KASP Molecular Markers Related to Cotton Fiber Length and Strength and Their Applications

Abstract

The present invention discloses a KASP molecular marker related to cotton fiber length and strength, and its application. The molecular marker is an R / G mutation at the 691bp position of the upstream promoter of the GhPAP gene, where R represents A+G. When detecting the genome to be tested with SEQ ID NO.9 and SEQ ID NO.10, if the amplified fragment is A+G at the 691bp position, it is a long and strong fiber strain line; if the amplified fragment is G at the 691bp position, it is a short and weak strain line. According to this sequence difference, a KASP marker is developed, which can significantly distinguish fiber length and strength in a secondary fiber quality segregation population containing 207 strain lines. Identifying it in 96 cotton elite parents further confirms that this marker has a significant effect in identifying fiber length and strength, and can be used for molecular marker-assisted breeding of high-quality cotton to achieve synchronous improvement of the two traits.

Description

Technical Field

[0001] The invention relates to the technical field of cotton fiber length and strength related molecular marker screening, and more specifically to a KASP molecular marker related to cotton fiber length and strength and an application thereof. Background Art

[0002] Cotton is an important economic crop in the world and the most important source of natural fiber for the textile industry. Among them, the annual output of upland cotton (G.hirsutum L.) accounts for about 95% of the world's total cotton output, and the quality of upland cotton directly determines the quality and value of textiles. In recent years, with the promotion of machine-picked cotton and the improvement of people's living standards, the market demand for high-quality cotton has been increasing. However, upland cotton currently generally has problems such as short fibers, low fiber strength, and high micronaire values. 95% of high-quality raw cotton suitable for spinning high-end cotton yarns above 60 counts relies on imports. Therefore, fiber quality improvement is crucial to the high-quality development of my country's cotton industry, and it is also an important goal for the future breeding of cotton varieties in my country.

[0003] The improvement of upland cotton fiber quality mainly involves traits such as length, strength and fineness. There is often a certain degree of negative correlation between these traits and yield, which makes the use of traditional breeding technology to improve cotton quality progress slow. With the rapid development of molecular biology and other fields, the use of sequencing and other technical means to explore and utilize fiber development-related genes and develop functional markers is an important means to improve cotton fiber quality.

[0004] Competitive allele specific PCR (KASP) technology is based on the specific matching of primer terminal bases to make accurate biallelic judgments on SNP sites. It has high accuracy in SNP typing and has the advantages of low cost and high throughput. It can be used for variety identification, genetic map construction, germplasm genetic diversity analysis, molecular marker-assisted breeding, etc. Currently, there are few KASP markers developed for genes related to upland cotton fiber development.

[0005] Therefore, mining the allelic variation of genes related to fiber development and how to develop KASP molecular markers related to cotton fiber length and strength based on sequence differences are issues that technicians in this field need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides KASP molecular markers related to cotton fiber length and strength and applications.

[0007] On the one hand, an embodiment of the present invention provides a KASP molecular marker related to cotton fiber length and strength, wherein the molecular marker is an R / G mutation at the 691 bp position of the upstream promoter of the GhPAP gene, wherein R represents A+G.

[0008] In the second aspect of the embodiments of the present invention, there is provided an application of the described KASP molecular marker in identifying cotton fiber length and strength.

[0009] In a preferred embodiment, the genomic DNA to be tested is detected with SEQ ID NO.9 and SEQ ID NO.10. If the amplified fragment is A+G at 691bp, it is a long and strong fiber line; if the amplified fragment is G at 691bp, it is a short and weak line.

[0010] Primer Seq Adllele X: GAAGGTGACCAAGTTCATGCTCACTTTGCCC ACCGTCACTACY, SEQ ID NO.9;

[0011] Primer Seq Adllele Y: GAAGGTCGGAGTCAACGGATTACTTTGCCCA CCGTCACTACC, SEQ ID NO.10.

[0012] In the third aspect of the embodiments of the present invention, there is provided an application of the described KASP molecular marker in cotton breeding.

