CN114622032A - Sweet potato weevil resistance sites SPWR1 and SPWR2, molecular markers and application thereof - Google Patents
Sweet potato weevil resistance sites SPWR1 and SPWR2, molecular markers and application thereof Download PDFInfo
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Abstract
The invention discloses sweet potato weevil resistance sites SPWR1 and SPWR2, molecular markers and application thereof. SPWR1 and SPWR2 are located on chromosome 7.326-7.462Mb and chromosome 7.23-30.30 Mb of sweet potato genome (reference sweet potato variety Taizhong No. 6 genome), respectively. The molecular marker SPWR1a closely linked with SPWR1 is located at the position 7372883bp of the chromosome 9 of the sweet potato genome, and is a Single Nucleotide Polymorphism (SNP) locus marker, the SNP genotype is homozygous at C/C base in a resistant variety, and is heterozygous at A/C base in a susceptible variety, and the SPWR2a closely linked with SPWR2 is located at the position 30306530bp of the chromosome 7 of the sweet potato genome, and is a polynucleotide insertion type (Indel) molecular marker of 120bp, the genotype is homozygous in the resistant variety, and the susceptible variety is heterozygous. The invention provides sweet potato weevil resistance loci SPWR1 and SPWR2, wherein the resistance loci are closely associated with the resistance phenotype of the sweet potato weevil, and are favorable for assisting sweet potato resistance screening molecular breeding.
Description
The technical field is as follows:
the invention belongs to the field of sweet potato insect resistance, and particularly relates to two sweet potato weevil resistance sites, a molecular marker and application thereof.
Background art:
sweet potatoes (Ipomoea batatas L.) are an important crop with wide planting area and high yield, have various varieties and wide application, and have higher production value and agricultural status. The main sweet potato production areas are concentrated in sub-africa and sub-tropical sweet potato production areas, wherein a sweet potato elephant nail (Cylas formicarius Fab.) disaster is very easy to outbreak in the tropical and sub-tropical sweet potato production areas.
Sweet potato weevil belongs to coleoptera and insects of the family of the armyworm and the weevil, is a worldwide quarantine pest and also a destructive pest in sweet potato production, usually reduces the yield of sweet potatoes by 20-50 percent, and even eliminates the yield in severe cases. The imago of the sweet potato weevil gnaws the cortex of the tender shoot tip, the stem and the petiole of the sweet potato and bites the root tuber into a plurality of small holes, thus influencing the growth and development of the sweet potato and the yield of the sweet potato. The larvae are boring into roots or vines, so that the growth and expansion of the potatoes can be inhibited, and excrement of the larvae is filled in a hidden channel to promote pathogen infection, so that the potatoes are rotten, mildewed, blacked and smelly, and the quality and storage of the potatoes are seriously affected.
In recent years, the frequent occurrence of the weevil of the sweet potato seriously threatens the planting of the sweet potato in China. At present, the prevention and control of the sweet potato weevil is mainly chemical prevention and control, the problems of pesticide abuse, environmental pollution and food safety exist, the selection of sweet potato insect-resistant germplasm resources is very difficult due to the lack of effective insect-resistant sites or molecular markers, and the breeding progress of the sweet potato weevil resistant molecules is slow. Therefore, digging and screening sweet potato self-resistance resources, resistance sites and molecular markers has important significance for breeding sweet potato weevil resistant varieties.
The invention content is as follows:
the invention aims to provide two molecular markers which can be used for detecting sweet potato weevil resistance sites and molecular markers for sweet potato molecular breeding and application thereof, and provides new genetic sites and available molecular markers for sweet potato insect resistance evaluation and breeding improvement.
The invention provides genetic loci SPWR1 and SPWR2 for sweet potato for detecting the resistance of sweet potato weevil, which are positioned on chromosome 9, 7.326-7.462Mb and chromosome 7, 30.23-30.30Mb of a sweet potato genome (refer to genome 6 in Taizhong sweet potato variety). The two insect-resistant sites have polymorphism in resistant variety (sweet potato variety N73 is selected as an example in the invention) and susceptible variety (sweet potato variety G87 is selected as an example in the invention), and the resistant sites are verified to exist in resistant material and be homozygous in hybrid offspring of susceptible and resistant parents and to be heterozygous in susceptible material.
