KASP molecular marker for identifying cotton thrip resistance and application of KASP molecular marker

Abstract

The invention relates to the technical field of molecular biology, and discloses a KASP molecular marker for identifying cotton thrip resistance and application of the KASP molecular marker. The invention relates to a KASP molecular marker for identifying cotton thrip resistance. The KASP molecular marker Thrips1 is a base TGTTG deleted after a 105731625 base G of a cotton A12 chromosome. An upstream primer sequence of the KASP molecular marker for identifying the cotton thrip resistance is as shown in SEQ ID NO.1, and a downstream primer sequence of the KASP molecular marker is as shown in SEQ ID NO.2 and SEQ ID NO.3. According to the KASP molecular marker for identifying the cotton thrip resistance and the application of the KASP molecular marker, genetic typing, selection of dominant genotype individuals, screening of cotton germplasm with high thrip resistance and early molecular marker-assisted selection of anti-thrip breeding can be carried out through the KASP molecular marker, and the KASP molecular marker has important breeding value and economic value.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to a KASP molecular marker for identifying cotton thrips resistance and an application thereof. Background Art

[0002] Cotton, a member of the genus Gossypium in the family Malvaceae, is one of the most important crops. There are more than 100 countries and regions that grow cotton in the world, and they are mainly concentrated in Asia and North America, accounting for 80% of the world's cotton planting. In China, cotton planting began in the Warring States Period more than 2,400 years ago. Cotton pests have always been an important factor affecting cotton yield and quality. There are many types of cotton pests, and more than 300 species have been recorded in my country, including cotton bollworm, thrips, cotton aphids, cotton blind bugs, etc. With climate change and the adjustment of planting structure, the large-scale promotion of genetically modified insect-resistant cotton, and the reduction in the use of pesticides, lepidopteran insects such as cotton bollworm have been reduced to secondary pests, while the damage caused by secondary pests such as cotton thrips is on the rise. Cotton thrips not only cause serious damage during the seedling stage, but also during the flowering stage. The damage rate is generally 10%-20%, and the more serious ones reach 30%-40%. The extremely serious plots can reach 60%, resulting in reduced cotton production and quality. Excessive use of pesticides will pollute the environment and soil, causing irreversible impacts on human health. In addition, agricultural pests will develop resistance to pesticides, and the efficacy of pesticides will continue to decline. Therefore, cultivating insect-resistant varieties and exploring insect-resistant genes is the most economical, effective, green and environmentally friendly way to increase cotton yield and quality.

[0003] Genome-wide association analysis (GWAS) is to detect the genetic variation polymorphism of multiple individuals in the whole genome to obtain genotypes, and then conduct statistical analysis on the genotypes and observable traits, i.e. phenotypes, at the population level. It is an effective method to mine excellent genes of crops based on statistics or P values, and has been widely used in various crops. In 2001, Risch and Merikangas first proposed the concept of GWAS, which is to use natural populations as research objects and use molecular markers and quantitative trait loci to locate QTLs in the whole genome. In recent years, with the development of DNA sequencing technology and computers, GWAS technology has been well applied in crops such as rice, corn, wheat, barley and soybeans, providing a new direction for analyzing plant gene functions and genetic mechanisms.

[0004] The purpose of the present invention is to provide a KASP marker related to cotton thrips resistance, screen out cotton germplasm with higher thrips resistance by genotyping the site, and perform early molecular marker-assisted selection for thrips-resistant breeding, which has important breeding value and economic value. Summary of the invention

[0005] The purpose of the present invention is to provide a KASP molecular marker for identifying cotton thrips resistance and an application thereof. The KASP molecular marker can be used to perform genotyping, select dominant genotype GG individuals, screen out cotton germplasm with higher thrips resistance, and perform early molecular marker-assisted selection for thrips-resistant breeding, which has important breeding value and economic value.

[0006] To achieve the above object, the present invention provides a KASP molecular marker for identifying cotton thrips resistance. The KASP molecular marker Thrips1 is a deletion of the base TGTTG after the base G at position 105731625 of the cotton A12 chromosome.

