KASP molecular markers associated with yield and fiber quality of upland cotton and breeding application

Abstract

The application discloses KASP molecular markers closely linked with cotton fiber length on chromosome A6, six cotton lint percentage and fiber quality, and a screening method thereof. The application uses hybrid cotton J1518 separation offspring as material, combines QTL positioning and high-throughput sequencing to identify multiple SNP difference sites on chromosome A6, screens the KASP molecular markers according to the difference sites, and further identifies the KASP molecular markers with remarkable screening effect on cotton lint percentage and fiber quality by using cotton backbone parents, so that the KASP molecular markers can be used for high-yield and high-quality cotton molecular breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to KASP molecular markers for cotton breeding, in particular to KASP molecular markers related to lint percentage and fiber quality of Gossypium hirsutum. BACKGROUND

[0002] Gossypium hirsutum L. is the most important natural fiber crop in the world. With the innovation of textile technology, the textile industry has increasingly high requirements for cotton fiber quality. At the same time, as one of the most important indicators of cotton yield, the improvement of lint percentage is crucial for the improvement of lint yield. At present, the main production varieties still have the problems of low lint percentage and quality deviation. Therefore, breeding high-lint high-quality varieties is one of the important tasks of cotton breeding.

[0003] Cotton fiber quality improvement mainly involves length, strength and fineness, etc. There is a certain degree of negative correlation between these traits and yield, which makes it slow to improve cotton quality by using traditional breeding techniques. With the rapid development of molecular biology and other fields, it has become possible to use modern biotechnology to improve the main target traits of cotton, especially the improvement of fiber quality. Precise identification of stably existing major quantitative trait loci (QTL) of fiber quality and mining of markers closely linked to cotton yield and quality for assisted selection are important means to realize cotton variety improvement. At present, although a large number of QTL and various markers have been mined through forward genetics, the screening effect of most of these markers on traits in breeding practice still needs to be verified.

[0004] The Kompetitive Allele specific PCR (KASP) technology is based on the specific matching of primer terminal bases to accurately determine the SNP site with double alleles. It has high accuracy in SNP typing, and has the advantages of low cost and high throughput, which can be used in variety identification, genetic map construction, germplasm genetic diversity analysis, molecular marker assisted breeding, etc. At present, the KASP markers developed for lint percentage, fiber development and other traits of Gossypium hirsutum are far from meeting the needs of molecular breeding. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a KASP molecular marker related to the fiber quality of Gossypium hirsutum, which is a base A / T mutation at the 76645707th base of chromosome A6 (KASP6).

[0006] Further, the primers for identifying the above-mentioned molecular marker include the sequences shown in SEQ ID NO. 1-SEQ ID NO. 3.

[0007] The application also provides application of the KASP molecular marker related to fiber quality of Gossypium hirsutum in identifying cotton fiber quality, which amplifies the genome of the sample to be tested by using the primer pair with the sequence shown as SEQ ID NO. 1-3, and the judgment standard is that if the 76645707th base is base T, it is a short fiber strain, and if the 76645707th site is base A, it is a long fiber strain.

[0008] The application also provides a kit for identifying the KASP molecular marker related to fiber quality of Gossypium hirsutum, which comprises the primer with the sequence shown as SEQ ID NO. 1-3.

[0009] The application also provides a KASP molecular marker related to fiber quality of Gossypium hirsutum, which is a mutation at the 1024897th base (KASP1), the 1451976th base (KASP2), the 1734500th base (KASP3), the 1954523th base (KASP4), the 5874422th base (KASP5) and the 12249923th base (KASP7) on the A6 chromosome.

[0010] The 1024897th base is a base C / G mutation, the 1451976th base is a base A / T mutation, the 1734500th base is a base C / T mutation, the 1954523th base is a base A / T mutation, the 5874422th base is a base C / T mutation, and the 12249923th base is a base C / Y (C+T) mutation.

[0011] Further, the markers at the 1024897th base, the 1451976th base, the 1734500th base and the 1954523th base are markers linked to lint percentage and micronaire value; the marker at the 5874422th base is a marker linked to lint percentage, fiber length and fiber strength; and the marker at the 12249923th base is a marker linked to lint percentage and fiber length.

[0012] Further, the primer for identifying the KASP molecular marker related to lint percentage and fiber quality of Gossypium hirsutum comprises:

[0013] such as the first group, SEQ ID NO. 4-SEQ ID NO. 6,

[0014] the second group, SEQ ID NO. 7-SEQ ID NO. 9,

[0015] the third group, SEQ ID NO. 10-SEQ ID NO. 12,

[0016] The fourth group is any one or several groups of sequences shown in SEQ ID NO. 13 to SEQ ID NO. 15.

[0017] The fifth group is any one or several groups of sequences shown in SEQ ID NO. 16 to SEQ ID NO. 18.

