CN117286286A - Molecular marker closely linked with cotton boll weight character and application thereof - Google Patents
Molecular marker closely linked with cotton boll weight character and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with the cotton boll weight character and application thereof, wherein the molecular marker closely linked with the cotton boll weight character is BB575 or/and BB578; the molecular markers BB575 and BB578 are adopted to carry out molecular marker selection in breeding populations related to Raymond cotton or L3077, so that the bell weight can be improved by more than 20%. The method of marker assisted selection can rapidly improve the boll weight of the existing upland cotton varieties, and greatly accelerate the cultivation and seed industrialization process of cotton high-yield new varieties in China.
Description
Technical Field
The invention belongs to the field of biotechnology breeding, and particularly relates to a molecular marker derived from tight linkage of Raymond cotton and cotton boll weight characteristics and application thereof.
Background
In the long-term cotton breeding practice, excellent genes of yield constituent factors are continuously mined, selected and utilized, the single yield level is steadily improved, the total yield is maintained relatively stable under the condition of reduced cotton area, the cotton demand of China is ensured, and the method also contributes to the expansion of area and the increase of total yield of food crops in China. However, due to the reduced genetic diversity, cotton yield improvement gradually enters a platform stage, and the excavation and utilization of heterologous excellent genes of cotton related species become an important way for breaking through the bottleneck of cotton high-yield breeding. When the total number of rings per unit area breaks through difficultly under the current high-density planting, the weight of the rings of the important factors of the yield becomes an important improvement target. In recent years, a large number of bell weight QTLs have been identified, but due to low genetic diversity, the QTL contribution rate is low and the gain effect is limited, in particular, the bell weight increasing major effective site or gene is still not found.
The patent applicant has studied for more than 10 years and utilized diploid wild cotton of Raymond's cotton @G. raimondii2n=2x=26=d5d5) and cultivar upland cotton @G. hirsutumDistant hybridization germplasm innovation is carried out by 2n=4x=52=aadd, the interspecific obstacle is overcome, an artificial heterohexaploid is created, continuous backcrossing is carried out with upland cotton, auxiliary selection is carried out by combining with developed molecular markers, a group of upland cotton-Raymond cotton chromosome segment substitution lines composed of 265 families such as BC3F2:10 is created, and 60% donor genome is covered. The phenotype examination result shows that the remarkable characteristics of the population are that the bolls are large, the single boll cotton weight of L3077 is 8.77 g on average for 3 years, and the single boll cotton weight is 22.3 percent higher than that of the receptor TM-1 of 7.17 g. Indicating that there are genetic loci or genes of Raymond cotton that significantly increase the weight of upland cotton bolls. By means of recombination mapping, the main effect QTL of the bell is finely positioned in the 380.0 kb interval marked by BB575 and BB578, the contribution rate of the QTL phenotype interpretation is as high as 19-25%, and the gene effect is obvious.
When the total number of rings per unit area breaks through difficultly under the current high-density planting, the weight of the rings of the important factors of the yield becomes an important improvement target. In recent years, a large number of boll weight QTLs are identified, but because of low genetic diversity of upland cotton, the QTL contribution rate is low, and the gain effect is limited, particularly, the main effective site or gene for boll weight increase is still not found.
The invention provides a molecular marker from closely linked Lemond cotton and boll weight of related species, which is used for molecular marker assisted selective breeding. By means of recombination mapping, the main effect QTL of the bell is finely positioned in the 380.0 kb interval marked by BB575 and BB578, the contribution rate of the QTL phenotype interpretation is as high as 19-25%, and the gene effect is obvious.
The invention can select plants with Raymond cotton characteristic strips from the molecular level, the bell weight of the selected single plants or strains is obviously improved, and then the large bell material can be rapidly screened for cotton high-yield breeding, the molecular marker auxiliary selection target is clear, the cost is low, and the breeding process is accelerated.
Disclosure of Invention
The invention aims at providing a molecular marker derived from Raymond cotton and closely linked with the bell weight character and application thereof.