[0007] Furthermore, the upstream primer sequence of the KASP molecular marker is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2 and SEQ ID NO.3.

[0008] The invention also provides a detection reagent, a kit and a chip comprising a KASP molecular marker for identifying cotton thrips resistance.

[0009] The invention also provides application of KASP molecular marker in identifying cotton thrips resistance.

[0010] The invention also provides application of KASP molecular marker in cotton genetic breeding.

[0011] The advantages and positive effects of the KASP molecular marker for identifying cotton thrips resistance and its application described in the present invention are:

[0012] 1. The KASP marker provided by the present invention is associated with cotton thrips resistance. By genotyping the site and selecting individuals with the dominant genotype GG, cotton germplasm with higher thrips resistance can be screened out, and early molecular marker-assisted selection for thrips-resistant breeding can be carried out, which has important breeding value and economic value.

[0013] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The thrips resistance phenotypic distribution and differences of 1081 germplasm resources in the embodiment of the present invention, wherein a is the DI index and coefficient of variation diagram of the Qingyuan population in 2020, b is the DI index and coefficient of variation diagram of the Hejian population in 2021, c is the DI index and coefficient of variation diagram of the Xinqu population in 2021, d is the DI index and coefficient of variation diagram of the Hejian population in 2022, e is the DI index and coefficient of variation diagram of the Xinqu population in 2022, and f is the average value of the DI index and coefficient of variation of 5 environments;

[0015] Figure 2 is the thrips resistance of the F2 single plant and the insect-resistant and insect-susceptible pool in the embodiment of the present invention, wherein a is the DI value of the insect-resistant material and the insect-susceptible material, and b is the insect population of the insect-resistant material and the insect-susceptible material;

[0016] Figure 3 : A Manhattan graph and a QQ graph based on SVs in an embodiment of the present invention, where a is a Manhattan graph and b is a QQ graph;

[0017] Figure 4 is the thrips resistance of the F2 individual plant and the resistant insect pool in the embodiment of the present invention, wherein a is the thrips resistance of the F2 individual plant, and b is the thrips resistance of the resistant insect pool;

[0018] Figure 5 The distribution of ED correlation values ​​on chromosomes in the embodiment of the present invention;

[0019] Figure 6 : is the distribution of SV-index association values ​​on chromosomes in the embodiment of the present invention, wherein a is the distribution of SNP-index of the insect-susceptible mixed pool on chromosomes, b is the distribution of SNP-index of the insect-resistant mixed pool on chromosomes, and c is the distribution of ΔSNP-index on chromosomes;

[0020] Figure 7 The cotton thrips resistance KASP marker development in the embodiment of the present invention, wherein a is the thrips resistance phenotype of the varieties corresponding to the two haplotypes, b is the sequence alignment of different thrips resistance materials, c is the genotyping of Thrips1 in the F2 segregating population, and d is a box plot comparing the DI difference of the two haplotypes of Thrips1 in the F2 segregating population. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] The cotton varieties used are all from germplasm resources collected and preserved in the laboratory, from the article "Ma ZY, et al. High-quality genome assembly and resequencing of modern cotton cultivarsprovide resources for crop improvement. Nature Genetics, 2021, 53(9): 1385-1391".

[0025] The present invention is based on 1081 upland cotton germplasm resources collected and sequenced in the laboratory, and the field resistance to thrips of the germplasm resources is identified. The SVs site significantly associated with thrips resistance is obtained through GWAS analysis. The SVs is located at 105731626-105731630 of the A12 chromosome. The haplotype of the thrips-sensitive material is GTGTTG, while the genotype of the thrips-resistant material is G, with a 5bp deletion, and the mutation is named Thrips1. An F2 segregation population is constructed with extremely insect-resistant materials as the female parent and extremely insect-susceptible materials as the male parent, and the resistance to thrips of the F2 individual plants is identified; based on the identification results, 30 insect-resistant individual plants are selected as the resistance pool, 30 insect-susceptible individual plants are selected as the sensitive pool, and two parents are subjected to BSA sequencing analysis. After the intersection is taken by two positioning association methods of ED and ΔSNP / ΔInDel-Index, the SVs are located in the 10.24Mb interval on the A12 chromosome. Combining GWAS and BSA results for joint analysis, Thrips1 is also located in the A12 chromosome related positioning interval located by BSA. Thrips1 was developed as a KASP marker and verified in the F2 segregation population, which was found to have a GTGTTG / G polymorphism. When the genotype of this site is GG, the resistance to thrips is significantly higher than that of the GTGTTGGTGTTG type.

