AU2021104355A4 - Molecular Marker-Assisted Breeding Primer for Growth Trait of Strongylocentrotus Intermedius and Screening Method - Google Patents
Molecular Marker-Assisted Breeding Primer for Growth Trait of Strongylocentrotus Intermedius and Screening Method Download PDFInfo
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Abstract
The present disclosure provides a molecular marker-assisted breeding primer for growth traits of
Strongylocentrotus intermedius and a screening method, and relates to the field of molecular
biology. In the present disclosure, a related single nucleotide polymorphism (SNP) marker of
growth traits of S. intermedius is mainly obtained by specific-locus amplified fragment sequencing
(SLAF-seq) technology and Kompetitive allele-specific PCR (KASP) technology, and the SNP
marker may be appropriate for early assisted selection of S. intermedius. In the present disclosure,
the SLAF-seq technology may enable HiSeq deep sequencing to guarantee accurate typing, reduce
sequencing costs through a simplified strategy, and guarantee an optimal number of markers
through a prediction of a previously simplified solution; the KASP technology may simplify the
operation time and steps, and reduce the cost.
Description
[0001] The present disclosure relates to the field of molecular biology, and in particular to a molecular marker-assisted breeding primer for growth traits of Strongylocentrotus intermedius and a screening method.
[0002] At present, S. intermedius is mostly bred by conventional breeding methods such as cross breeding and selective breeding. Both cross breeding and selective breeding select parents based on sea urchins with excellent growth traits. In these methods, it is necessary to measure the growth traits (shell height, shell diameter, and body weight) of the sea urchins regularly during the growth thereof, and these growth traits are used as criteria for stepwise screening. These selection modes not only take substantial manpower and material resources, but also lead to low output of progeny larvae with poor genotypes because genes of parents are not excellent. By contrast, molecular marker-assisted breeding can improve the efficiency of the selection process and reduce the impact of undesirable genes on selection.
[0003] CHANG Yaqing used HRM technology to screen genotypes at specific SNP loci for molecular assisted breeding in the patent "SNP Primer and Screening Method for Early Screening of Fine Breed of Strongylocentrotus Intermedius". However, this method only involves the detection of one single nucleotide polymorphism (SNP) locus associated with growth traits, where the loci for detection regarding desirable genes for controlling quantitative traits are few, thus having certain limitations in use.
[0004] To solve the problems in the prior art, the present disclosure provides a SNP marker related to growth traits of S. intermedius and a development method thereof based on a KASP technology, and the SNP marker may be appropriate for early assisted larval selection of S. intermedius.
[0005] The aim of the present disclosure is achieved by the following technical solutions:
[0006] The present disclosure provides a molecular marker-assisted breeding primer for growth traits of S. intermedius, wherein the primer is selected from the following KASP primer sets:
(1) SNP named Marker49152, marker named A007402, having the corresponding primer sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3;
(2) SNP named Marker112898, marker named A007399, having the corresponding primer sequences shown as SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6;
(3) SNP named Markerl40758, marker named A007397, having the corresponding primer sequences shown as SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9;
(4) SNP named Marker63934, marker named A007389, having the corresponding primer sequences shown as SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12;
(5) SNP named Marker203513, marker named A007388, having the corresponding primer sequences shown as SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15;
(6) SNP named Marker24433, marker named A007376, having the corresponding primer sequences shown as SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18;
(7) SNP named Marker72763, marker named A007374, having the corresponding primer sequences shown as SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21;
(8) SNP named Marker23360, marker named A007366, having the corresponding primer sequences shown as SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24;
(9) SNP named Markerl3442, marker named A007362, having the corresponding primer sequences shown as SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27;
(10) SNP named Marker6879, marker named A007358, having the corresponding primer sequences shown as SEQ ID NO. 28, SEQ ID NO. 29, and SEQ ID NO. 30.
