Cotton salt-tolerant QTL site screened based on linkage QTL positioning and QTL-seq technology, molecular marker and application

Abstract

The invention discloses a cotton salt-tolerant QTL site screened based on linkage QTL positioning and a QTL-seq technology, a molecular marker and application, and belongs to the technical field of cotton molecular breeding. The cotton salt-tolerant QTL loci screened on the basis of linkage QTL positioning and the QTL-seq technology in a combined manner comprise qSAA05.1, qSAD08.1 and qSAD10.1; the qSAA05.1 is located in A05: 105.99 to 106.26 Mb, and the interpretable phenotypic variation rate of the qSAA05.1 is up to 10.42%; the qSAD08.1 is located at D08: 46.62 to 48.95 Mb, and the explainable phenotypic variation rate of the qSAD08.1 is up to 8.99%; the qSAD10.1 is located at D10: 2.28-3.21 Mb, and the explainable phenotypic variation rate of the qSAD10.1 is up to 15.36%. By integrating multi-environment QTL positioning and QTL-seq analysis, the stable salt-tolerant QTL locus and the linkage marker are accurately obtained, the method can be used for cotton salt tolerance rapid identification and molecular marker-assisted breeding, the salt-tolerant variety breeding process is remarkably accelerated, and important genetic resources and technical support are provided for cotton salt-tolerant breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cotton molecular breeding, and particularly relates to a cotton salt-tolerant QTL site, a molecular marker and application based on combined screening of linkage QTL positioning and QTL-seq technology. BACKGROUND

[0002] Soil salinization is one of the main abiotic stress factors restricting global agricultural production, especially in arid and semiarid regions, the problem of salinization is becoming increasingly serious. Cotton, as an important economic crop, has a certain salt tolerance and is known as the "pioneer crop" of saline-alkali land, but salt stress will still cause its growth to be hindered, fiber quality to be reduced, yield to be reduced, and seriously affect the sustainability of cotton production. With the continuous expansion of the global saline-alkali land area, breeding salt-tolerant cotton varieties has become a research hotspot in the field of cotton breeding.

[0003] Traditional cotton salt-tolerant breeding mainly relies on phenotypic selection, but it has long cycle, low efficiency, and is greatly affected by environmental factors. The molecular marker assisted selection (MAS) technology selects based on molecular markers closely linked to the target traits, has the advantages of being fast, accurate, and not affected by the environment, and can significantly accelerate the breeding process. The QTL (quantitative trait loci) of precisely positioning the salt-tolerant traits and its linked markers are the premise of carrying out MAS breeding.

[0004] At present, many cotton salt-tolerant QTLs have been identified by different genetic populations, but most of the QTL sites have poor stability in different environments, small effects, and lack of effective linked markers, which are difficult to apply to actual breeding. The QTL positioning method can detect the sites related to the traits, but the resolution is low; the QTL-seq technology can quickly locate the target QTL based on whole genome resequencing, but the results need to be further verified. Therefore, integrating QTL positioning and QTL-seq technology, mining stable salt-tolerant QTL sites and developing linked markers have important significance for cotton salt-tolerant molecular breeding. SUMMARY

[0005] The first purpose of the application is to provide a cotton salt-tolerant QTL site and a molecular marker based on combined screening of linkage QTL positioning and QTL-seq technology to overcome the technical problems of poor stability of cotton salt-tolerant QTL sites and lack of linked markers in the prior art.

[0006] To achieve the above purpose, the application adopts the following technical solutions:

[0007] A salt-tolerant QTL site of cotton screened based on linkage QTL positioning and QTL-seq technology combined screening, including qSAA05.1, qSAD08.1 and qSAD10.1; the qSAA05.1 is located at A05: 105.99-106.26 Mb, and can explain a phenotypic variation rate of 10.42%; the qSAD08.1 is located at D08: 46.62-48.95 Mb, and can explain a phenotypic variation rate of 8.99%; and the qSAD10.1 is located at D10: 2.28-3.21 Mb, and can explain a phenotypic variation rate of 15.36%.

