KASP molecular marker related to lint content and fiber quality of upland cotton and breeding application

Abstract

The invention discloses 1 KASP molecular markers closely linked with cotton fiber length, 6 cotton lint and fiber quality on an A6 chromosome, and a screening method thereof. A plurality of SNP difference sites are identified on an A6 chromosome by using a hybrid cotton Ji 1518 segregation offspring as a material in combination with QTL positioning and high-throughput sequencing, the KASP molecular marker is obtained by screening according to the difference sites, and the KASP molecular marker with a remarkable cotton lint / fiber quality screening effect is further identified by using a cotton backbone parent. The method can be used for high-yield high-quality cotton molecular breeding.

Description

Technical Field

[0001] The invention relates to a KASP molecular marker for cotton breeding, in particular to a KASP molecular marker related to upland cotton lint content and fiber quality. Background Art

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

[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 techniques to improve cotton quality progress slow. With the rapid development of fields such as molecular biology, it has become possible to use modern biotechnology to improve the main target traits of cotton, especially the improvement of fiber quality. Accurately identifying the stable main effect quantitative trait loci (QTL) of fiber quality and mining markers that are closely linked to cotton yield and quality for auxiliary selection are important means to achieve cotton variety improvement. Although a large number of QTLs and various markers have been discovered through forward genetics, the screening effect of most of these markers on traits in breeding practice has yet to be verified.

[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. The KASP markers currently developed for upland cotton lint content, fiber development and other traits are far from sufficient to meet the needs of molecular breeding. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a KASP molecular marker related to upland cotton fiber quality, wherein the molecular marker is a base A / T mutation at base 76645707 on chromosome A6 (KASP6).

[0006] Furthermore, the primers for identifying the above molecular markers include sequences shown as SEQ ID NO.1 to SEQ ID NO.3.

[0007] The present invention also provides an application of the above-mentioned KASP molecular marker related to upland cotton fiber quality in identifying cotton fiber quality, wherein the genome of the sample to be tested is amplified using primers with sequences as shown in SEQ ID NO.1 to SEQ ID NO.3, and the judgment standard is: 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 present invention also provides a kit for identifying the KASP molecular marker related to upland cotton fiber quality, wherein the kit comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO.3.

[0009] The present invention also provides KASP molecular markers related to upland cotton lint content and fiber quality, wherein the molecular markers are mutations at bases 1024897 (KASP1), 1451976 (KASP2), 1734500 (KASP3), 1954523 (KASP4), 5874422 (KASP5) and 12249923 (KASP7) on chromosome A6;

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

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

[0012] Further, primers for identifying KASP molecular markers related to upland cotton lint content and fiber quality are provided, wherein the primers include:

[0013] For example, the first group, SEQ ID NO.4 to SEQ ID NO.6,

[0014] The second group, SEQ ID NO.7 to SEQ ID NO.9,

[0015] The third group, SEQ ID NO.10 to SEQ ID NO.12,

[0016] The fourth group, SEQ ID NO.13 to SEQ ID NO.15,

[0017] The fifth group, SEQ ID NO.16 to SEQ ID NO.18,

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

[0019] The present invention also provides the use of the above-mentioned KASP molecular marker related to upland cotton lint content and fiber quality in identifying cotton lint content and fiber quality, including:

[0020] For example, the first group, SEQ ID NO.4 to SEQ ID NO.6,

[0021] The second group, SEQ ID NO.7 to SEQ ID NO.9,

[0022] The third group, SEQ ID NO.10 to SEQ ID NO.12,

[0023] The fourth group, SEQ ID NO.13 to SEQ ID NO.15,

[0024] The fifth group, SEQ ID NO.16 to SEQ ID NO.18,

[0025] The sixth group, any one or several groups of the sequences shown in SEQ ID NO.19 to SEQ ID NO.21 are primers for amplifying the genome of the sample to be tested, and the judgment criteria are:

[0026] If the 1024897th marker site is base C, it is a strain with high lint content and high micronaire value; if the 1024897th marker site is base G, it is a strain with low lint content and low micronaire value;

[0027] 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;

[0028] If the 1734500th position is base C, it is a strain with high lint content and high micronaire value; if the 1734500th position is base T, it is a strain with low lint content and low micronaire value;

[0029] If the 1954523rd position is base T, it is a strain with high lint content and high micronaire value. If the 1954523rd position is base A, it is a strain with low lint content and low micronaire value.

[0030] 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;

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

[0032] The present invention also provides a kit for identifying the degree of cotton fiber and fiber quality of upland cotton, characterized in that the kit comprises the first group, SEQ ID NO.4 to SEQ ID NO.6,

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

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

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

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

[0037] The sixth group, any one or several groups of primers among the sequences shown in SEQ ID NO.19 to SEQ ID NO.21.