[0007] The present disclosure further provides a molecular marker-assisted breeding and screening method for growth traits of S. intermedius using the above primer, including the following steps: (1) screening growth trait-related SLAF tags from an established genetic map of S. intermedius according to QTL mapping results, and selecting SLAF tags for designing the KASP primers from the growth trait-related SLAF tags. In the present disclosure, specific locus amplified fragment sequencing (SLAF-seq) technology is used for gene sequencing of S. intermedius; substantial SLAF tags are obtained in sequencing, and the SLAF tags can be used to construct a genetic map; the genetic map is used for correlation analysis with phenotypic traits (shell height, shell diameter, and body weight) of S. intermedius; quantitative trait loci (QTL) are mapped, and growth trait (shell height, shell diameter, and body weight)-related SLAF tags can be deduced reversely by screening QTL of target traits (shell height, shell diameter, and body weight). Because SLAF tag sequences are obtained by simplified genome sequencing and 2-3 tag sequences are obtained after cluster analysis, the SLAF tag sequences can directly display base types of SNP loci;
(2) finding SNP loci for designing the KASP primers from the SLAF tags for designing the KASP primers, i.e., polymorphic SNP markers, and using Allele Specific (forward) and reverse primers to conduct PCR amplification; typing resulting PCR products by FAM and VIC fluorescence scanning, and verifying designed KASP primers according to the fluorescence typing results. At the same time, detection results of the KASP markers in a verification population will give a specific base type of each marker in each individual in the form of text. When a genotype at a SNP locus can be divided into 2-3 genotypes, it is indicated that the designed KASP primer can perform SNP genotyping;
(3) conducting genotyping verification on DNAs of the verification population in the LCG SNP-line genotyping platform using the KASP primer sets to finally obtain a KASP primer set for accurately typing candidate SNP loci, that is, growth trait-related KASP markers;
(4) according to the phenotypic data of growth traits of the verification population (shell height, shell diameter, and body weight) and the typing results of growth trait-related KASP markers, using a linear model for each growth trait (shell height, shell diameter, or body weight) to analyze the correlation between each marker and the phenotypic data separately; using significantly correlated markers with P<0.05 and the phenotypic data for each growth trait to establish a linear model, and optimizing the linear model using stepwise AIC to obtain molecular markers significantly correlated with growth traits (P<0.1) and finally prediction models for each growth trait. After prediction models for each growth trait are obtained, compressing each predicted value of each growth trait to an interval of 0-1 to facilitate the calculation of a comprehensive predictive value of individual traits. According to the weight of each trait, finally establishing a molecular marker-based growth prediction model of sea urchins and a comprehensive predictive value formula for sea urchin traits.
[0008] According to the above technical solution, preferably, the growth traits may include shell height, shell diameter, and body weight. The body weights of the present disclosure may be live weight.
[0009] According to the above technical solution, preferably, a screening criterion for the growth trait-related SLAF tags in step (1) may be as follows: when a significance threshold exceeds a critical value (LOD = 2-3), a quantitative trait loci (QTL) may be considered to exist.
[00010] According to the above technical solution, preferably, selection criteria for the SLAF tags for designing the KASP primers in step (1) may be as follows: (1 absence of insertion/deletion (InDel) polymorphism loci in the SLAF tags; only one SNP locus in a primer sequence; and @ controlling annealing temperature within a temperature range required by PCR according to base content, wherein the annealing temperature may be 55-65°C.
[00011] According to the above technical solution, preferably, the PCR amplification in step 2 may be conducted at 55-65°C, and the PCR product may have a length less than 200 bp.
[00012] According to the above technical solution, preferably, the criterion for obtaining the growth trait-related KASP marker in step (3) may be as follows: the marker may be considered available when 2-3 genotypes of an SNP locus are divided.
[00013] The embodiments may have the following beneficial effects:
[00014] In the present disclosure, the growth traits of sea urchin were marked by the KASP markers, and 10 SNP loci related to growth traits (shell height, shell diameter, and body weight) were obtained by the KASP technology. Multi-locus marker screening may be more accurate than single-locus screening. In HRM technology, when the small fragment primer method is used in the genotyping of G/C (class III mutation) or A/T mutation (class IV mutation), because of insufficient difference in Tm value, usually, it is not easy to separate wild type homozygotes from mutant homozygotes. Moreover, the small fragment primer method may lead to a chaotic melting curve due to the difference in PCR product yield caused by the inhomogeneity of a DNA template, seriously influencing the accuracy of genotyping. The KASP tags have a lower requirement for the DNA template, and accurate genotyping may be conducted as long as the template falls within the required range. In addition, the KASP markers may have lower detection costs than those of HRM.