[0018] The sixth group is any one or several groups of sequences shown in SEQ ID NO. 19 to SEQ ID NO. 21.

[0019] The application also provides application of the KASP molecular markers related to lint percentage and fiber quality of upland cotton in identifying lint percentage and fiber quality of cotton, which comprises:

[0020] The first group is any one or several groups of sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6.

[0021] The second group is any one or several groups of sequences shown in SEQ ID NO. 7 to SEQ ID NO. 9.

[0022] The third group is any one or several groups of sequences shown in SEQ ID NO. 10 to SEQ ID NO. 12.

[0023] The fourth group is any one or several groups of sequences shown in SEQ ID NO. 13 to SEQ ID NO. 15.

[0024] The fifth group is any one or several groups of sequences shown in SEQ ID NO. 16 to SEQ ID NO. 18.

[0025] The sixth group is any one or several groups of sequences shown in SEQ ID NO. 19 to SEQ ID NO. 21.

[0026] If the 1024897 locus is base C, it is a high-lint high-micronaire value strain, and if the 1024897 locus is base G, it is a low-lint low-micronaire value strain.

[0027] If the 1451976 locus is base A, it is a high-lint high-micronaire value strain, and if the 1451976 locus is base T, it is a low-lint low-micronaire value strain.

[0028] If the 1734500 locus is base C, it is a high-lint high-micronaire value strain, and if the 1734500 locus is base T, it is a low-lint low-micronaire value strain.

[0029] If the 1954523 locus is base T, it is a high-lint high-micronaire value strain, and if the 1954523 locus is base A, it is a low-lint low-micronaire value strain.

[0030] If the 5874422 site is base T, it is a high lint fiber short weak strain, if the 5874422 site is base C, it is a low lint fiber long strong strain;

[0031] If the 12249923 site is base Y (C+T), it is a long fiber, low lint strain, if the 12249923 site is base C, it is a short fiber, high lint strain.

[0032] The application also provides a kit for identifying the lint degree and fiber quality of Gossypium hirsutum, characterized in that the kit comprises any one or more of the primers shown in the following sequences:

[0033] The second group, SEQ ID NO. 7-SEQ ID NO. 9,

[0034] The third group, SEQ ID NO. 10-SEQ ID NO. 12,

[0035] The fourth group, SEQ ID NO. 13-SEQ ID NO. 15,

[0036] The fifth group, SEQ ID NO. 16-SEQ ID NO. 18,

[0037] The sixth group, SEQ ID NO. 19-SEQ ID NO. 21.

[0038] The screening method of the above-mentioned KASP marker of Gossypium hirsutum comprises the following steps:

[0039] 1) Using J1518 to separate F2 and F2:3 populations to locate QTL sites related to yield and fiber quality on chromosome A6;

[0040] 2) Screening two near-isogenic lines of J1518 F2:11, high-quality strain Af and high lint strain Fy for whole genome resequencing, the above-mentioned two strains have 866 gene sequences different on chromosome A6, among which 335 genes have both SNP and InDel differences;

[0041] 3) According to the above-mentioned SNP and InDel difference sites, according to the physical position and genome sequence alignment results, a plurality of sequence differences are developed into molecular markers;

[0042] 4) Using the lint and fiber quality data of 94 cotton core parents in 10 environments, using T-test two-tailed test function to detect the correlation between the markers and the lint and fiber quality, 1 KASP molecular marker with significant screening effect on fiber quality and 6 KASP molecular markers with significant screening effect on cotton lint and fiber quality are identified.

[0043] The beneficial effects of the present invention are:

[0044] This study used F2 and F2:3 populations of Ji 1518 to locate multiple QTL loci related to yield and fiber quality on chromosome A6, thus selecting chromosome A6 as the research focus. Two near-isogenic lines of Ji 1518 F2:11—the high-quality line Af and the high-lint percentage line Fy—were selected for whole-genome resequencing. These two lines shared 866 gene sequence differences on chromosome A6, 335 of which contained both single-nucleotide polymorphisms (SNPs) and indels. This study leveraged these sequence differences to develop a set of KASP molecular markers. Furthermore, using lint percentage and fiber quality data from 94 cotton backbone parents across 10 environments, seven KASP molecular markers on chromosome A6 with significant screening effects on lint percentage and fiber quality were identified, providing an effective resource for molecular design breeding of cotton.

[0045] The physical locations of the SNP mutation sites and specific primers used as the basis for the KASP markers in the present invention are shown in Table 1.