[0038] The above-mentioned screening method for upland cotton KASP marker comprises the following steps:

[0039] 1) Using Ji 1518 to separate F2 and F2:3 populations, we located QTL loci related to yield and fiber quality on chromosome A6;

[0040] 2) Two near-isogenic lines of Ji 1518F2:11, the high-quality line Af and the high-lint line Fy, were selected for whole-genome resequencing. A total of 866 gene sequences on chromosome A6 differed between the two lines, of which 335 genes had both SNP and InDel differences.

[0041] 3) Based on the above SNP and InDel differential sites, multiple sequence differences were screened to develop molecular markers according to physical location and genomic sequence alignment results;

[0042] 4) Using the lint percentage and fiber quality data of 94 cotton backbone parents in 10 environments, the T-test two-tailed test function was used to detect the correlation between markers and lint percentage and fiber quality, and one KASP molecular marker with significant screening effect on fiber quality and six KASP molecular markers with significant screening effect on cotton lint percentage and fiber quality were identified.

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

[0044] In this study, multiple QTL loci related to yield and fiber quality were located on chromosome A6 using F2 and F2:3 populations of Ji 1518, so chromosome A6 was selected as the research focus. Two near-isogenic lines of Ji 1518 F2:11, the high-quality line Af and the high-lint line Fy, were screened for whole-genome resequencing. There were 866 gene sequence differences between the two lines on chromosome A6, of which 335 genes had both SNP and InDel differences. This study used the above sequence differences to develop a batch of KASP molecular markers, and further used the lint and fiber quality data of 94 cotton backbone parents in 10 environments to identify 7 KASP molecular markers on chromosome A6 that had significant screening effects on cotton lint and fiber quality, providing effective resources for cotton molecular design breeding.

[0045] The physical location of the SNP mutation site and the specific primers based on the KASP marker in the present invention are shown in Table 1.

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

[0047]

[0048]

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 JI1518F2、F 2:3 Construction of chromosome genetic map of segregating progeny A6 and location of QTLs for 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, are: 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 Schematic diagram of KASP6 marker typing;

[0058] Fig. 9 Schematic diagram of KASP7 marker typing;

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

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

[0061] Example 1 QTLs related to lint percentage, fiber length, fiber strength, micronaire, etc. were located on chromosome A6. QTLs related to yield and fiber quality were located on chromosome A6 using F2 and F2:3 populations separated by Ji 1518.

[0062] The offspring of Ji 1518 were hybridized to construct F2 and F2:3 segregation populations containing 244 individual plants / lines. The plant height, number of fruit branches, number of bolls, disease resistance, etc. of F2 and F2:3 families (20 plants each) were investigated in the field. Single plants were collected for F2, and 30 bolls were collected for F2:3 families. Boll weight, lint percentage, and seed index were tested indoors. Fiber quality traits include fiber length, fiber strength, micronaire value, elongation, and uniformity. The CTAB method was used to extract total DNA from young leaves of individual plants of the F2 generation. 3989 pairs of SSR primers were screened to construct a linkage map, and Joinmap3.0 was used to determine the linkage relationship of molecular markers. QTLIciMapping3.2 software was used to locate QTLs for yield and quality data, and ICIM ADD mapping analysis was used, with an LOD value of 2.5. The mapping software Map Chart2.2 was used to draw a distribution map of QTLs on linkage groups. Figure 1 As shown, the linden region (qYF-) ( Figure 1 A) and fiber length (qFL-), fiber strength (qFS-), micronaire value (qFM-) ( Figure 1 B) and other related QTLs.

[0063] Example 2: Alignment of A6 chromosome differential sites and development of KASP markers

[0064] The high-generation (F2:11) segregation population of Ji 1518 was used to screen two near-isogenic lines with superior quality (Af) and high lint percentage (Ay). Whole-genome resequencing (sequencing depth 20×) was performed and it was found that Af and Ay had 375,986 SNP variant sites and 243,897 InDel sequences ( Figure 2 ), there are 673 genes with SNP variation sites on chromosome A6, 528 genes with insertion and deletion sequences, and 335 genes with both SNP and InDel sequence differences. Based on the above SNP and InDel difference sites and genome sequence comparison results, sequence differences were screened and a batch of KASP markers were 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, using Figure 1 The upstream and downstream primers of SSR were compared with the reference genome of upland cotton (Gossypiumhirsutum v1.1, https: / / phytozome.jgi.doe.gov) by BLAST (basic local alignment search tool), and the physical information of the associated sites was predicted. The specific method was as follows: the e-value was 1e-10, and the physical position range of the fiber quality and yield QTL sites was preliminarily predicted. The whole genome sequencing information of two near-isogenic lines with high quality (Af) and high lint percentage (Ay) was referenced to screen SNP sites in the target area. The similarity comparison was performed based on the 50bp sequences upstream and downstream of the SNP difference site, and sites with less than 3 similar sequences were selected for subsequent KASP marker development.