[00015] The present disclosure mainly adopts SLAF-seq technology and KASP technology. The SLAF-seq technology is a set of simplified genome sequencing technology. By designing a proper restriction enzyme digestion solution, double-end 2 x 100 bp effective genome read length is used to select specific fragments that are uniformly distributed throughout the genome and avoid repeat domains for high-throughput sequencing, and the most complete variation images (SNPs and InDels) in the whole genome can be obtained. The SLAF seq technology has the following advantages: HiSeq deep sequencing may ensure accurate typing, a simplified strategy reduces sequencing costs, and a prediction of a previously simplified solution guarantees an optimal number of markers.
[00016] The KASP is the latest means of genetic analysis. The KASP uses the specific matching of bases at the end of the primers to genotype the SNPs and determine alleles thereof. The KASP may be characteristic of low marker development cost, high conversion rate, low demand for DNA samples, accurate typing, high throughput, high detection sensitivity, high specificity, excellent reproducibility, easy operation, and wide application range; compared with conventional SNP mutation analysis method and quantitative probe method, the KASP may simplify the operation time and steps and reduce the cost.
[00017] The following non-limiting example is provided to enable a person of ordinary skill in the art to understand the present disclosure more fully, but not to limit the present disclosure in any way.
[00018] In the following example, the SLAF-seq technology was provided by Beijing Biomarker Technologies Corporation, and the KASP technology was provided by China Golden Marker (Beijing) Biotech Co., Ltd.
Example
1. Screening of SLAF tags of growth traits
[00019] In the present disclosure, SLAF-seq technology was used for gene sequencing of S. intermedius; substantial SLAF tags were obtained in sequencing, and the SLAF tags could be used to construct a high-density genetic map; the genetic map was used for correlation analysis with phenotypic traits (shell height, shell diameter, and body weight) of S. intermedius; quantitative trait loci (QTL) were mapped, and growth trait (shell height, shell diameter, and body weight)-related SLAF tags could be deduced reversely by screening QTL of target traits (shell height, shell diameter, and body weight). Herein, the genetic map was developed and constructed by using the SLAF-seq technology and HighMap software, and the QTL were mapped by using the interval mapping (IM) algorithm in MapQTL and the composite interval mapping (CIM) algorithm in RQTL software. Specifically, the method for constructing the genetic map of S. intermedius and mapping the QTL can refer to the disclosure of the Chinese Patent CN 105925698A. Based on the QTL mapping results, 61 growth trait-related SLAF tags were screened from the established high-density genetic map of S. intermedius (when a significance threshold exceeds a critical value (LOD), a QTL may be considered to exist; when LOD>2.5 was set during the actual screening, there was a QTL), and 51 SLAF tags for designing the KASP primers were selected therefrom (selection criteria are as follows: 1: absence of InDel polymorphism loci in the SLAF tags; 2, only one SNP locus included in a primer sequence; and 3, controlling annealing temperature within a temperature range required by PCR according to base content, where the annealing temperature may be 55-65°C). Because SLAF tag sequences were obtained by simplified genome sequencing and 2-3 tag sequences were obtained after cluster analysis, the SLAF tag sequences could directly display base types of SNP loci.
2. Measurement of phenotypic values of growth traits and DNA extraction
[00020] For 201 individuals in the verification population, the shell height and shell diameter were measured by an electronic digital display vernier caliper (with an accuracy of 0.01 mm); the body weight was measured by an electronic balance (with an accuracy of 0.01 g). The tube foot DNA of sea urchin was extracted using a marine animal genomic DNA kit. The S. intermedius in the verification population was selected from F6 generation S. intermedius selected by the Key Laboratory of Mariculture and Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University.
3. Screening of polymorphic SNP markers, design of KASP primers, SNP genotyping and acquisition of KASP markers
[00021] Fifty-one SNP loci for designing KASP primers could be found from 51 finally screened SLAF tags. The KASP reaction system was composed of three parts: (1) a KASP primer set; (2) fluorescence sequences; and (3) a DNA template. Allele Specific (forward) and reverse primers were designed near the SNP loci using Premier 5.0 software (Tm values were controlled at 55-65°C, and PCR products had a length of <200 bp); the reverse primer was a reverse universal primer, and the Allele Specific primer included two forward primer sequences. In the LGCSNPline platform, when gradient PCR amplification was conducted at 65-55°C, terminal bases of forward primer sequences could specifically match with alleles at the SNP loci; moreover, during PCR, the fluorescence sequences were added to the PCR products, and two different fluorescence sequences were added to different PCR products amplified by forward primers; different types of bases at the SNP loci finally produced different fluorescence due to different fluorescence sequences. Subsequently, the PCR products were typed on a microplate reader by FAM and VIC fluorescence scanning, and the designed KASP primers were verified according to the typing results. The base type of each marker would be given in the output result of marker detection of each individual, which could accurately detect the base type for typing. At the same time, detection results of the KASP markers in the verification population would give a specific base type of each marker in each individual in the form of text. When a genotype at a SNP locus could be divided into 2-3 genotypes, it was indicated that the designed KASP primer could perform SNP genotyping.