[0013] As can be seen from the above technical solutions, compared with the prior art, the present invention uses a fiber quality segregating population of hybrid cotton Ji 1518, and uses bulked segregant resequencing to perform association analysis on fiber quality. A total of 4.54 Mb of candidate regions were obtained on chromosomes A6 and D6, which contained 187 candidate genes. At the same time, transcriptome sequencing was performed on the fibers of the long and strong line Adh and the short and weak line Adl of the segregating progeny of Ji 1518. Among the above 187 genes, it was found that a PAP_fibrillin gene GhPAP was lowly expressed during the fiber development of the long and strong line Adh, but highly expressed during the fiber development of the short and weak line Adl. Moreover, there was a sequence difference (R / G) of one base in the 691bp promoter region upstream of this gene between Adh and Adl. When this site was base R, it was a long and strong line; when this site was base G, it was a short and weak line. According to this sequence difference, a KASP marker was developed, which could significantly distinguish fiber length and strength in a secondary fiber quality segregating population containing 207 lines. Identifying it in 96 cotton elite parents further confirmed that this marker had a significant effect in identifying fiber length and strength, and could be used for molecular marker-assisted breeding of high-quality cotton to achieve synchronous improvement of the two traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0015] Figure 1 The drawing is an amplification diagram of the coding region (CDS) and upstream promoter (PRO) of the gene GhPAP.

[0016] Figure 2 The drawing is the structural analysis of the GhPAP gene.

[0017] Figure 3 The drawing is the relative expression levels of GhPAP at different fiber development stages in the long and strong strain Adh and the short and weak strain Adl.

[0018] Figure 4 The drawing is the GhPAP-R / G genotyping map of the secondary F 2:3 population by the KASP marker of the present invention.

[0019] Figure 5 The drawing is the extremely significant correlation diagram of GhPAP-R / G genotyping with fiber length (A) and fiber strength (B) in the secondary population.

[0020] Figure 6 The drawing is the GhPAP-R / G genotyping map of the backbone parents by the KASP marker of the present invention.

[0021] Figure 7 The drawing is the extremely significant correlation diagram of GhPAP-R / G genotyping with fiber length (A) and fiber strength (B) in the cotton backbone parents. Detailed implementation manners

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0023] Example 1 Identification of the GhPAP gene related to cotton fiber development

[0024] (1) Population construction and phenotype determination

[0025] Using the parents Ji 228 × Ji 567 for hybridization to obtain the F1 generation (Ji 1518). The fiber quality data of the above materials are shown in Table 1. Individual plants of F1 were self-crossed to construct an F2 segregating population containing 244 individual plants. At the harvest stage, 15 g fiber samples of each of the parents and 244 individual plants were harvested and sent to the Cotton Quality Supervision and Inspection Test Center of the Ministry of Agriculture and Rural Affairs to detect fiber length and fiber strength.

[0026] Table 1 Fiber quality data of parents and F1 (average of three years)

[0027] Material Fiber length Fiber strength Micronaire value Ji 228 31.24±0.11 33.50±0.21 4.43±0.11 Ji 567 28.20±0.20 28.90±0.44 4.94±0.07 <![CDATA[F1 (Ji 1518)]]> 30.73±0.14 32.53±0.48 4.95±0.07

[0028] (2) Construction of fiber length / strength extreme material bulk pools and resequencing

[0029] Extract the leaf DNA of parents and individual plants. From 244 F2 population materials, 20 extreme plants with long fiber length and high strength and 20 extreme plants with short fiber length and low strength were screened respectively to construct bulk pools. At the same time, bulk pool resequencing was performed on the parents Ji 228, Ji 567 and two fiber length / strength bulk pools. The sequencing depth was 10×, and the analysis was carried out with reference to cotton genome information (WHU-updated v1, Huang et Adl., 2020).

[0030] (3) Transcriptome sequencing of fiber development stages of long fiber length and high strength line Adh and short fiber length and low strength line Adl

[0031] In the high-generation (F 2:10 ) segregating population of Ji 1518 (F1), the long fiber length and high strength line Adh and the short fiber length and low strength line Adl were screened (Table 2). Fibers of Adh and Adl at different fiber development stages (0 DPA, 5 DPA, 10 DPA, 15 DPA, 20 DPA and 30 DPA) were taken for transcriptome sequencing. Sequence alignment was performed with reference to cotton genome information (WHU-updated v1, Huang et Adl., 2020). The DESeq R software package (version 1.10.1) was used to identify differentially expressed genes with the parameter |log2FoldChange| > 1.5, and qRT-PCR was used to verify the spatio-temporal specific expression of the differentially expressed genes.

[0032] Table 2 Fiber quality data of long fiber length and high strength line A dh and short fiber length and low strength line A dl (average of three years)

[0033]

[0034] Research results: Resequencing was performed on the mixed pools of 228, Ji 567, and extreme materials for fiber length / strength. Using the ED distance and SNP-index association algorithms, three candidate regions for fiber length and strength, totaling 4.54 Mb, were obtained. One of them is located on chromosome A6, and two are on chromosome D6, containing a total of 187 candidate genes (Table 3). According to the transcriptome and sequencing results of the fiber development stages of the long and strong strain Adh and the short and weak strain Adl for fiber quality, among the above 187 genes, only one gene, GhPAP, encoding PAP_fibrillin, was differentially expressed during fiber development in the long and strong strain Adh and the short and weak strain Adl (Table 4), and the role of this gene or its homologous genes in fiber development has not been reported.