[0008] The second object of the application is a screening method of a salt-tolerant QTL site of cotton based on linkage QTL positioning and QTL-seq technology combined screening.

[0009] The screening method of the salt-tolerant QTL site of cotton based on linkage QTL positioning and QTL-seq technology combined screening comprises the following steps:

[0010] S1: a recombinant inbred line population and a BC1F2 population are constructed by taking Gossypium hirsutum TM-1 as a female parent and Gossypium barbadense Hai7124 as a male parent, and the BC1F2 population is selfed to obtain a BC2F 2:3 line;

[0011] S2: a recombinant inbred line population and a salt stress treatment group and a blank control group of parents are constructed in at least three different environments, salt-tolerant related traits of the recombinant inbred line population and the parents are determined, and a ratio of the salt-tolerant related traits in the salt stress treatment group to the blank control group is taken as phenotypic data; the salt-tolerant related traits include plant fresh weight, plant dry weight, leaf SPAD value, leaf water content and leaf conductivity;

[0012] S3: genotypic analysis is performed on the recombinant inbred line population by using a molecular marker, a genetic linkage map is constructed, QTL positioning is performed by using QTL IciMapping 4.2 software and adopting an ICIM-ADD method, and a salt-tolerant QTL site is obtained;

[0013] S4: salt-tolerant related traits of the BC1F 2:3 line are determined, a comprehensive salt-tolerant evaluation value is calculated through membership function values and weights, a comprehensive evaluation is performed on each line, 20 lines with the highest D values and 20 lines with the lowest D values are selected to construct a salt-tolerant pool and a salt-sensitive pool respectively;

[0014] S5: whole genome resequencing is performed on the salt-tolerant pool and the salt-sensitive pool, a salt-tolerant related genomic region is obtained through Δ(SNP-index) and G' value analysis;

[0015] S6: the salt-tolerant QTL site obtained in S3 is integrated with the genomic region obtained in S5, and an overlapping QTL site is screened, and the salt-tolerant QTL site is obtained.

[0016] Further, the different environments in S3 include spring greenhouse, summer artificial climate chamber and autumn greenhouse; the comprehensive salt tolerance evaluation value in S4 is obtained by integrating the membership function values and weights of aboveground fresh weight, aboveground dry weight, leaf SPAD value, leaf water content and leaf conductivity.

[0017] The application of a cotton salt-tolerant QTL site screened based on linkage QTL mapping and QTL-seq technology in the research of cotton salt-tolerant molecular mechanism.

[0018] The third object of the application is to provide a molecular marker linked to the cotton salt-tolerant QTL site screened based on linkage QTL mapping and QTL-seq technology.

[0019] The molecular marker linked to the cotton salt-tolerant QTL site screened based on linkage QTL mapping and QTL-seq technology, the molecular marker linked to qSAA05.1 is NAU934 and HAU1496, qSAA05.1 is located in the genetic interval 123.41-128.46 cM between NAU934 and HAU1496; the molecular marker linked to qSAD08.1 is NAU2292 and TMD05, qSAD08.1 is located in the genetic interval 65.77-72.37 cM between NAU2292 and TMD05; the molecular marker linked to qSAD10.1 is NAU2776 and NAU5359, qSAD10.1 is located in the genetic interval 0-3.86 cM between NAU2776 and NAU5359.

[0020] Further, the forward primer sequence of the NAU934 is SEQ ID NO. 1, and the reverse primer sequence is SEQ ID NO. 2; the forward primer sequence of the HAU1496 is SEQ ID NO. 3, and the reverse primer sequence is SEQ ID NO. 4; the forward primer sequence of the NAU2292 is SEQ ID NO. 5, and the reverse primer sequence is SEQ ID NO. 6; the forward primer sequence of the TMD05 is SEQ ID NO. 7, and the reverse primer sequence is SEQ ID NO. 8; the forward primer sequence of the NAU2776 is SEQ ID NO. 9, and the reverse primer sequence is SEQ ID NO. 10; the forward primer sequence of the NAU5359 is SEQ ID NO. 11, and the reverse primer sequence is SEQ ID NO. 12.