[00022] The KASP primer set was used to verify the DNAs of the verification population (201 sea urchin individuals) in the LCGSNPline typing platform through two genotypings, and 23 KASP primer sets for accurately typing candidate SNP loci (the marker was considered available when the KASP markers could divide genotypes at an SNP locus into 2-3 types) were finally obtained, that is, 23 growth trait-related KASP markers associated were obtained.
4. Molecular marker-based growth trait prediction model of S. intermedius
[00023] Combined with the phenotypic data (shell height, shell diameter, and body weight) of 201 verification populations and the typing results of KASP markers, a linear model was used for each trait (shell height, shell diameter, or body weight) to analyze the correlation between each marker and the phenotypic data separately; significantly correlated markers with P<0.05 and the phenotypic data were used for each trait to establish a linear model, and the linear model was optimized by using stepwiseAIC to obtain molecular markers significantly correlated with growth traits (P<0.1) and finally prediction models for each trait. After prediction models for each trait were obtained, each predicted value of each growth trait was compressed to an interval of 0-1 to facilitate the calculation of a comprehensive predictive value of individual traits. According to the weight of each trait (shell height * 0.3, shell diameter * 0.3, and body weight * 0.4), a molecular marker-based growth prediction model of sea urchins and a comprehensive predictive value formula of sea urchin traits were finally established. (NOTE: The analysis software used in method 5 was from R Programming Language provided by China Golden Marker (Beijing) Biotech Co., Ltd.)
[00024] The markers included in each trait prediction model are shown in Table 1
(including a total of 10 markers), and correlations between each marker and traits are shown in Table 2. Finally, 10 sets of KASP primer sequences related to the shell height, shell diameter, and body weight of S. intermedius were selected. The results are shown in Table 3.
[00025] Calculation formula: Comprehensive predictive value of individual traits = (shell height score x 0.3) + (shell diameter score x 0.3) + (body weight score x 0.4).
[00026] Table 1. Results of correlation analysis between SNP loci and growth traits.
Name of KASP marker Shell height Shell diameter Body weight A007358 0.07355 0.1598 A007362 0.02870 A007366 0.01375 0.0226 A007374 0.09826 0.0836 A007376 0.00708 0.00916 0.0274 A007388 0.02396 A007389 0.07661 0.0831 A007397 0.03537 A007399 0.02396 A007402 0.0205
NOTE: P<0.1 is defined as significantly correlated.
[00027] Table 2. Molecular marker-based growth trait prediction model.
Trait Markers included in the prediction model Shell height A007362+ A007366 + A007376 + A007388 + A007399 Shell diameter A007358 + A007374 + A007376 + A007389 + A007397 Body weight A007358 + A007366 + A007374 + A007376 + A007389 + A007402
[00028] Table 3. The information on primers of KASP markers developed based on 10 SNPs.