The invention provides molecular markers SPWR1a and SPWR2a for identifying resistance sites SPWR1, SPWR2, the molecular markers SPWR1a, SPWR2a are closely linked to the corresponding resistance sites;
the molecular marker SPWR1a is located at the 7372883bp position of a chromosome 9 of a sweet potato genome and is a Single Nucleotide Polymorphism (SNP) locus marker, the SNP genotype in a resistant variety is homozygous at C/C bases, the genotype in a susceptible variety is heterozygous at A/C bases, the SPWR2a is located at the 30306530bp position of a chromosome 7 of a sweet potato genome and is a 120bp polynucleotide insertion type (Indel) molecular marker, the genotype in the resistant variety is homozygous, the susceptible variety is heterozygous, and the 120bp polynucleotide is 'TGCCAAAGACCTTGTGGTCAAGCGGCACCCGGTGTACACTACTTTGAGAAAGTAGCTATGAACAGATACTACATTCTAACCGAGTCAGTAGAACTCAAAAAAAAAAATTAAACTCTTATA', and is specifically shown in SEQ ID No. 1.
The second objective of the invention is to provide a detection primer set of the molecular markers SPWR1a and SPWR2 a:
the detection primer sequence of the molecular marker SPWR1a comprises: f1 primer: 5' -CTCGATAGGAGCCCCGACAT; r1 primer: 5' -GATACCTTGGAGTTTCTCAC, the PCR amplification product is about 800bp, which includes a Single Nucleotide Polymorphism (SNP) locus mark, the SNP genotype is C/C base homozygous in resistant variety, and A/C base heterozygous in inductive variety; the detection primer sequence of the molecular marker SPWR2a comprises: f2 primer: 5' -CCAAGTAAGTCTATCTGAAG; r2 primer: 5' -GATCCAGACTGAATGCCCACA, the PCR amplification product with homozygous resistant locus is about 835bp in length, and comprises a 120bp polynucleotide insertion type (Indel) molecular marker, wherein the genotype is homozygous in resistant varieties, and the genotype is heterozygous in sensitive varieties.
The third purpose of the invention is to provide the application of the molecular markers SPWR1a, SPWR2a or the detection primer group thereof in assisting sweet potato breeding or sweet potato resistance germplasm resource identification. Namely, molecular markers SPWR1a and SPWR2a or a detection primer group thereof are used for assisting in screening the resistant sweet potato materials.
Preferably, the application is the application of the sweet potato variety breeding method for resisting the sweet potato weevil. That is, by using the molecular markers SPWR1a and SPWR2a, the variety containing the resistant loci SPWR1 and SPWR2 and the variety not containing the resistant loci SPWR1 and SPWR2 are selected as parents to be crossed, and the lines containing the resistant loci SPWR1 and SPWR2 are selected again from the filial generation of the cross which shows excellent agronomic characters, so that the excellent agronomic line with the resistance of the sweet potato Xiaoxiangjia can be obtained.
The fourth purpose of the invention is to provide a method for identifying the genotypes of the sweet potato resistance loci SPWR1 and SPWR2, namely, the genotypes of the resistance loci SPWR1 and SPWR2 are judged by identifying the genotypes of the molecular markers SPWR1a and SPWR2a in the variety.
Preferably, the specific identification steps are as follows:
carrying out PCR amplification on the genomic DNA of the sweet potato sample to be detected by respectively using detection primer sets of molecular markers SPWR1a and SPWR2a to obtain amplification products;
DNA sequencing is carried out on the amplified product of the molecular marker SPWR1a, whether a single nucleotide polymorphism site marker exists or not is judged, SNP genotype in resistant varieties is C/C base homozygous, A/C base heterozygous in inductive varieties is judged, and strains with resistant sites are screened;
and (3) carrying out agarose gel electrophoresis on the amplification product of the molecular marker SPWR2a, wherein the length of the PCR amplification product with the homozygous resistance locus is 835bp, and screening the strain with the resistance locus.
Preferably, the PCR amplification reaction is performed in a system of 10. mu.L, and comprises: 1 mu L of Taq DNA Polymerase Mix 5 mu L, DNA template, 0.5 mu L of each of upstream primer and downstream primer and 3 mu L of deionized water, and the PCR amplification reaction program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30 sec; annealing at 55-60 deg.C for 30 sec; extension at 72 ℃ for 60-90sec for 35 cycles; extension at 72 ℃ for 5 min.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides sweet potato weevil resistance loci SPWR1 and SPWR2, wherein the resistance loci are closely associated with the resistance phenotype of the sweet potato weevil, and are favorable for assisting sweet potato resistance screening molecular breeding.
2. The invention provides molecular markers SPWR1a and SPWR2a which can identify the SPWR1 and SPWR2 sites. By utilizing the genotype information of the molecular marker PCR amplification product, the sweet potato resistant material can be effectively and quickly identified, resistant germplasm resources are obtained, and the breeding efficiency is improved.