[0026] Example 1 Phenotypic variation of thrips resistance in 1081 germplasm resources

[0027] A total of 1081 germplasm resources were identified for field resistance to thrips in five environments over three years in Qingyuan, Baoding, Xinji, Shijiazhuang, and Hejian, Cangzhou. Figure 1 As shown, the pest index (DI) of the entire Qingyuan group in 2020 had the largest variation range, with a maximum DI of 98.33, a minimum of 1.67, and a coefficient of variation of 57.82% ( Figure 1 a); In 2021, the maximum DI of Hejian is 63.33, the minimum is 0.00, and the coefficient of variation is 48.72% ( Figure 1 b); In 2021, the maximum DI of Xinji is 100.00, the minimum is 3.75, and the coefficient of variation is 37.95% ( Figure 1c); the maximum DI value of Hejian in 2022 is 53.41, the minimum value is 0.00, and the coefficient of variation is 35.34% ( Figure 1 d); In 2022, the maximum DI of Xinji is 46.67, the minimum is 3.50, and the coefficient of variation is 34.49% ( Figure 1 In 2021, the overall thrips damage in Xinji was the most serious, with an average DI of 33.23, and the thrips damage in Hejian was the lightest in 2021, with an average DI of 22.02. After averaging the phenotypic data of the five environments, the coefficient of variation of the average DI was 24.31%, the maximum DI was 56.50, and the minimum was 7.08 ( Figure 1 f). The above results show that the thrips resistance and the degree of damage caused by thrips in cotton vary significantly between years due to the influence of environmental climate and other factors. At the same time, the genetic resistance of 1081 germplasm resources to thrips is widely distributed.

[0028] Example 2 Indoor insect resistance identification of extremely resistant germplasm

[0029] In order to further verify the accuracy of the field survey results, 10 susceptible materials and 10 resistant materials were selected for indoor insect resistance identification according to the field survey results. The pest survey was conducted 20 days after the inoculation of thrips. The DI of the 10 resistant materials was between 28.23-50.69, with an average DI of 43.76. The indoor DI of the 10 susceptible materials was between 60.93-80.90, with an average DI of 72.47. The average DI of the resistant materials was significantly lower than that of the susceptible materials ( Figure 2 a); At the same time, the average insect population per plant was also counted. The insect population of the insect-resistant materials was between 0.83 and 6.00, with an average insect population of 3.45 per plant. The insect population of the insect-susceptible materials was between 6.00 and 11.58, with an average insect population of 8.79 per plant. Similarly, the average insect population per plant of the insect-resistant materials was significantly lower than that of the insect-susceptible materials ( Figure 2 (b) The indoor identification results are highly consistent with the field investigation results, indicating the accuracy of our field investigation results.

[0030] Example 3 GWAS mining of SVs significantly associated with resistance to thrips

[0031] Based on the 304,630 SVs obtained from the laboratory's preliminary resequencing results, a GWAS analysis was performed using a mixed linear model using the mean DI values ​​from five environmental surveys, and a total of 193 significantly associated SVs (-log 10P ≥ 5.5), except for 1 SVs on chromosome A11, 2 SVs on chromosome D10, and 3 SVs on chromosome D11, the remaining 187 significantly associated SVs loci were located on chromosomes A12 and D12, of which chromosome A12 contained 142 SVs and chromosome D12 contained 45 SVs, forming two significant peaks ( Figure 3 shown).