[0021] The application of a molecular marker in cotton salt-tolerance identification or salt-tolerant cotton breeding.

[0022] The fourth object of the application is to provide a cotton salt-tolerance identification method.

[0023] A salt tolerance identification method of cotton, comprising the following steps:

[0024] S1: extracting genomic DNA of the cotton material to be identified;

[0025] S2: using the genomic DNA extracted in S1 as a template, performing PCR amplification on the above-mentioned molecular markers NAU934 and HAU1496, NAU2292 and TMD05, NAU2776 and NAU5359 respectively to obtain PCR amplification products;

[0026] S3: detecting the PCR amplification products, if the cotton material to be identified contains at least one pair of marker amplification fragments identical to Gossypium barbadense Hai7124, the material has the corresponding salt tolerance characteristics; the more pairs of markers contained, the stronger the salt tolerance.

[0027] The fifth object of the present application is to provide a salt-tolerant cotton breeding method.

[0028] A salt-tolerant cotton breeding method, comprising the following steps:

[0029] S1: using Gossypium barbadense Hai7124 as a donor parent and a target cotton variety as a receptor parent to perform hybridization and backcrossing to obtain a progeny population;

[0030] S2: using the above-mentioned molecular markers to perform molecular marker assisted selection on the progeny population to screen individuals containing at least one pair of marker amplification fragments identical to Gossypium barbadense Hai7124;

[0031] S3: performing salt tolerance identification and agronomic trait selection on the screened individuals to cultivate a salt-tolerant cotton variety.

[0032] The QTL site qSAA05.1 is mainly related to leaf SPAD value and leaf water content. The QTL site qSAD08.1 is mainly related to aboveground fresh weight. The QTL site qSAD10.1 is mainly related to leaf SPAD value.

[0033] The beneficial effects of the present application are:

[0034] (1) The present application integrates multi-environment QTL positioning and QTL-seq analysis, and accurately identifies stable cotton salt-tolerant QTL sites qSAA05.1, qSAD08.1 and qSAD10.1 for the first time, which are stable in different populations and different environments and have significant effects.

[0035] (2) The application provides molecular markers closely linked to qSAA05.1, qSAD08.1 and qSAD10.1, which can be directly used for rapid identification of salt tolerance and molecular assisted breeding, greatly shortening the screening period and improving breeding efficiency.

[0036] (3) The application integrates QTL positioning and QTL-seq technology, and takes into account accuracy and efficiency, providing a reusable paradigm for QTL mining of complex traits in other crops.

[0037] (4) The salt-tolerant QTL site and molecular marker of the application can be directly used for breeding salt-tolerant varieties, which can improve the salt tolerance of cotton while retaining excellent traits. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 2 is a diagram of the distribution of salt-tolerant QTLs on chromosomes obtained by QTL positioning based on a recombinant inbred line population, wherein the red dotted line is an overlapping QTL.

[0039] Figure 2 Figure 3 is a diagram of the distribution of salt-tolerance-related traits of two extreme pools screened based on BC1F2, wherein D value is a comprehensive salt-tolerance evaluation value, PFW is fresh weight above ground, PDW is dry weight above ground, SPAD is SPAD value, EC is leaf conductivity, and WC is leaf water content.

[0040] Figure 3 Figure 4 is a diagram of six candidate gene regions related to salt tolerance of cotton identified by QTL-seq technology of the BC1F2 population, wherein A is the value of △(SNP-index), and B is the value of G'. DETAILED DESCRIPTION

[0041] The application will be further described below in conjunction with the embodiments of the application and the accompanying drawings.