3. The resistance locus provided by the invention is stably inherited in offspring, and genetic verification proves that an insect-resistant phenotype stably occurs in filial generations, the resistance locus does not have obvious linkage with part of excellent agronomic traits, and a new strain with resistance is easily obtained in genetic improvement of high-yield and high-quality varieties.
Description of the drawings:
FIG. 1: QTL mapping analysis chart of the resistance trait of the sweetpotato weevil, and arrows indicate the resistance loci SPWR1 and SPWR 2.
FIG. 2: resistance loci SPWR1, SPWR2 genotype polymorphism model examples, red font indicates resistance genotype and blue font is non-resistance genotype.
FIG. 3: SPWR1a molecular marker DNA sequencing peak maps of resistant variety N73 and susceptible variety G87 are illustrated, arrows indicate the SNP site positions of SPWR1, resistant genotypes refer to the N73 peak map, and non-resistant genotypes refer to the G87 peak map.
FIG. 4: SPWR2a molecular marker agarose gel electrophoresis images of resistant variety N73 and susceptible variety G87 illustrate the resistant genotype reference N73 electrophoresis band and the non-resistant genotype reference G87 electrophoresis band.
FIG. 5: comparison of resistance phenotypes between the susceptible, resistant parent and the hybrid progeny with or without partial carrying of the resistance loci, the SPWR1, SPWR2 lines. The abscissa, from left to right, is the perceptual parent G87, the resistant parent N73, the offspring (001) without the resistant locus and the offspring (002) with the resistant locus respectively, and the ordinate is the area of the sweetpotato weevil eating the sweet potato leaves under the same experimental conditions. The parental resistance phenotype was counted in 10 replicates and both progeny were randomly sampled in the F1 generations.
The specific implementation mode is as follows:
the following examples are intended to further illustrate the invention without limiting it, and the scope of the invention is not limited to the specific examples below.
Unless otherwise defined, all terms used in the following examples have the same meaning as they are commonly understood in the art, and the use of all terms is not intended to limit the scope of the present invention. All reagent materials and instrumentation used, except where specifically noted, are materials and instrumentation common to the art, either commercially available or self-prepared.
Example 1:
acquisition of sweet potato weevil resistance QTL
Resistant variety N73 and susceptible variety G87 with distinct phenotypes are obtained by screening in various sweet potato germplasm resources, 225F 1 filial generations are obtained by taking the resistant variety N73 and the susceptible variety G87 as parents through hybridization, and parent resequencing and SLAF sequencing of the filial generations are carried out. SNP markers were developed in both parents and progeny by evaluation of parental resequencing and 225 progeny SLAF sequencing data.
And comparing the developed SNPs, filtering out the SNP labels of which the filial generation has no polymorphism among parents and has excessive deletion and biased expression in the filial generation group, and carrying out genotype coding on the rest SNP labels. And (3) establishing a linkage group by comparing No. 6 reference genomes in Taizhong to separate the SNP markers, and analyzing the linear arrangement sequence of the markers in the linkage group and the genetic distance between adjacent markers to finally obtain a neutral genetic map.
The constructed sweet potato genetic map is combined with phenotype data of two-time evaluation of the resistance of the sweet potato weevil by using the filial generation of the F1 population, and the genetic linkage map of the resistance of the sweet potato weevil is constructed by a Composite Interval Mapping (CIM) method. The LOD value is determined to be 3 by PT Test (Permutation Test, P <0.05), namely the probability of the linkage of the marker and the gene is more than 95 percent, and finally the QTLs related to the resistance of the weevil of the sweet potato is obtained. Through the analysis, the phenotype data obtained by the repeated insect-resistant experiments of the offspring of the F1 population locate the QTL related to the resistance of the sweet potato weevil at two sites of the chromosome 7 and the chromosome 9 (arrows in figure 1 indicate two QTL sites).
Example 2
Acquisition of resistance sites SPWR1 and SPWR2 and development of linked molecular markers
Designing molecular marker primers for fine positioning according to SNP data obtained by resequencing of susceptible and resistant parents, obtaining genotypes of all molecular markers in filial generations through PCR amplification and product sequencing, simultaneously calculating genetic intervals linked with resistance phenotypes by combining the resistance phenotypes of the filial generations, and finally determining the genetic intervals of two QTL sites to 30.23-30.30Mb of chromosome 7 and 7.326-7.462Mb of chromosome 9 respectively. According to the sequencing result of the linked molecular markers and the corresponding resistance phenotype, two resistance loci are obtained and are finally named as SPWR1 (chromosome 9) and SPWR2 (chromosome 7). The two loci have polymorphism in a susceptible variety G87 and a resistant variety N73, wherein the SPWR1 locus is a Single Nucleotide Polymorphism (SNP) locus marker, shows A/C base heterozygosity in the susceptible variety and shows C/C base homozygosity in the resistant variety; the SPWR2 locus is a 120bp polynucleotide insertion type (Indel) locus marker, the insertion sequence is 'TGCCAAAGACCTTGTGGTCAAGCGGCACCCGGTGTACACTACTTTGAGAAAGTAGCTATGAACAGATACTACATTCTAACCGAGTCAGTAGAACTCAAAAAAAAAAATTAAACTCTTATA', the insertion type is shown as a heterozygous type in a sensitive variety, and the insertion type is shown as a homozygous type in a resistant variety (figure 2).