[0067] Next, 94 backbone parents of upland cotton from different sources and backgrounds were screened, and the backbone parents were genotyped using the above-mentioned KASP markers. The accurate yield and quality data of the backbone parents (lint percentage, boll weight, boll number, fiber length, fiber strength, micronaire value, fiber elongation, and fiber uniformity) (data after BLUP analysis of 10 environments) were used to detect the correlation between the above-mentioned KASP markers and yield and quality indicators, and 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 ( Figure 3-Figure 9 ) and KASP molecular markers with significant screening effects on cotton lint content and fiber quality ( Fig.10), if the KASP1 marker site is base C, it is a high lint content and high micronaire value strain, if the site is base G, it is a low lint content and low micronaire value strain; if the KASP2 marker site is base A, it is a high lint content and high micronaire value strain, if the site is base T, it is a low lint content and low micronaire value strain; if the KASP3 marker site is base C, it is a high lint content and high micronaire value strain, if the site is base T, it is a low lint content and low micronaire value strain; if the KASP4 marker site is base T, it is If the base at the site is A, the strain with high lint percentage and high micronaire value is low lint percentage and low micronaire value; if the KASP5 marker site is T, it is a strain with high lint percentage and short and weak fibers, and if the site is C, it is a strain with low lint percentage and long and strong fibers; if the KASP6 marker site is T, it is a strain with short fibers, and if the site is A, it is a strain with long fibers; if the KASP7 marker site is Y, it is a strain with long fibers and low lint percentage, and if the site is C, it is a strain with short fibers and high lint percentage. In breeding practice, especially when screening hybrid offspring, these markers can be combined according to different breeding purposes, which helps to achieve directional improvement of specific traits of parents.

Claims

1. KASP molecular markers related to upland cotton fiber quality, characterized in that: The molecular marker is the base A / T mutation at the 76645707th base on chromosome A6.

2. A primer for identifying the molecular marker according to claim 1, characterized in that: The primers include sequences shown as SEQ ID NO.1 to SEQ ID NO.

3.

3. The use of the KASP molecular marker related to upland cotton fiber quality according to claim 1 in identifying cotton fiber quality, characterized in that: The genome of the sample to be tested was amplified using primers with sequences as shown in SEQ ID NO.1 to SEQ ID NO.3, and the judgment standard was: if the 76645707th base was base T, it was a short-fiber strain, and if the 76645707th base was base A, it was a long-fiber strain.

4. 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.

5. KASP molecular markers related to upland cotton lint and fiber quality, characterized in that: The molecular markers 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.

6. The KASP molecular marker associated with upland cotton lint and fiber quality according to claim 5, characterized in that: The markers at the 1024897th base, 1451976th base, 1734500th base, and 1954523rd base are all markers linked to the lint percentage and micronaire value; the marker at the 5874422nd base is a marker linked to the lint percentage, fiber length, and fiber strength; and the marker at the 12249923rd base is a marker linked to the lint percentage and fiber length.

7. A primer for identifying the KASP molecular marker related to lint content and fiber quality of upland cotton according to claim 5, 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: any one or several groups of sequences shown in SEQ ID NO.19 to SEQ ID NO.

21.

8. The use of the KASP molecular marker related to upland cotton lint content and fiber quality in identifying cotton lint content and fiber quality according to claim 5, characterized in that: To 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 for amplifying the genome of the sample to be tested, and the judgment criteria are: If the 1024897th marker site is base C, it is a strain with high lint content and high micronaire value; if the 1024897th marker site is base G, it is a strain with low lint content and low micronaire value; If the 1451976th position is base A, it is a strain with high lint content and high micronaire value; if the 1451976th position is base T, it is a strain with low lint content and low micronaire value; If the 1734500th position is base C, it is a strain with high lint content and high micronaire value; if the 1734500th position is base T, it is a strain with low lint content and low micronaire value; If the 1954523rd position is base T, it is a strain with high lint content and high micronaire value. If the 1954523rd position is base A, it is a strain with low lint content and low micronaire value. 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 percentage strain. If the 12249923rd position is base C, it is a short-fiber, high-lint percentage strain.

9. 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, any one or several groups of primers among the sequences shown in SEQ ID NO.19 to SEQ ID NO.

21.

10. The method for screening KASP markers of upland cotton according to claim 1 or 5, characterized in that: The steps include: 1) Using Ji 1518 to separate F2 and F2:3 populations, we located QTL loci related to yield and fiber quality on chromosome A6; 2) Two near-isogenic lines of Ji 1518F2:11, the high-quality line Af and the high-lint line Fy, were selected for whole-genome resequencing. A total of 866 gene sequences on chromosome A6 differed between the two lines, of which 335 genes had both SNP and InDel differences. 3) Based on the above SNP and InDel differential sites, multiple sequence differences were screened to develop molecular markers according to physical location and genomic sequence alignment results; 4) Using the lint percentage and fiber quality data of 94 cotton backbone parents in 10 environments, the T-test two-tailed test function was used to detect the correlation between markers and lint percentage and fiber quality, and one KASP molecular marker with significant screening effect on fiber quality and six KASP molecular markers with significant screening effect on cotton lint percentage and fiber quality were identified.

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