[0032] Example 4 Identification of the thrips resistance phenotype of F2 isolate population

[0033] With the thrips-resistant material SW923 as the female parent and the thrips-susceptible material SW016 as the male parent, an F2 segregation population of 381 individual plants was constructed for indoor thrips resistance identification. A single-plant insect survey was conducted 20 days after thrips inoculation, and it was found that the thrips resistance of the F2 population conformed to the normal distribution ( Figure 4 In a), there are 5 plants with average pest levels of 0 and 4. We further selected 30 insect-resistant plants as the insect-resistant pool and 30 insect-susceptible plants as the insect-susceptible pool. The average insect pest level of the insect-resistant pool was 0.81, and the average insect pest level of the insect-susceptible pool was 3.38. The resistance of the two insect-resistant and insect-susceptible pools was significantly different ( Figure 4 In b), parents SW923 and SW016 were selected simultaneously to construct a parent pool.

[0034] Example 5 BSA localization

[0035] Thirty insect-resistant plants and 30 insect-susceptible plants were selected from the F2 plants for DNA extraction based on the CTAB method, and the DNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific). Equal amounts of DNA were mixed to produce insect-resistant pools and insect-susceptible pools. These two DNA pools were used together with the DNA of the two parents to construct a 350bp sequencing library, which was sequenced on the Illumina / BGI sequencing platform to generate a 150bp double-end sequence. The raw data obtained by sequencing was quality controlled, variant detected, and annotated using the Biomarker bioinformatics analysis platform. The 122,378 InDels sites obtained were used for association analysis using the same analysis method as for SNP association analysis, and the following results were obtained: Figure 5 When the association threshold was 0.01, three regions stained with A11, A12 and D12 were obtained, with a total length of 21.03 Mb.

[0036] Using the SV-index method for analysis, when the confidence level was 90, a total of 10 regions were obtained. Although there were as many as 7 regions stained by A11, they were basically single InDel sites, while chromosome A12 had a region of 10.24 Mb, chromosome D12 had a region of 6.76 Mb, and the total association length was 17.01 Mb ( Figure 6 Similarly, the chromosome regions obtained by the two association methods are intersected, and the final region obtained is the same as the region obtained by the SV-index method.

[0037] Example 6 KASP Marker Development

[0038] In order to verify the accuracy of the associated loci and apply the located genetic loci related to thrips resistance to molecular selection breeding. 10 (P) = 15.8, Ref: GTGTTG; Alt: G) designed KASP primers. This SV locus can divide the 1081 natural population into two haplotypes. The two haplotypes have significant differences in thrips resistance ( Figure 7 In a), 5 cotton varieties of two haplotypes were selected for PCR amplification and sequencing of the SV locus. The results showed that the SV locus (5 bp deletion) was indeed present, which was consistent with the resequencing results ( Figure 7 b). The designed KASP primers were used to perform genotyping in the F2 segregating population, and 353 out of 381 individual strains were successfully typed ( Figure 7 In the middle (c), the thrips resistance of the F2 plants with 80 G haplotypes was significantly better than that of the thrips resistance of the 78 GTGTTG haplotypes ( Figure 7 d). The KASP marker was named Thrips1, indicating that the developed KASP marker can effectively distinguish the thrips resistance of cotton and is of great significance for the molecular marker-assisted selection of new thrips-resistant cotton varieties.

[0039] Therefore, the present invention adopts the above-mentioned KASP molecular marker for identifying cotton thrips resistance and its application, and can perform genotyping through the KASP molecular marker, select dominant genotype GG individuals, screen out cotton germplasm with higher thrips resistance, and perform early molecular marker-assisted selection for thrips-resistant breeding, which has important breeding value and economic value.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Identification of KASP molecular markers for cotton thrips resistance, characterized in that: The KASP molecular marker Thrips1 is a deletion of the base TGTTG after the G at position 105731625 of the cotton chromosome A12.

2. The KASP molecular marker for identifying cotton thrips resistance according to claim 1, characterized in that: The upstream primer sequence of the KASP molecular marker is shown in SEQ ID NO.1, and the downstream primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.

3.

3. A detection reagent, a kit and a chip comprising the KASP molecular marker for identifying cotton thrips resistance according to claim 1 or 2.

4. Use of the KASP molecular marker as claimed in claim 1 or 2 in identifying cotton thrips resistance.

5. Use of the KASP molecular marker as claimed in claim 1 or 2 in cotton genetic breeding.

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