[0046] Table 1 Related information of the 7 KASP markers in the present invention

[0047]

[0048]

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 J1518F2, F 2:3 Construction of chromosome genetic map of segregating progeny A6 and QTL mapping of quality (A) and yield (B) related traits;

[0051] Figure 2 The distribution of structural variations in the Af and Ay genomes, from outside to inside, is as follows: chromosome, SNP, InDel;

[0052] Figure 3 Schematic diagram of KASP1 marker typing;

[0053] Figure 4 Schematic diagram of KASP2 marker typing;

[0054] Figure 5 Schematic diagram of KASP3 marker typing;

[0055] Figure 6 Schematic diagram of KASP4 marker typing;

[0056] Figure 7 Schematic diagram of KASP5 marker typing;

[0057] Figure 8 KASP6 marker genotyping schematic diagram;

[0058] Figure 9 KASP7 marker genotyping schematic diagram;

[0059] Figure 10 Screening effect of 7 KASP markers on 94 core parent yield and fiber quality traits (P < 0.01). DETAILED DESCRIPTION

[0060] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0061] Example 1 Positioning of QTLs related to lint percentage and fiber length, fiber strength, micronaire on chromosome A6

[0062] Positioning of QTLs related to yield and fiber quality on chromosome A6 using J1518 F2 and F2:3 populations Positioning of QTLs related to yield and fiber quality on chromosome A6 using J1518 F2 and F2:3 populations J1518 offspring were crossed to construct F2 and F2:3 separation populations containing 244 single plants / strains. The plant height, fruit branch number, boll number, disease resistance, etc. of F2 and F2:3 families (20 plants each) were investigated in the field. F2 single plants were collected, and F2:3 family plants were mixed and collected 30 bolls. Boll weight, lint percentage, seed index, fiber length, fiber strength, micronaire, elongation rate, and uniformity were measured indoors. The total DNA of F2 generation single plants was extracted from young leaves using the CTAB method. 3989 pairs of SSR primers were screened to construct a linkage map, and the linkage relationship of molecular markers was determined using Joinmap3.0. QTL IciMapping3.2 software was used for QTL positioning of yield and quality data, ICIM ADD mapping analysis was used, and the LOD value was 2.5. Map Chart 2.2 software was used to draw the distribution map of QTLs on the linkage group. As shown in Table 1, QTLs related to lint percentage (qYF-) (A) and fiber length (qFL-), fiber strength (qFS-), micronaire (qFM-) (B) were positioned on chromosome A6. Figure 1 Figure 1 Figure 1

[0063] Example 2 Comparison of different sites on chromosome A6 and development of KASP markers

[0064] ​​​The high-generation (F2:11) segregating population of Ji 1518 was used to screen two near-isogenic lines with high quality (Af) and high lint content (Ay). Whole-genome resequencing (sequencing depth 20×) was performed, and it was found that Af and Ay had a total of 375,986 SNP variant sites and 243,897 InDel sequences ( Figure 2 ) On chromosome A6, 673 genes harbored SNP variants, 528 genes harbored insertion / deletion sequences, and 335 genes harbored both SNP and InDel sequence differences. Based on these SNP and InDel differential sites and genomic sequence alignment results, a series of KASP markers were screened for sequence differences and developed.

[0065] Example 3 Development and identification of KASP molecular markers linked to cotton lint percentage, fiber quality and other traits

[0066] according to Figure 1 QTL mapping results were obtained using Figure 1 BLAST (basic local alignment search tool) alignments of SSR upstream and downstream primers were performed against the upland cotton reference genome (Gossypium hirsutum v1.1, https: / / phytozome.jgi.doe.gov) to predict the physical location of the associated loci. The specific method was to use an e-value of 1e-10 to preliminarily predict the physical location range of fiber quality and yield QTL loci. Using whole-genome sequencing data from two near-isogenic lines with superior quality (Af) and high lint percentage (Ay), SNPs were screened in the target regions. Sequences within 50 bp upstream and downstream of the SNP differential loci were then compared for similarity, and sites with fewer than 3 similar sequences were selected for subsequent KASP marker development.

[0067] Next, 94 backbone parents of upland cotton from different backgrounds were screened and genotyped using the above-mentioned KASP markers. The accurate yield and quality data (lint percentage, boll weight, boll number, fiber length, fiber strength, micronaire value, fiber elongation, and fiber uniformity) of the backbone parents (data after BLUP analysis of 10 environments) were used to detect the correlation between the above-mentioned KASP markers and yield and quality indicators. The practical effect of the above-mentioned KASP markers on the traits was evaluated using the T-test two-tailed test function (P<0.01).

