermedius and application
Technical Field
The invention belongs to the field of molecular breeding, and particularly relates to a molecular marker assisted breeding primer group for the fatty acid content of echinus intermedius and an application method thereof.
Background
The Strongylocentrotus intermedius is the most important mariculture species in China, and the main nutrition of the Strongylocentrotus intermedius is that gonads (commonly called echinus yellow) of the Strongylocentrotus intermedius are rich in highly unsaturated fatty acid. The characteristic polyunsaturated fatty acid eicosapentaenoic acid (EPA) in the gonad is polyunsaturated fatty acid which is necessary for human body, and can regulate blood fat, reduce blood viscosity, prevent formation and development of atherosclerosis, and prevent cardiovascular diseases such as cerebral thrombosis, cerebral hemorrhage, hypertension, etc. Arachidonic acid (AA or ARA) is all cis-5, 8,11, 14-eicosatetraenoic acid, a direct precursor of prostaglandin E2(PGE2), prostacyclin (PGI2), thromboxane a2(TXA2), and leukotrienes and C4(LTC 4). These bioactive substances have important regulating effects on lipid protein metabolism, hemorheology, vascular elasticity, leukocyte function, platelet activation, etc., and are also important substances in brain development and optic nerve development.
How to improve the content of highly unsaturated fatty acid in the gonad of sea urchin, especially the content of characteristic highly unsaturated fatty acid, is an important subject to be paid attention to in the genetic breeding work of sea urchin, because the gonad character of sea urchin belongs to complicated quantitative characters, the heritage parameter is difficult to estimate accurately, it is difficult to develop traditional selective breeding, and molecular marker assisted breeding utilizes the characteristic that the molecular marker is closely linked with the gene determining the target character, and the existence of the target gene can be detected by detecting the molecular marker, thereby achieving the purpose of selecting the target character, and having the advantages of rapidness, accuracy and no interference of environmental conditions. Is widely applied to a plurality of fields of animal genetic breeding, resource protection and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an SNP marker related to fatty acid of echinacea and a development method thereof based on a KASP typing technology, and the SNP marker is suitable for auxiliary seed selection of the fatty acid content of echinacea.
The invention is realized by the following technical scheme:
a primer group for molecular marker assisted breeding of fatty acids of Strongylocentrotus intermedius, which is shown in Table 1:
TABLE 1 SNP-based molecular marker assisted breeding primers for gonadal fatty acid content of Strongylocentrotus intermedius
Figure BDA0003180619210000021
The invention also provides a breeding method by using the primer group, which selects sea urchins which simultaneously meet the requirements that the A007359 marker genotype is CC, the A012370 marker genotype is CC, the A012381 marker genotype is TT and the A012386 marker genotype AA as parents to carry out seedling cultivation.
Compared with the prior art, the invention has the beneficial effects that:
the primer of the invention can identify individuals with high fatty acid content in gonad of the sea urchin Strongylocentrotus intermedius as breeding parents, thereby breeding individuals with high fatty acid content in gonad.
Detailed Description
The following non-limiting examples will allow those of ordinary skill in the art to more fully understand the present invention, but in any way limit the invention.
Example 1
1. Screening of a related SLAF label of gonadal fatty acid shape of the intermediate strongylocentrotus intermedius and design of a KASP primer.
The invention utilizes the SLAF-seq technology to prepare the high-density genetic linkage map of the Strongylocentrotus intermedius, adopts an Interval Mapping (IM) algorithm of MapQTL software and a Composite Interval Mapping (CIM) algorithm of RQTL software to carry out QTL positioning on the fatty acid character of the Strongylocentrotus intermedius (when the significance threshold value exceeds a critical value (LOD), a QTL locus is considered to exist, and when the LOD is set to be more than 2.5 during actual screening, a QTL locus exists), and screens out the SLAF label 287 strips related to the fatty acid.
Eliminating (1) copy number > 2; (2) too many repetitive sequences near the SNP site; (3) too high of a GC content; (4) there were other fragments that did not meet the primer design rules, and the SLAF tag 92 strips that met the design rules were obtained and the corresponding KASP tags were designed.
2. Kasp marker typing related to gonadal fatty acid status of sea urchin medicina intermedia
160 healthy intermediate strongylocentrotus intermedius (taken from the sea area of Longwangtang pond in Dalian city) with mature gonads are selected for KASP marker typing related to gonad fatty acid characters, and the content of fatty acids in the strongylocentrotus intermedius is detected by gas chromatography to obtain the total fatty acid content of the gonads, the content of polyunsaturated fatty acids and the content of AA + EPA. The result showed that the total amount of gonadal fatty acids in sea urchin was 131613.81mg/kg at the highest, 38990.66mg/kg at the lowest, and 99314.74mg/kg on average. The highest total amount of highly unsaturated fatty acids is 68479.32mg/kg, the lowest value is 15394.45mg/kg, and the average value is 43056.38 mg/kg. The maximum AA + EPA content was 20001.81mg/kg, the minimum 1349.84mg/kg, and the average was 9986.65 mg/kg.
Extracting the tube foot DNA of the sea urchin according to the operation procedure of the marine animal genome DNA extraction kit, taking DNA Maker with 5000bp as a reference substance, detecting by using 1% agarose gel electrophoresis, and using the DNA with a single and clear main band for subsequent KASP typing.
And (3) carrying out gradient PCR amplification at 61-55 ℃ on an LGC SNpline platform by using the extracted sea urchin DNA as a template and using a designed KASP primer, and after the PCR reaction program is finished, respectively placing the sample on an Omega fluorescent signal reader and Araya for fluorescent signal conversion and carrying out numerical analysis. Genotyping was performed using analysis software Kraken TM provided by LGC, where X: X represents homozygous type including A: A, C: C, -: minus three types; x and Y represent heterozygote, and the heterozygote comprises three types A, G, C, T, A; -represents no signal or a weak signal; uncallable represents a signal but no explicit typing; finally, successfully typing to obtain 25 KASP markers related to the gonadal traits of the intermediate strongylocentrotus intermedius.
Combining 160 pieces of intermediate strongylocentrotus intermedius gonadal fatty acid shape data (total unsaturated fatty acid amount, total fatty acid amount, EPA + AA), utilizing a linear model to perform relevance typing of markers and phenotype data, establishing the linear model by using a significant relevance marker with P <0.05 and the phenotype data for each character, then optimizing the linear model by using stepwiseAIC to obtain molecular markers (P <0.1) significantly related to fatty acid, and obtaining markers related to the fatty acid shape of the strongylocentrotus intermedius as shown in Table 4 (comprising 9 markers in total). Finally, 9 sets of KASP primer sequences related to the fatty acids (total unsaturated fatty acids, total fatty acids, EPA + AA) of Strongylocentrotus intermedius were selected, and the results are shown in Table 3.
TABLE 3 correlation analysis results of SNP sites and fatty acid traits
Figure BDA0003180619210000051
Note: let P <0.1 be associated significance
3. Mark verification
Taking 30 urchins of the urchin Strongylocentrotus intermedius cultured in a laboratory, taking the tubular feet of the urchin Strongylocentrotus intermedius, extracting DNA, simultaneously measuring the total fatty acid amount, the total unsaturated fatty acid amount and the EPA + AA content of the urchin Strongylocentrotus by using a gas chromatography, analyzing the gonadal fatty acid characters of the urchin Strongylocentrotus of different genotypes, and showing the results in a table 4, wherein the trend is consistent with the correlation results. The differences among the markers A007359, A012370, A012381 and A012386 are the most significant, and finally the 3 KASP markers are used as molecular markers of the gonadal fatty acid content of the intermediate strongylocentrotus intermedius.
TABLE 4 Total fatty acid content of Strongylocentrotus intermedius, Total polyunsaturated fatty acid content and average AA + EPA content correlation with markers (mg/kg)
Figure BDA0003180619210000052
Figure BDA0003180619210000061
4. Practical application case
Taking tube feet of 100 sea urchins reserved with parents according to the steps, extracting DNA, selecting A007359, A012370, A012381 and A012386KASP primers for amplification, reserving 8 sea urchins which simultaneously meet the marking genotype of A007359 as CC, marking genotype of A012370 as CC, marking genotype of A012381 as TT and marking genotype AA of A012386 as parents (3 male and 5 female), and performing population fertilization breeding; randomly selecting 8 sea urchins (3 male and 5 female) from the rest individuals for group fertilization propagation (as a control group), feeding under the same environment and bait conditions, detecting the gonad fatty acid state after 1.5 years, and respectively increasing the total fatty acid content, the polyunsaturated fatty acid content and the AA + EPA content by 12%, 18% and 23% compared with the control group, wherein the marker assisted selection effect is better.
Sequence listing
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Claims (2)