[0042] Example 1

[0043] Genetic population construction

[0044] A recombinant inbred line (RIL) population of 186 was constructed using the method of single seed descent, with upland cotton TM-1 as the female parent and sea island cotton Hai7124 as the male parent. The process is as follows: 186 F2 single plants were obtained by 1 cross and 1 selfing, and the offspring derived from each F2 single plant was planted into F 2:3 generation, and selfing was continuously performed to F 2:5 generation. Then, one plant was randomly selected from each F 2:5 generation, and seeds were collected and planted to form F 5:6 generation, and seeds were collected and planted to form F 5:7and their offspring were considered as RIL population, and a large amount of seeds were collected for subsequent QTL mapping studies. Similarly, with Gossypium hirsutum TM-1 as the receptor and Gossypium barbadense Hai7124 as the donor, a BC1F2 population was constructed through one cross and backcross, and the corresponding BC2F 2:3 strain was selfed to produce a BC2F

[0045] Example 2

[0046] Recombinant inbred line salt tolerance identification

[0047] In 2022, the RIL population and parents were subjected to salt stress treatment in three environments: spring greenhouse (E1), summer artificial climate chamber (E2), and autumn greenhouse (E3). After surface sterilization, the seeds were sown, and the seedlings were treated with 70 mL of 400 mM NaCl solution (salt stress treatment group) and an equal amount of water (blank control group) at the two-leaf stage. After one week, the aboveground fresh weight (PFW), aboveground dry weight (PDW), leaf SPAD value, leaf water content (WC), and leaf conductivity (EC) were measured, and the ratio of the salt stress treatment group to the blank control group was calculated as the phenotypic data. Table 1 shows the basic parameters of the five traits under three environmental conditions. Under three environmental conditions, the five traits showed a large variation range. The coefficient of variation (CV) of WC was the lowest (11.76%-13.02%), while the CV of PFW was significantly higher than that of other traits (16.24%-20.20%), indicating that PFW was more susceptible to environmental factors.

[0048] Table 1 Basic information of salt tolerance traits of RIL population under three environments

[0049]

[0050] Example 3

[0051] Genetic linkage map construction and QTL mapping

[0052] Young leaves of each RIL and parent were taken, and DNA was extracted using the CTAB method. 392 pairs of polymorphic SSR marker primers were obtained from the CottonGen database, and these primers were used for genotyping of the RIL population. Using JoinMap 4.0 software with a threshold value of LOD 3.0, a genetic linkage map was constructed. A genetic map containing 392 markers was constructed, covering 3101.50 cM, with an average marker interval of 7.91 cM Figure 1). Then the salt tolerance phenotypic data in three environments were combined with the constructed genetic linkage map to locate QTLs using the inclusive composite interval mapping (ICIM-ADD) method of QTL IciMapping v4.2 software. The results showed that a total of 16 QTLs were detected. Among them, 4 QTLs were associated with PFW, qPFW-D01-1 and qPFW-D06-1 could be detected in two environments. There were 3 QTLs associated with PDW. There were 4 QTLs associated with SPAD, qSPAD-A05-1 could be detected in two environments. There were 3 QTLs associated with WC. There were 2 QTLs associated with EC, and qEC-A03-1 could be detected in two environments.

[0053] Example 4

[0054] Construction of extreme pools and QTL-seq analysis

[0055] The same salt stress treatment was carried out on the BC1F 2:3 strains, and the above-mentioned five phenotypic indexes were measured. The weight of each trait was calculated by using the membership function method combined with principal component analysis, and then the comprehensive salt tolerance evaluation value (D value) was obtained. The calculation formula is D = ∑[U(Xij)Wj] (j = 1, 2, 3, …, n), where Xij represents the measured value of the ith strain for the jth trait, U(Xij) is the corresponding membership function value, and Wj is the weight of the jth trait.

[0056] The 20 strains with the highest D value were selected to construct the salt-tolerant pool (ST), and the 20 strains with the lowest D value were selected to construct the salt-sensitive pool (SS). Compared with the sensitive group, the group with stronger tolerance showed significantly higher PFW, PDW, SPAD and WC, but significantly lower EC Figure 2 ), indicating that the constructed extreme pool is suitable for QTL-seq analysis. Then, the two pools were subjected to genome resequencing with a sequencing depth of 20x. The method of calculating Δ(SNP-index) and G' value was used to identify salt tolerance QTL intervals. The results showed that there were 6 genomic regions Figure 3 A and B) related to the salt tolerance of cotton. These regions are distributed on five chromosomes, and the genomic interval ranges from 198.11 kb to 19.99 Mb.