The aim of the invention can be achieved by the following technical scheme:
a molecular marker closely linked with the cotton boll weight character, wherein the molecular marker closely linked with the cotton boll weight character is BB575 or/and BB578;
the primer pair for detecting the molecular marker BB575 is as follows:
forward primer: GTCTCAAGATAGGGCTCCCC (SEQ ID NO. 1),
reverse primer: TTTCGAGTACCCCCAAGTTC (SEQ ID NO. 2);
the primer pair for detecting the molecular marker BB578 is:
forward primer: TGAGTTGTGGTTTCATTTTTCG (SEQ ID NO. 3),
reverse primer: CAAAGACAGCCCTCAAAAGG (SEQ ID NO. 4).
A primer group for detecting a molecular marker closely linked with the cotton boll weight trait, wherein the molecular marker closely linked with the cotton boll weight trait is BB575 or/and BB578; the primer set comprises at least one of a primer pair for detecting the molecular marker BB575 and a primer pair for detecting the molecular marker BB578;
the primer pair for detecting the molecular marker BB575 is as follows:
forward primer: GTCTCAAGATAGGGCTCCCC, reverse primer: TTTCGAGTACCCCCAAGTTC;
the primer pair for detecting the molecular marker BB578 is as follows:
forward primer: TGAGTTGTGGTTTCATTTTTCG, reverse primer: CAAAGACAGCCCTCAAAAGG.
A kit is used for detecting molecular markers closely linked with the boll weight character of cotton or for molecular marker-assisted breeding of cotton varieties with increased boll weight, and comprises the primer group.
The molecular marker closely linked with the cotton boll weight character is applied to auxiliary breeding of new cotton germplasm with increased boll weight.
The primer group for detecting the molecular marker closely linked with the cotton boll weight character is applied to auxiliary breeding of new cotton germplasm with increased boll weight.
The kit is applied to auxiliary breeding of new cotton germplasm with increased bell weight.
The molecular marker assisted breeding method of cotton varieties with increased bell weight adopts the primer group or the kit to carry out molecular detection on single plants or strains of target groups; and analyzing the detection result, and selecting plants with characteristic bands (167 bp or/and 242 bp) to obtain cotton varieties with remarkably improved bell weight.
According to the upland cotton boll weight auxiliary breeding method, the molecular marker selection is carried out on the boll weight characters in breeding populations related to the Raymond cotton and L3077 by using the specialization molecular markers BB575 and BB578 from the Raymond cotton control boll weight QTL, so that the boll weight of the upland cotton can be remarkably improved. The molecular markers BB575 and BB578 used in the method, the QTL related to the bell weight character is named qBW-chr.D09, and is derived from Raymond cotton, the contribution rate to the improvement of the bell weight is 19.46%, and the additive effect is 0.89 g (figure 2). The invention provides a new gene locus for molecular marker assisted breeding, and lays a foundation for gene cloning. The invention can select plants with Raymond cotton characteristic strips from molecular level, the bell weight of the selected single plants or strains is obviously improved, and further, the materials of the large bell cotton can be rapidly screened for cotton yield breeding, and the method has definite targets, low cost and quick breeding process during auxiliary breeding.
The invention has the beneficial effects that:
the invention provides a ringtone weight trait specialization molecular marker derived from Raymond cotton and application thereof. The molecular markers are BB575 and BB578, and the molecular marker selection is carried out in breeding populations related to Raymond cotton or L3077, so that the bell weight can be improved by more than 20%. The method of marker assisted selection can rapidly improve the boll weight of the existing upland cotton varieties, and greatly accelerate the cultivation and seed industrialization process of cotton high-yield new varieties in China.
Description of the drawings:
fig. 1: digging a linkage marking process of the bell weight QTL;
wherein, TM-1, upland cotton genetic standard line; l3077, derived from the Raymond cotton introgression line and boll weight was significantly greater than that of the upland cotton standard line TM-1.F (F) 1 Progeny resulting from the crossing of TM-1 and L3077; f (F) 2 、F 3 、F 4 、F 5 Is a offspring population generated by successive selfing year by year.