1. A primer group for molecular marker assisted breeding of fatty acid content of echinus intermedia, which is characterized in that the primer group is shown in Table 1:
TABLE 1 SNP-based molecular marker assisted breeding primer set for gonadal fatty acid content of Strongylocentrotus intermedius
Figure FDA0003180619200000011
2. A method of breeding by using the primer set of claim 1, wherein sea urchins satisfying the A007359 marker genotype CC, the A012370 marker genotype CC, the A012381 marker genotype TT and the A012386 marker genotype AA at the same time are selected as parents to carry out the seedling cultivation.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073074A2 (en) * 2000-03-24 2001-10-04 Millennium Pharmaceuticals, Inc. 18806, a novel trypsin serine protease-like molecule and uses thereof
CN105925698A (en) * 2016-06-03 2016-09-07 大连海洋大学 SNP primers for early screening strongylocentrotus intermedius fine variety and screening method
CN108660225A (en) * 2018-07-18 2018-10-16 大连海洋大学 Molecular mark primer and screening technique for Strongylocentrotus intermedius growth traits

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073074A2 (en) * 2000-03-24 2001-10-04 Millennium Pharmaceuticals, Inc. 18806, a novel trypsin serine protease-like molecule and uses thereof
CN105925698A (en) * 2016-06-03 2016-09-07 大连海洋大学 SNP primers for early screening strongylocentrotus intermedius fine variety and screening method
CN108660225A (en) * 2018-07-18 2018-10-16 大连海洋大学 Molecular mark primer and screening technique for Strongylocentrotus intermedius growth traits

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YAQING CHANG ET AL.: ""SLAF-based high-density genetic map construction and QTL mapping for major economic traits in sea urchin Strongylocentrotus intermedius"", 《SCIENTIFIC REPORTS》 *
赵文飞等: ""中间球海胆脂肪酸含量的QTL 定位及基因注释"", 《2018 年中国水产学会学术年会论文摘要集》 *

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