NameofSNP Name of marker Primer sequence FAM primer sequence: TTTGGAAAAACATGTTCAAATTGAGTTATGTC Marker49152 A007402 VIC primer sequence: TTTGGAAAAACATGTTCAAATTGAGTTATGTT COM primer sequence: TCGCGTTGAAATGTGCTAAAGCTATACAAA FAM primer sequence: AGTTGATTCAGAGCCATATCCACATA Marker112898 A007399 VIC primer sequence: AGTTGATTCAGAGCCATATCCACATT COM primer sequence: CCCTGTTCCTTCGCCTATGATTACAT FAM primer sequence: GTCAGACTGTGAAATTTGAAGCCTTG Marker140758 A007397 VIC primer sequence: GTCAGACTGTGAAATTTGAAGCCTTA COM primer sequence: GTTGGGGTAGGCTCACCAGCAA FAM primer sequence: TGGTTCCTCACTGGACTGTCTAA Marker63934 A007389 VIC primer sequence: GTTCCTCACTGGACTGTCTAG COM primer sequence: ACAAACTCTTCTATTGTGTTATTCCTTAGT FAM primer sequence: GAGAAGTTGYATAAGATATTGTTCTCAATC Marker203513 A007388 VIC primer sequence: GAGAAGTTGYATAAGATATTGTTCTCAATT COM primer sequence: ACTTTAAGGGAAATATCGGAGCAAATTCAT FAM primer sequence: CCCATTAATGGTATTTCCACCAAATTTAG Marker24433 A007376 VIC primer sequence: CCCATTAATGGTATTTCCACCAAATTTAA COM primer sequence: CATTCGATCTTCACGGTTTCATAGTAAGTT FAM primer sequence: GTTGAATGTATCTGACCAAAAGCGAA Marker72763 A007374 VIC primer sequence: TGTATATTCTCGCAATATCAAGTTTCTCAA COM primer sequence: TGTATATTCTCGCAATATCAAGTTTCTCAA FAM primer sequence: CAAATACAGCATCAACATAGAATAAGG Marker23360 A007366 VIC primer sequence: GCAAATACAGCATCAACATAGAATAAGT COM primer sequence: TTTGAGCCTTAATATTAATTTCCAGGTGCA FAM primer sequence: ATCTATAATATTTTGATGAAAATTACATTTTC Marker13442 A007362 VIC primer sequence: ATCTATAATATTTTGATGAAAATTACATTTTT COM primer sequence: AATAAGCAAAAATTCTGATATTTCATTACAGT FAM primer sequence: TGTGTTATTTCTAAAATACAATATACCTTC Marker6879 A007358 VIC primer sequence: TGTGTTATTTCTAAAATACAATATACCTTT COM primer sequence: CATAACATTGAGCATGAGAGGATAATCTA
[00029] Table 4. The information on primers of KASP markers developed based on 10 SNPs.
Name of marker Allele to be detected Primer ID C SEQIDNO.1 A007402 T SEQIDNO.2 SEQ ID NO. 3 A SEQ ID NO. 4 A007399 T SEQIDNO.5 SEQ ID NO. 6 C SEQ ID NO. 7 A007397 T SEQIDNO.8 SEQ ID NO. 9 A SEQ ID NO. 10 A007389 G SEQ ID NO. 11 SEQ ID NO. 12 C SEQIDNO.13 A007388 T SEQ ID NO. 14 SEQ ID NO. 15 C SEQIDNO.16 A007376 T SEQ ID NO. 17 SEQ ID NO. 18 A SEQ ID NO. 19 A007374 T SEQ ID NO. 20 SEQ ID NO. 21 G SEQ ID NO. 22 A007366 T SEQ ID NO. 23 SEQ ID NO. 24 G SEQ ID NO. 25 A007362 A SEQ ID NO. 26 SEQ ID NO. 27 C SEQ ID NO. 28 A007358 T SEQ ID NO. 29 SEQ ID NO. 30
5. Comprehensive predictive values of traits of parent individuals of S. intermedius
[00030] According to the genotyping results of breeding individuals in method 4, the comprehensive predictive value of traits of sea urchin individuals was calculated by using the molecular marker-based growth trait prediction model established in method 5. The specific process was as follows: sea urchin tube foot DNAs were extracted by method 2, the genotypes of the 201 sea urchin individuals were obtained by method 4, and the prediction results of each growth trait were obtained by the growth trait prediction model obtained by method 5; the prediction results of each trait were multiplied by the weight of the respective trait (shell height x 0.3, shell length x 0.3, and body weight x 0.4) to obtain the comprehensive predictive values of the traits of the parent individuals.
6. Selection scheme
[00031] Using the comprehensive predictive values of the traits of the parent individuals obtained in Method 6, the comprehensive predictive values of the traits of the parent individuals were arranged in a descending manner, and the sea urchins were divided into an excellent selection group, a medium selection group, and a lower selection group. The selection method was as follows: individuals with the scores of the comprehensive predictive values of the traits ranked in the top 30% were assigned to the excellent selection group, those with the scores of the comprehensive predictive values of the traits ranked in 40% of the middle interval were assigned to the medium selection group, and those with the scores of the comprehensive predictive values of the traits ranked in the last 30% were assigned to the lower selection group. Using the phenotypic data obtained in method 2 and combined with the grouping results, a significant analysis was conducted of the growth traits of the sea urchins, as shown in Table 5.