[0035] Table 3 Candidate regions for fiber length / strength obtained by resequencing of mixed pools

[0036]

[0037] Table 4 Differential expression and annotation information of GhPAP

[0038]

[0039] Example 2 Development and verification of KASP markers

[0040] (1) Amplification, verification of differential expression, and obtaining of differential sites of the GhPAP gene

[0041] The full-length gene GhPAP was cloned from the cDNA of the leaves of Adh and Adl using homologous cloning. At the same time, the promoter region approximately 1800 bp upstream of GhPAP was amplified using the DNA of the leaves of Adh and Adl, respectively. The amplification primers are as follows:

[0042] CDS-F: ATGGCAGCTTGTTCCCTC, SEQ ID NO.1;

[0043] CDS-R: TTAAACGTTGGTTGAGCCTCC, SEQ ID NO.2;

[0044] PRO-F: AAGAAGTAGCTTTGACAGTTGTG, SEQ ID NO.3;

[0045] PRO-R: GGCCGCCGGATATCCCAAA, SEQ ID NO.4.

[0046] Specific method: Primers were designed using Primer Premier 6.25 (PREMIER Biosoft International, San Francisco, USA), and the GhPAP gene was amplified. The amplification results are shown in Figure 1 ; After purification of the PCR product, the product was ligated into the TOPO-TA / Blunt simple vector PT0102 (LABLE AD, Beijing, China) and transformed into competent Escherichia coli TOP10 cells. Eight monoclonal clones were randomly selected from each gene and then subjected to Sanger sequencing (Sangon Biotech, Shanghai, China). The sequences of the genes were aligned using MEGA 7.0 (Kumar et al., 2016).

[0047] Result analysis: The full-length ORF of the GhPAP gene is 708 bp, encoding 236 amino acids. There is no difference in the coding region of this gene between Adh and Adl ( Figure 2 ); At the same time, it was found that there is a base difference 691 bp upstream of the GhPAP gene (physical position D0626664158). It is R (A+G) in Adh of long and strong fibers and G in Adl of short and weak fibers ( Figure 2 ).

[0048] ORF: >GhPAP[mRNA] locus = D06:26664849:26665955:+

[0049] ATGGCAGCTTGTTCCCTCACTCTATCGTCCTCCTTGCCCCAACCTAAACCCTCATTTTTTGGATCGAAACCCTCTCATCTATCCTTACACAGCACTTCGTTTGCGCTCAAATCCCAGTGTTTCAGGGTTTCCTCAAGTTCCGTTTCCATCTCCAGTCGCCCCGCCGATGATCTAGTTGCATCTCTTCTCTCCAAGGTGATAAAAACGGATGGTGGAGTTTCACTCACCACAAAACAGCACCAAGAGGTAGCTCAAGTGGCTAATGAGTTGAACAAATATTGCGTTGATGAACCAGTCAAATGCCCTTTAATCTTCGGAGATTGGGATGTGGTTTACTGTTCGAATCCCACGTCACCAGGAGGCGGCTACAGAAGTGCATTGGGGCGCCTTTTCTTCAAGACCAAGGACATGGTCCAGGCTGTTGAAGCTCCTGACTCCGTACGAAACAAAGTCTCCTTCTCTGTTTTTGGGTTTCTTGAGGGAGAGGTCTCCTTGAAAGGAAAGCTGAAGGTCTTGGATCATCAATGGATTCAAGTCATTTTTCAGCCACCTGAACTGAGGGTAGGAGCAATGGACTTCCAGTATGGTGGCGAGAGTGAGGTCAAGCTACAGATCACGTATATTGACGAGAAGATCAGATTAGGAAAGGGCTCTAGAGGTTCTTTATTTGTATTTCGAAGGCGGCACGGAGGCTCAACCAACGTTTAA, SEQ ID NO.5。

[0050] Amino acids: >GhPAP [mRNA] locus=D06:26664849:26665955:+

[0051] MAACSLTLSSSLPQPKPSFFGSKPSHLSLHSTSFALKSQCFRVSSSSVSISSRPADDLVASLLSKVIKTDGGVSLTTKQHQEVAQVANELNKYCVDEPVKCPLIFGDWDVVYCSNPTSPGGGYRSALGRLFFKTKDMVQAVEAPDSVRNKVSFSVFGFLEGEVSLKGKLKVLDHQWIQVIFQPPELRVGAMDFQYGGESEVKLQITYIDEKIRLGKGSRGSLFVFRRRHGGSTNV*, SEQ ID NO.6。