[0057] Example 5

[0058] Comparison analysis of linkage QTL mapping and QTL-seq analysis results

[0059] The two QTL positioning methods were compared and analyzed, as shown in Table 2, and the results showed that the three genomic intervals determined in the QTL-seq analysis were consistent with the above linkage QTL positioning results. Among them, qSAA05.1 overlaps with qSPAD-A05-2 and qWC-A05-1, covering a genomic interval of 260.3Kb, located within the genetic interval of 123.41-128.46cM, and the linked molecular markers are NAU934 and HAU1496. qSAD08.1 overlaps with qPDW-D08-1, with a genomic interval of 2297.6Kb, located within the genetic interval of 65.77-72.37cM, and the linked molecular markers are NAU2292 and TMD05. qSAD10.1 overlaps with qSPAD-D10-1, covering a genomic interval of 899.42Kb, located within the genetic interval of 0-3.86cM, and the linked molecular markers are NAU2776 and NAU5359. The primer sequences of the linked molecular markers are shown in Table 3.

[0060] Table 2 Comparison of linkage QTL positioning results and QTL-seq analysis results

[0061]

[0062] Note: PVE (%): QTL explains phenotypic variation. Among them, qSAA05.1, qSAD08.1, qSAD10.1 are QTL loci overlapping two QTL positioning methods.

[0063] Table 3 Nucleic acid sequences of molecular linkage marker primers

[0064]

[0065] Other embodiments of the present disclosure will be readily apparent to those skilled in the art upon considering the description set forth herein and the practice of the present disclosure. The present disclosure is intended to cover any variations, uses, or adaptations of the present disclosure following the general principles thereof and including modifications and equivalents of the present disclosure that are obvious to those skilled in the art. The specification and examples are to be regarded as exemplary only, and the true scope and spirit of the present disclosure are indicated by the claims.

[0066] The above is only the preferred embodiment of the present application, and is not used to limit the present application, the patent protection scope of the present application is subject to the claims, any equivalent structural changes made by using the content of the specification of the present application, and the same reason should be included in the protection scope of the present application.

Claims

1. A salt-tolerant QTL site of cotton based on combined screening of linkage QTL positioning and QTL-seq technology, characterized in that, The qSAA05.1 is located at A05: 105.99-106.26 Mb, and can explain 10.42% of the phenotypic variation rate; the qSAD08.1 is located at D08: 46.62-48.95 Mb, and can explain 8.99% of the phenotypic variation rate; and the qSAD10.1 is located at D10: 2.28-3.21 Mb, and can explain 15.36% of the phenotypic variation rate.

2. The screening method of claim 1, wherein the screening method is characterized in that, The method comprises the following steps: S1: Recombinant inbred line population and BC1F2 population were constructed with upland cotton TM-1 as female parent and sea island cotton Hai7124 as male parent, and BC1F2 population was selfed to obtain BC2F 2:3 lines; S2: constructing a recombinant inbred line population and parent salt stress treatment groups and blank control groups in at least three different environments, and determining salt tolerance related traits of the recombinant inbred line population and the parents, and taking the ratio of the salt tolerance related traits in the salt stress treatment groups and the blank control groups as the phenotypic data; the salt tolerance related traits include plant fresh weight, plant dry weight, leaf SPAD value, leaf water content and leaf conductivity; S3: performing genotype analysis on the recombinant inbred line population by using molecular markers, constructing a genetic linkage map, and performing QTL positioning by using the QTL IciMapping 4.2 software and the ICIM-ADD method to obtain salt tolerance QTL sites; S4: Determination of BC1F 2:3 The salt-tolerant related traits of the strains were comprehensively evaluated by membership function value and weight calculation, and the 20 strains with the highest D values and the 20 strains with the lowest D values were selected to construct the salt-tolerant pool and the salt-sensitive pool, respectively. S5: performing whole genome resequencing on the salt tolerance pool and the salt sensitive pool, and obtaining salt tolerance related genomic regions through Δ(SNP-index) and G' value analysis; S6: integrating the salt tolerance QTL sites obtained in S3 and the genomic regions obtained in S5, and screening overlapping QTL sites.