[0068] The results showed that a total of 7 marker loci were successfully identified and typed ( Figures 3-9 ) and has a significant effect on cotton lint content and fiber quality screening KASP molecular markers ( Figure 10), if the KASP1 marker site is base C, it is a high lint percentage and high micronaire value line, if the site is base G, it is a low lint percentage and low micronaire value line; if the KASP2 marker site is base A, it is a high lint percentage and high micronaire value line, if the site is base T, it is a low lint percentage and low micronaire value line; if the KASP3 marker site is base C, it is a high lint percentage and high micronaire value line, if the site is base T, it is a low lint percentage and low micronaire value line; if the KASP4 marker site is base T, it is a high lint percentage and high micronaire value line, if the site is base A, it is a low lint percentage and low micronaire value line; if the KASP5 marker site is base T, it is a high lint percentage and short weak fiber line, if the site is base C, it is a low lint percentage and long strong fiber line; if the KASP6 marker site is base T, it is a short fiber line, if the site is base A, it is a long fiber line; if the KASP7 marker site is base Y, it is a long fiber and low lint percentage line, if the site is base C, it is a short fiber and high lint percentage line. In breeding practice, especially in screening of hybrid offspring, these markers can be combined according to different breeding purposes, which is helpful to realize the directional improvement of specific traits of parents.

Claims

1. Primers for KASP molecular markers related to upland cotton fiber quality, characterized in that: The primers include sequences shown as SEQ ID NO. 1 to SEQ ID NO.

3.

2. A kit for identifying the KASP molecular marker associated with upland cotton fiber quality according to claim 1, characterized in that: The kit includes primers with sequences shown as SEQ ID NO. 1 to SEQ ID NO.

3.

3. A primer for identifying KASP molecular markers related to lint content and fiber quality in upland cotton, characterized in that: The primers include: For example, the first group, SEQ ID NO. 4 to SEQ ID NO. 6, The second group, SEQ ID NO. 7 to SEQ ID NO. 9, The third group, SEQ ID NO. 10 to SEQ ID NO. 12, The fourth group, SEQ ID NO. 13 to SEQ ID NO. 15, The fifth group, SEQ ID NO. 16 to SEQ ID NO. 18, The sixth group includes any one or more of the sequences shown in SEQ ID NO. 19 to SEQ ID NO.

21.

4. Application of primers for KASP molecular markers related to upland cotton lint content and fiber quality in identifying cotton lint content and fiber quality, characterized in that: include: For example, the first group, SEQ ID NO. 4 to SEQ ID NO. 6, The second group, SEQ ID NO. 7 to SEQ ID NO. 9, The third group, SEQ ID NO. 10 to SEQ ID NO. 12, The fourth group, SEQ ID NO. 13 to SEQ ID NO. 15, The fifth group, SEQ ID NO. 16 to SEQ ID NO. 18, The sixth group, any one or more of the sequences shown in SEQ ID NO. 19 to SEQ ID NO. 21 are primers, The KASP molecular markers related to upland cotton lint content and fiber quality are mutations at bases 1024897, 1451976, 1734500, 1954523, 5874422, and 12249923 on chromosome A6; wherein base 1024897 is a C / G mutation, base 1451976 is an A / T mutation, base 1734500 is a C / T mutation, base 1954523 is an A / T mutation, base 5874422 is a C / T mutation, and base 12249923 is a C / Y mutation; The markers at bases 1024897, 1451976, 1734500, and 1954523 are all markers linked to lint percentage and micronaire value; the marker at base 5874422 is a marker linked to lint percentage, fiber length, and fiber strength; the marker at base 12249923 is a marker linked to lint percentage and fiber length; The genome of the sample to be tested is amplified, and the judgment criteria are: If the 1024897th marker site is a C, it is a high lint content and high micronaire value strain; if the 1024897th marker site is a G, it is a low lint content and low micronaire value strain; If the 1451976th position is base A, it is a high lint content and high micronaire value strain; if the 1451976th position is base T, it is a low lint content and low micronaire value strain; If the 1734500th position is a C, it is a high lint content and high micronaire value strain; if the 1734500th position is a T, it is a low lint content and low micronaire value strain; If the 1954523rd position is a T, it is a high lint content and high micronaire value strain; if the 1954523rd position is an A, it is a low lint content and low micronaire value strain; If the 5874422nd position is base T, it is a strain with high lint content and short and weak fibers; if the 5874422nd position is base C, it is a strain with low lint content and long and strong fibers; If the 12249923rd position is base Y, it is a long-fiber, low-lint-content strain; if the 12249923rd position is base C, it is a short-fiber, high-lint-content strain; The reference genome of upland cotton is Gossypium hirsutum v1.

1.

5. A kit for identifying the degree of cotton clothing and fiber quality, characterized in that: The kit comprises: For example, the first group, SEQ ID NO. 4 to SEQ ID NO. 6, The second group, SEQ ID NO. 7 to SEQ ID NO. 9, The third group, SEQ ID NO. 10 to SEQ ID NO. 12, The fourth group, SEQ ID NO. 13 to SEQ ID NO. 15, The fifth group, SEQ ID NO. 16 to SEQ ID NO. 18, The sixth group includes any one or more primers selected from the group consisting of SEQ ID NO. 19 to SEQ ID NO. 21.

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