[00032] Table 5. The statistical analysis of growth traits of breeding population.
Trait Excellent selection group Medium selection group Lower selection group Quantity (N) 61 80 60 Shell height 30.76 ±2.498 29.77± 2. 8 5b 28.73 2.87c Shell diameter 53.93 ±3.898 52.75 ±3.858 50.49 3.2b Body weight 54.8 29.958 49.83± 9. 3 6b 45.95 8.71c Predictive value of 0.64 0.108 0.47± 0. 06b 0.28 0.03c parent NOTE: The same letter in the same line represents no significant difference between groups, and different letters in the same line represent significant differences between groups.
[00033] The analysis results showed that there were significant differences in growth traits (shell height, shell diameter, and body weight) and comprehensive predictive values of the traits of the parents among the three groups (all P<0.05), and in the phenotypic traits, the three groups ranked in the following order: excellent selection group > medium selection group > lower selection group.
7. Verification of progeny growth traits
[00034] In October 2017, according to the grouping results in method 7, sea urchins were induced to spawn, and progeny lines were established according to the spawning inducement results. When sea urchin progenies developed to 4 or 5 months old, a statistical analysis was conducted on the growth traits of the sea urchin progenies. The specific process was as follows: when the sea urchins developed to 4 months old, 100 sea urchins were randomly selected from the sea urchin lines in the excellent, medium and lower selection groups, respectively; each line of sea urchins were placed in separate cages and raised in 1.4 m3 aquaculture ponds. Meanwhile, 100 sea urchins of the same specification (with a shell diameter of about 7 mm) were selected from the sea urchin lines in the excellent, medium and lower selection groups, respectively; each line of sea urchins were placed in separate cages and raised in 1.4 m3 aquaculture ponds. After raising for 2 months, a comparative analysis was conducted on the growth traits and specific growth rates of the progeny sea urchin, as shown in Tables 6, 7, and 8.
[00035] Table 6. The statistical analysis on the growth traits of three populations of sea urchins aged 4 months (time: March 14, 2018).
Trait Excellent selection Medium selection Lower selection group group group Quantity (N) 200 300 200 Body weight (g) 0.18 ±0.1la 0.09± 0.05 0.04 ±0.03c Shell diameter (mm) 7.65 ±1.88a 6.62 1.4 4.78 ±1.01c
NOTE: The same letter in the same line represents no significant difference between groups, and different letters in the same line represent significant differences between groups.
[00036] Table 7. The statistical analysis on the growth traits of three populations of sea urchins aged 5 months (time: April 4, 2018).
Trait Excellent selection Medium selection Lower selection group group group Quantity (N) 190 292 195 Body weight (g) 0.61 0.38a 0.33 ± 0.18 ±0.13c Shell diameter (mm) 10.33 2.45a 8.49± 1.95 6.81 ±1.62c
NOTE: The same letter in the same line represents no significant difference between groups, and different letters in the same line represent significant differences between groups.
[00037] Table 8. The statistical analysis on the specific growth rate of three populations of sea urchins aged 4-5 months (time: from March 14, 2018 to April 14, 2018).
Trait Excellent selection Medium selection Lower selection group group group Quantity (N) 195 285 195 Body weight SGR 4.57% 4.33% 4.24% Shell diameter SGR 1.34% 1.18% 1.05%
[00038] Results:
[00039] Among three groups of randomly selected sea urchins, after two comparative analyses of the growth traits, phenotypic measurement results of all growth traits (shell height, shell diameter, and body weight) showed that the excellent selection group was significantly higher than the medium selection group (P<0.05), and the medium selection group was significantly higher than the lower selection group (P<0.05). The analysis results of the specific growth rate of body weight and shell diameter of the three groups of sea urchins with the same initial specification (shell diameter) showed: excellent selection group > medium selection group > lower selection group (P<0.05). After the analysis of the phenotypic data of the parents and the analysis of the growth traits of the progenies, the results showed that the 10 KASP markers selected had a good application value in the early selection of S. intermedius.
[00040] Any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the invention. It should not be taken as an admission that any of the material forms a part of the prior art base or the common general knowledge in the relevant art in Australia on or before the priority date of the disclosure herein.
[00041] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other features, integers, steps, components to be grouped therewith.