Fig. 2: a cotton boll weight QTL fine positioning result and a boll weight QTL linkage marking process are mined;
wherein A represents F 2 Genetic map of population construction; b represents F 3 Constructing a genetic map; c represents F 4 Constructing a genetic map; d, F is represented by 5 Population displacement mapping, group1-4 represents four recombinants and corresponding boll weight phenotype data differential analysis.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Unless otherwise indicated, all of the experimental materials, reagents, and equipment used in the examples of the present invention are commercially available, and unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 marker positioning linked to the weight-bell QTL
(1) Selecting a parent: screening of the introgression line L3077, which has a significantly better bell weight than TM-1, from the introgression line of Raymond cotton with upland cotton TM-1 as background revealed by phenotypic examination that the single bell cotton of L3077 was 8.77 g on average over 3 years, 22.3% higher than the 7.17 g of receptor TM-1 (Table 1):
TABLE 1L 3077 and TM-1 yield trait variance analysis
| BW bell weight | |
| L3077 | 8.77±0.17** |
| TM-1 | 7.17±0.16 |
(2) Group construction: taking upland cotton TM-1 as a male parent, taking an introgression line L3077 as a female parent, and hybridizing to obtain F 1 Instead of F, selfing to obtain F 2 The population, 2019, was planted in the Henan mall and contained 1241 individual plants. The genome of the individual young leaves of the parents and populations was extracted by the CTAB method (Paterson et al 1993).
（3）F 2 Population molecular identification: the 8 pairs of primers on the L3077 introgression fragment (Table 2) were used to molecular identify the 1241 individual strain of the F2 population. The amplification reaction system of all the primers was 10. Mu.l, of which 2. Mu.l of ultrapure water, 2X Hieff PCR Master Mix. Mu.l, 1. Mu.l of forward primer (10. Mu.m/. Mu.l), 1. Mu.l of reverse primer (10. Mu.m/. Mu.l) and 1.0. Mu.l of template DNA were used. The amplification reaction procedure is the first step: pre-denaturation at 98 ℃ for 5 min; and a second step of: denaturation at 98℃for 30s, annealing at 58℃for 30s, extension at 72℃for 1min,30 cycles; and a third step of: extending at 72℃for 8min. The amplified products were electrophoresed in a 9% polypropylene gel, silver stained according to the method of Zhang Jun (2000), read and record the results.
TABLE 2L 3077 introgression fragment region 10 polymorphic markers
| Tag name | Forward primer sequence (5 '-3') | Reverse primer sequence Reverse (5 '-3') | Marking type |
| BB228 | TTGAAAAAGAGAATCCCACGA | GCCTTTTTGCCTCTTCCAAT | InDel |
| BB227 | AAGGGGAAGGGGTAGATTTTT | ATTCAGAACACGAACATGCG | InDel |
| BB573 | TGCAAGGCAATTGTTCTCAA | TGGCCAAATTGAGTAACTAGGA | InDel |
| BB575 | GTCTCAAGATAGGGCTCCCC | TTTCGAGTACCCCCAAGTTC | InDel |
| BB396 | ATAGAGGGGAGCAAGGTGGT | CATCACGTGAGTTGTCAAGAGTT | InDel |
| BB578 | TGAGTTGTGGTTTCATTTTTCG | CAAAGACAGCCCTCAAAAGG | InDel |
| BB397 | TTGGATGCCCTTTTGCTATC | CGATAGGTACTCTTCTCCGTGG | InDel |
| BB398 | CAAATTGTTACCTTCGGTGGA | TCCTTTGTGTTTGTCATCCCT | InDel |
(4) Bell relocation: by F 2 The phenotype data and genotype data of the bell weight character of the population are subjected to positioning analysis by adopting QTL IcinMapping, the QTL related to the bell weight character is detected and is positioned on a D09 chromosome, the QTL is named as qBW-chr.D09, the contribution rate of the phenotype interpretation of the QTL is as high as 20-25%, and the gene effect is obvious (Table 3). According to the detected QTL interval mark, adopting a single seed transmission method to lead the derived secondary segregation population F 3 、F 4 、F 5 Is planted in Henan dune year by year from 2020 to 2022. The refined localization of the bell-weight major QTL was performed using recombinant mapping, and the bell-weight major QTL was refined to the 380.0 kb interval of the BB575 and BB578 markers, which are considered to be closely linked markers qBW-chr.d09 (see fig. 1 and 2). The marking information is shown in table 4.