The resistance locus is named as SPWR1a and SPWR2a by using the minimum interval linkage molecular markers obtained by fine positioning, and positioning primers are designed according to the molecular markers SPWR1a and SPWR2 a: the detection primer sequence of the molecular marker SPWR1a comprises: f1 primer: 5' -CTCGATAGGAGCCCCGACAT; r1 primer: 5' -GATACCTTGGAGTTTCTCAC, the obtained PCR amplification product is about 800bp and can be used for detecting SNP locus molecular markers; the detection primer sequence of the molecular marker SPWR2a comprises: f2 primer: 5' -CCAAGTAAGTCTATCTGAAG; r2 primer: 5' -GATCCAGACTGAATGCCCACA, wherein the PCR amplification product with homozygous resistance sites is approximately 835bp in length, can be used to detect inserted Indel molecular markers.
The method comprises the following steps of (1) extracting the genome DNA of a sweet potato sample by adopting a CTAB method:
1. taking 300mg of fresh tissue of a sweet potato sample, putting the fresh tissue into a 1.5mL centrifuge tube, putting the centrifuge tube into liquid nitrogen, quickly freezing the fresh tissue and grinding the fresh tissue into powder by a grinder.
2. Adding 750 μ L of preheated CTAB extract at 60 deg.C into the centrifuge tube, mixing by vortex, placing in 65 deg.C metal bath for 30min, and mixing by inversion every 10 min.
3. Adding equal volume of chloroform/isoamyl alcohol mixture (volume ratio 24:1), reversing and mixing evenly, and centrifuging at 10000rpm for 10min at room temperature.
4. Adding 750 μ L of preheated CTAB extract at 60 deg.C into the centrifuge tube, mixing by vortex, placing in 65 deg.C metal bath for 30min, and mixing by inversion every 10 min. Centrifuging at 10000rpm for 15min at room temperature, discarding the supernatant, and keeping the DNA precipitate.
5. Adding 1mL of 75% ethanol into the precipitated DNA, shaking up and down to wash the DNA, centrifuging at 10000rpm for 1min at room temperature, discarding the supernatant, and retaining the DNA precipitate. And step 5 is repeated once.
6. After placing the centrifuge tube on a clean bench or airing at room temperature, 200. mu.L of deionized water was added, and DNA was dissolved by vortex shaking.
PCR amplification is carried out on the sample genome DNA by using detection primers marked by SPWR1a and SPWR2a molecules, the system of PCR amplification reaction is 10 mu L, and the PCR amplification reaction specifically comprises the following steps: 1 μ L of Taq DNA Polymerase Mix 5 μ L, DNA template, 0.5 μ L of each of the upstream and downstream primers, and 3 μ L of deionized water. The PCR amplification reaction program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30 sec; annealing at 55-60 deg.C for 30 sec; extension at 72 ℃ for 60-90sec (with reference to the actual reaction efficiency of the user's DNA polymerase), 35 cycles; extension at 72 ℃ for 5 min.
The amplification products obtained were used for DNA sequencing and agarose gel electrophoresis using 3% agarose for electrophoresis time 15-30min at 135v (for reference).
And finally, analyzing whether the sweet potato material carries the resistance locus or not according to the resistance locus sequencing and electrophoresis results of the comparison example. Examples of the SPWR1 and SPWR2 locus genotyping of the susceptible and resistant parents are shown in FIG. 2, an example of the base signal peak at the molecular marker SPWR1a is shown in FIG. 3, and an example of the electrophoretic band at the molecular marker SPWR2a is shown in FIG. 4.
Example 3
Application of resistance sites SPWR1 and SPWR2 in resisting sweetpotato elephant beetle
The method comprises the steps of hybridizing a parent N73 with homozygous resistance sites SPWR1 and SPWR2 and a parent G87 with heterozygous sites (with agronomic characters) to obtain F1 generation filial generation, screening a strain carrying the resistance sites by using molecular markers SPWR1a and SPWR2a on the basis of sweet potato filial generation with excellent agronomic characters, wherein the strain can be used as a seedling for breeding, the resistance phenotype of the filial generation bred by cutting is stable, and the resistance phenotype of the filial strain carrying the resistance sites is obvious (figure 5).