SEQUENCE LISTING Jul 2021
<110> Dalian Ocean University
<120> MOLECULAR MARKER-ASSISTED BREEDING PRIMER FOR GROWTH TRAIT OF STRONGYLOCENTROTUS INTERMEDIUS AND SCREENING METHOD
<130> GWP202105434
<160> 30 2021104355
<170> PatentIn version 3.5
<210> 1 <211> 32 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker49152, marker named A007402
<400> 1 tttggaaaaa catgttcaaa ttgagttatg tc 32
<210> 2 <211> 32 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker49152, marker named A007402
<400> 2 tttggaaaaa catgttcaaa ttgagttatg tt 32
<210> 3 <211> 30 <212> DNA <213> Artificial Sequence
<220>
<223> COM primer for SNP named Marker49152, marker named A007402 Jul 2021
<400> 3 tcgcgttgaa atgtgctaaa gctatacaaa 30
<210> 4 <211> 26 <212> DNA 2021104355
<213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker112898, marker named A007399
<400> 4 a g t t g a t t c a g a g c c a t a t c c a c a t a 26
<210> 5 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker112898, marker named A007399
<400> 5 a g t t g a t t c a g a g c c a t a t c c a c a t t 26
<210> 6 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker112898, marker named A007399
<400> 6 c c c t g t t c c t t c g c c t a t g a t t a c a t
<210> 7 Jul 2021
<211> 26 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker140758, marker named A007397
<400> 7 2021104355
g t c a g a c t g t g a a a t t t g a a g c c t t g 26
<210> 8 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker140758, marker named A007397
<400> 8 g t c a g a c t g t g a a a t t t g a a g c c t t a 26
<210> 9 <211> 22 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker140758, marker named A007397
<400> 9 g t t g g g g t a g g c t c a c c a g c a a 22
<210> 10 <211> 23 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker63934, marker named A007389 Jul 2021
<400> 10 t g g t t c c t c a c t g g a c t g t c t a a 23
<210> 11 <211> 21 2021104355
<212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker63934, marker named A007389
<400> 11 g t t c c t c a c t g g a c t g t c t a g 21
<210> 12 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker63934, marker named A007389
<400> 12 acaaactctt ctattgtgtt attccttagt 30
<210> 13 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker203513, marker named A007388
<400> 13 gagaagttgy ataagatatt gttctcaatc
<210> 14 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker203513, marker named A007388 2021104355
<400> 14 gagaagttgy ataagatatt gttctcaatt 30
<210> 15 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker203513, marker named A007388
<400> 15 actttaaggg aaatatcgga gcaaattcat 30
<210> 16 <211> 29 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker24433, marker named A007376
<400> 16 cccattaatg gtatttccac caaatttag 29
<210> 17 <211> 29 <212> DNA <213> Artificial Sequence
<220> Jul 2021
<223> VIC primer for SNP named Marker24433, marker named A007376
<400> 17 cccattaatg gtatttccac caaatttaa 29
<210> 18 2021104355
<211> 30 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker24433, marker named A007376
<400> 18 cattcgatct tcacggtttc atagtaagtt 30
<210> 19 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker72763, marker named A007374
<400> 19 g t t g a a t g t a t c t g a c c a a a a g c g a a 26
<210> 20 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker72763, marker named A007374
<400> 20 g t t g a a t g t a t c t g a c c a a a a g c g a t
<210> 21 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker72763, marker named A007374 2021104355
<400> 21 tgtatattct cgcaatatca agtttctcaa 30
<210> 22 <211> 27 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker23360, marker named A007366
<400> 22 c a a a t a c a g c a t c a a c a t a g a a t a a g g 27
<210> 23 <211> 28 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker23360, marker named A007366
<400> 23 g c a a a t a c a g c a t c a a c a t a g a a t a a g t 28
<210> 24 <211> 30 <212> DNA <213> Artificial Sequence
<220> Jul 2021
<223> COM primer for SNP named Marker23360, marker named A007366
<400> 24 tttgagcctt aatattaatt tccaggtgca 30
<210> 25 2021104355
<211> 32 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker13442, marker named A007362
<400> 25 atctataata ttttgatgaa aattacattt tc 32
<210> 26 <211> 32 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker13442, marker named A007362
<400> 26 atctataata ttttgatgaa aattacattt tt 32
<210> 27 <211> 32 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker13442, marker named A007362
<400> 27 aataagcaaa aattctgata tttcattaca gt
<210> 28 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> FAM primer for SNP named Marker6879, marker named A007358 2021104355
<400> 28 tgtgttattt ctaaaataca atataccttc 30
<210> 29 <211> 30 <212> DNA <213> Artificial Sequence
<220> <223> VIC primer for SNP named Marker6879, marker named A007358