[0052] Furthermore, using the cDNA of Adh and Adl leaves as templates, amplification was carried out using qRT-PCR primers. The primer information is as follows:

[0053] F: AATCTTCGGAGATTGGGATGTG, SEQ ID NO.7;

[0054] R: ACCTTCAGCTTTCCTTTCAAGG, SEQ ID NO.8.

[0055] Verify that GhPAP is lowly expressed during the formation of fiber length and strength in the long and strong line Adh, while highly expressed in the short and weak line Adl at the same period ( Figure 3 ).

[0056] (2) Development and preliminary verification of KASP markers

[0057] For the SNP difference site 691 bp upstream of the GhPAP gene, KASP marker sites were developed. Using Primer3, PCR 3'-end amplification primers were designed based on the SNP site. The primer Tm value was 63°C. Two SNP-specific primers and one common primer were designed. The sequence information is as follows:

[0058] Primer Seq Adllele X: GAAGGTGACCAAGTTCATGCTCACTTTGCCC ACCGTCACTACY, SEQID NO.9;

[0059] Primer Seq Adllele Y: GAAGGTCGGAGTCAACGGATTACTTTGCCCA CCGTCACTACC, SEQID NO.10;

[0060] Primer Seq common: GTGGCTTGGACTTGGCATGTGTTAT, SEQ ID NO.11.

[0061] Construct a secondary F population containing 207 lines by crossing Adh and Adl, plant it in a total of 4 environments at 2 locations for 2 years, and measure the fiber length and strength of each material respectively according to the above method. Use BLUP (Best Linear Unbiased Prediction) analysis to obtain the phenotypic data of fiber length and strength to reduce the influence of different environments on fiber quality. At the same time, extract the DNA of the above 207 lines for KASP genotyping detection. The KASP-PCR reaction is carried out on the Douglas Array 2:3 Platform, read the fluorescence signal on the ARAYA fluorescence reader, and then import the results into the database. Perform sample SNP genotyping on the Douglas Scientific Dashboard according to the principles of clear typing and no specific amplification in NTC (no sample negative control). The results show that: KASP in the present invention can accurately identify the GhPAP-R / G genotype ( ), and the above genotype is significantly correlated with fiber length and length ( Figure 4 ). Figure 5 )

[0062] (3) Further verify the screening effect of KASP markers on the fiber length and strength of cotton backbone parents

[0063] Screen a total of 92 backbone parent materials of various types, perform BLUP analysis based on the fiber quality data from 2013 to 2023, use the above KASP markers to genotype the backbone parents, and verify the effects of the above KASP markers on fiber length and strength. The results show that this marker can accurately identify the GhPAP-R / G genotype in the backbone parents ( Figure 6 ), and it has a significant effect on identifying fiber length and strength in the backbone parents ( Figure 7 ). Therefore, the KASP markers developed according to fiber development-related genes in the present invention have relatively wide applicability, can be used for molecular marker-assisted breeding of high-quality cotton, and are expected to achieve simultaneous improvement of the two traits.

[0064] Each embodiment in this specification is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same and similar parts among the embodiments can be referred to each other. The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer pair for identifying KASP molecular markers related to cotton fiber length and strength, characterized in that the KASP molecular marker is an R / G mutation at the 691bp of the upstream promoter of the GhPAP gene, where R represents A+G; the nucleotide sequences of the primer pair are as follows: Primer Seq Adllele X: GAAGGTGACCAAGTTCATGCTCACTTTGCCCACCGTCACTACY, SEQ IDNO.9; Primer Seq Adllele Y: GAAGGTCGGAGTCAACGGATTACTTTGCCCACCGTCACTACC, SEQ IDNO.10; Primer Seq common: GTGGCTTGGACTTGGCATGTGTTAT, SEQ ID NO.11; the cotton is upland cotton.

2. Use of the primer pair according to claim 1 in identifying the length and strength of cotton fibers, characterized in that, When detecting the genome to be tested with SEQ ID NOs. 9 to 11, if the 691bp of the upstream promoter of the amplified GhPAP gene is A+G, it is a long and strong fiber strain line; if the 691bp of the upstream promoter of the amplified GhPAP gene is G, it is a short and weak strain line; the cotton is upland cotton.

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