3. The screening method of claim 2, wherein the salt-tolerant QTL site of cotton is screened based on linkage QTL mapping and QTL-seq technology. The different environments in S3 include a spring greenhouse, a summer artificial climate chamber and an autumn greenhouse; and the comprehensive salt tolerance evaluation value in S4 is obtained by integrating the membership function values and weights of the aboveground fresh weight, the aboveground dry weight, the leaf SPAD value, the leaf water content and the leaf conductivity.

4. Application of the cotton salt tolerance QTL site screened based on the combined linkage QTL positioning and QTL-seq technologies in claim 1 in the study of cotton salt tolerance molecular mechanisms.

5. A molecular marker linked to the salt tolerance QTL site of cotton screened in combination with the QTL linkage and QTL-seq technology according to claim 1. The molecular markers linked to the qSAA05.1 are NAU934 and HAU1496, the qSAA05.1 is located in the genetic interval of 123.41-128.46 cM between NAU934 and HAU1496; the molecular markers linked to the qSAD08.1 are NAU2292 and TMD05, the qSAD08.1 is located in the genetic interval of 65.77-72.37 cM between NAU2292 and TMD05; and the molecular markers linked to the qSAD10.1 are NAU2776 and NAU5359, the qSAD10.1 is located in the genetic interval of 0-3.86 cM between NAU2776 and NAU5359. The qSAA05.1 is located at A05: 105.99-106.26 Mb, and can explain 10.42% of the phenotypic variation rate; the qSAD08.1 is located at D08: 46.62-48.95 Mb, and can explain 8.99% of the phenotypic variation rate; and the qSAD10.1 is located at D10: 2.28-3.21 Mb, and can explain 15.36% of the phenotypic variation rate.

6. The molecular marker of claim 5, wherein The forward primer sequence of the NAU934 is SEQ ID NO. 1, and the reverse primer sequence is SEQ ID NO. 2; the forward primer sequence of the HAU1496 is SEQ ID NO. 3, and the reverse primer sequence is SEQ ID NO. 4; the forward primer sequence of the NAU2292 is SEQ ID NO. 5, and the reverse primer sequence is SEQ ID NO. 6; the forward primer sequence of the TMD05 is SEQ ID NO. 7, and the reverse primer sequence is SEQ ID NO. 8; the forward primer sequence of the NAU2776 is SEQ ID NO. 9, and the reverse primer sequence is SEQ ID NO. 10; the forward primer sequence of the NAU5359 is SEQ ID NO. 11, and the reverse primer sequence is SEQ ID NO.

12.

7. Use of the molecular marker of claim 5 in identifying salt tolerance of cotton or breeding salt-tolerant cotton.

8. A method for identifying salt tolerance in cotton plants, characterized by, The method comprises the following steps: S1: extracting genomic DNA of the cotton material to be identified; S2: using the genomic DNA extracted in S1 as a template, performing PCR amplification using the molecular markers NAU934 and HAU1496, NAU2292 and TMD05, and NAU2776 and NAU5359 of claim 5, respectively, to obtain PCR amplification products; S3: detecting the PCR amplification products, and if the cotton material to be identified contains at least one pair of marker amplification fragments identical to those of Gossypium barbadense Hai7124, the material has the corresponding salt tolerance; the more pairs of marker amplification fragments contained, the stronger the salt tolerance.

9. A method of breeding salt tolerant cotton plants, comprising the steps of, The method comprises the following steps: S1: crossing Gossypium barbadense Hai7124 as a donor parent with a target cotton variety as a receptor parent to obtain a progeny population; S2: using the molecular markers of claim 5 to perform molecular marker-assisted selection on the progeny population to screen individuals containing at least one pair of marker amplification fragments identical to those of Gossypium barbadense Hai7124; S3: identifying the salt tolerance of the screened individuals and selecting their agronomic traits to breed a salt-tolerant cotton variety.

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