<400> 29 tgtgttattt ctaaaataca atataccttt 30
<210> 30 <211> 29 <212> DNA <213> Artificial Sequence
<220> <223> COM primer for SNP named Marker6879, marker named A007358
<400> 30 cataacattg agcatgagag gataatcta
Claims (5)
1. A molecular marker-assisted breeding primer for growth traits of Strongylocentrotus intermedius, wherein the primer is selected from the following KASP primer sets:
(1) SNP named Marker49152, marker named A007402, having the corresponding primer sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; (2) SNP named Marker112898, marker named A007399, having the corresponding primer sequences shown as SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6; (3) SNP named Marker140758, marker named A007397, having the corresponding primer sequences shown as SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9; (4) SNP named Marker63934, marker named A007389, having the corresponding primer sequences shown as SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12; (5) SNP named Marker203513, marker named A007388, having the corresponding primer sequences shown as SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15; (6) SNP named Marker24433, marker named A007376, having the corresponding primer sequences shown as SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18; (7) SNP named Marker72763, marker named A007374, having the corresponding primer sequences shown as SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21; (8) SNP named Marker23360, marker named A007366, having the corresponding primer sequences shown as SEQ ID NO. 22, SEQ ID NO. 23, and SEQ ID NO. 24; (9) SNP named Marker13442, marker named A007362, having the corresponding primer sequences shown as SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27; (10) SNP named Marker6879, marker named A007358, having the corresponding primer sequences shown as SEQ ID NO. 28, SEQ ID NO. 29, and SEQ ID NO. 30.
2. A molecular marker-assisted breeding and screening method for growth traits of Strongylocentrotus intermedius using the primer according to claim 1, comprising the following steps:
(1) screening growth trait-related SLAF tags from an established genetic map of Strongylocentrotus intermedius according to QTL mapping results, and selecting SLAF tags for designing the KASP primers from the growth trait-related SLAF tags;
(2) finding SNP loci for designing the KASP primers from the SLAF tags for designing KASP primers, and using primers to conduct PCR amplification; typing resulting PCR products by fluorescence scanning, and verifying designed KASP primers according to the typing results;
(3) conducting genotyping verification on DNAs of a verification population using the KASP primer sets to finally obtain a KASP primer set for accurately typing candidate SNP loci, that is, growth trait-related KASP markers; and,
(4) according to the phenotypic data of the growth traits of the verification population and the typing results of growth trait-related KASP markers, conducting correlation analysis to obtain molecular markers markedly associated with the growth traits, and finally prediction models for each growth trait; calculating a comprehensive predictive value of individual growth traits through each predictive value, and finally establishing a molecular marker-based growth prediction model of Strongylocentrotus intermedius.
3. The molecular marker-assisted breeding and screening method for growth traits of Strongylocentrotus intermedius according to claim 2, wherein the growth traits comprise shell height, shell diameter, and body weight.
4. The molecular marker-assisted breeding and screening method for growth traits of Strongylocentrotus intermedius according to claim 2 or claim 3, wherein a screening criterion for the growth trait-related SLAF tags in step (1) is as follows: when a significance threshold exceeds a critical value of limit of detection (LOD), a quantitative trait locus (QTL) is considered to exist, wherein LOD = 2-3; a selection criteria for the SLAF tags for designing the KASP primers in step (1) are as follows: 0 absence of insertion/deletion (InDel) polymorphism loci in the SLAF tags;
only one SNP locus in a primer sequence; and controlling annealing temperature within a temperature range required by PCR according to base content, wherein the annealing temperature is -65 0 C.
5. The molecular marker-assisted breeding and screening method for growth traits of Strongylocentrotus intermedius according to any one of claims 2 to 4, wherein the PCR amplification in step (2) is conducted at 55-65 0C, and the PCR product has a length of <200 bp; a criterion for obtaining the growth trait-related KASP marker in step (3) is as follows: the marker is considered available when 2-3 genotypes of an SNP locus are divided.
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