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aacagatact acattctaac cgagtcagta gaactcaaaa aaaaaaatta aactcttata 120
Claims (10)
1. The sweet potato weevil resistance loci SPWR1 and SPWR2 are characterized in that SPWR1 has a tightly-linked effective molecular marker SPWR1a on chromosome 9 of the sweet potato genome, and SPWR2 has a tightly-linked effective molecular marker SPWR2a on chromosome 7 of the sweet potato genome.
2. The molecular markers SPWR1a and SPWR2a which are closely linked with the sweet potato weevil resistance locus SPWR1 and SPWR2 as claimed in claim 1, wherein the molecular marker SPWR1a is located at the 7372883bp position of chromosome 9 of the sweet potato genome and is a single nucleotide polymorphism locus marker, the SNP genotype is C/C base homozygous in the resistant variety and A/C base heterozygous in the susceptible variety, the SPWR2a is located at the 30306530bp position of chromosome 7 of the sweet potato genome and is a polynucleotide insertion type molecular marker of 120bp, the genotype is homozygous in the resistant variety and heterozygous in the susceptible variety, and the polynucleotide of 120bp is TGCCAAAGACCTTGTGGTCAAGCGGCACCCGGTGTACACTACTTTGAGAAAGTAGCTATGAACAGATACTACATTCTAACCGAGTCAGTAGAACTCAAAAAAAAAAATTAAACTCTTATA.
3. A detection primer set for identifying the molecular markers SPWR1a and SPWR2a of claim 2, wherein the detection primers of the molecular markers SPWR1a are: f1 primer: 5' -CTCGATAGGAGCCCCGACAT; r1 primer: 5' -GATACCTTGGAGTTTCTCAC; the detection primers of the molecular marker SPWR2a are as follows: f2 primer: 5' -CCAAGTAAGTCTATCTGAAG; r2 primer: 5' -GATCCAGACTGAATGCCCACA.
4. Use of the molecular markers SPWR1a, SPWR2a of claim 2 or the detection primer set of claim 3 for assisting in sweet potato breeding or in identification of sweet potato resistant germplasm.
5. The use of claim 4, wherein the molecular markers SPWR1a and SPWR2a or their detection primer sets are used to assist in the screening of resistant sweetpotato material.
6. The use of claim 4, wherein the use is in a method for breeding a sweet potato variety resistant to sweetpotato elephant beetle.
7. The use of claim 6, wherein the molecular markers SPWR1a and SPWR2a are used to obtain the excellent agronomic line with resistance to sweetpotato elephant beetle by selecting the variety with resistance sites SPWR1 and SPWR2 as parent and crossing with the variety without resistance sites SPWR1 and SPWR2, and re-selecting the lines with resistance sites SPWR1 and SPWR2 from the filial generation of the cross with excellent agronomic character.
8. A method for identifying the genotypes of the sweet potato resistance loci SPWR1 and SPWR2, which is characterized in that the genotypes of the resistance loci SPWR1 and SPWR2 are judged by identifying the genotypes of the molecular markers SPWR1a and SPWR2a in the variety as claimed in claim 2.
9. The method according to claim 8, characterized in that the specific identification steps are as follows:
carrying out PCR amplification on the genomic DNA of a sweet potato sample to be detected by respectively using the detection primer sets of the molecular markers SPWR1a and SPWR2a of claim 3 to obtain amplification products;
DNA sequencing is carried out on the amplified product of the molecular marker SPWR1a, whether a single nucleotide polymorphism site marker exists or not is judged, SNP genotype in resistant varieties is C/C base homozygous, A/C base heterozygous in inductive varieties is judged, and strains with resistant sites are screened;
and (3) carrying out agarose gel electrophoresis on the amplification product of the molecular marker SPWR2a, wherein the length of the PCR amplification product with the homozygous resistance locus is 835bp, and screening the strain with the resistance locus.
10. The method of claim 9, wherein the PCR amplification is performed in a 10 μ L system comprising: 1 mu L of Taq DNA Polymerase Mix 5 mu L, DNA template, 0.5 mu L of each upstream primer and downstream primer and 3 mu L of deionized water, and the PCR amplification reaction program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30 sec; annealing at 55-60 deg.C for 30 sec; extension at 72 ℃ for 60-90sec for 35 cycles; extension at 72 ℃ for 5 min.
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