TABLE 3 quantitative F2 population QTL positioning results
| QTL | Left side mark | Right side mark | LOD | Contribution ratio (%) | Additive effect (g) |
| qBW-Chr. D09 | BB575 | BB578 | 21.4954 | 19.4573 | 0.8929 |
Table 4: information of two markers BB575 and BB578 closely linked to qBW-chr. D09
| Tag name | Forward primer sequence (5 '-3') | Reverse primer sequence (5 '-3') | Product size |
| BB575 | GTCTCAAGATAGGGCTCCCC | TTTCGAGTACCCCCAAGTTC | 167 bp |
| BB578 | TGAGTTGTGGTTTCATTTTTCG | CAAAGACAGCCCTCAAAAGG | 242bp |
Example 2 molecular marker assisted selection method to increase land boll weight
Molecular marker selection was performed in a breeding population associated with raymond cotton or L3077 using the molecular markers BB575 and BB578 obtained in example 1, comprising the steps of:
1) Using L3077 (upland cotton-Raymond cotton introgression line) and upland cotton TM-1 as parent, and hybridizing to obtain F 1 Post-self-mating configuration F 2 Population and F 3 、F 4 、F 5 A derivative system. The CTAB method is adopted to extract and isolate individual plant or strain DNA.
2) Molecular markers BB575 and BB578 are used for carrying out molecular marker detection on genotypes of individual plants or plant lines of the population, materials with characteristic strips of Raymond cotton are selected, the bell weight can reach 8.58-8.96 g, and the improvement range is 19.7-23.1%.
3) The upland cotton variety line with improved bell weight can be obtained by the method, and the cotton yield breeding process is accelerated.
The invention provides a new locus for molecular marker assisted breeding and lays a foundation for gene cloning. The invention can select plants with Raymond cotton characteristic strips from molecular level, the selected single plants or strains are obviously improved in boll weight, further boll weight materials can be rapidly screened for cotton yield breeding, and the method has definite targets, low cost and quick breeding process during auxiliary breeding.
Claims (8)
1. The molecular marker closely linked with the cotton boll weight character is characterized in that the molecular marker closely linked with the cotton boll weight character is BB575 or/and BB578;
the primer pair for detecting the molecular marker BB575 is as follows:
forward primer: GTCTCAAGATAGGGCTCCCC, reverse primer: TTTCGAGTACCCCCAAGTTC;
the primer pair for detecting the molecular marker BB578 is:
forward primer: TGAGTTGTGGTTTCATTTTTCG, reverse primer: CAAAGACAGCCCTCAAAAGG.
2. The primer group for detecting the molecular marker closely linked with the cotton boll weight trait is characterized in that the molecular marker closely linked with the cotton boll weight trait is BB575 or/and BB578; the primer set comprises at least one of a primer pair for detecting the molecular marker BB575 and a primer pair for detecting the molecular marker BB578;
the primer pair for detecting the molecular marker BB575 is as follows:
forward primer: GTCTCAAGATAGGGCTCCCC, reverse primer: TTTCGAGTACCCCCAAGTTC;
the primer pair for detecting the molecular marker BB578 is as follows:
forward primer: TGAGTTGTGGTTTCATTTTTCG, reverse primer: CAAAGACAGCCCTCAAAAGG.
3. A kit for detecting a molecular marker closely linked to the boll weight trait of cotton or for molecular marker-assisted selection of cotton varieties with increased boll weight, the kit comprising the primer set of claim 2.
4. The use of a molecular marker closely linked to the boll weight trait of cotton as claimed in claim 1 in the assisted selection of new cotton germplasm with increased boll weight.
5. The use of a primer set for detecting molecular markers closely linked to the boll weight trait of cotton according to claim 2 in the assisted selection of new cotton germplasm with increased boll weight.
6. Use of the kit according to claim 3 for assisting in breeding new cotton germplasm with increased bell weight.
7. The molecular marker assisted breeding method of cotton varieties with increased bell weight is characterized in that the primer set of claim 2 or the kit of claim 3 is adopted to carry out molecular detection on single plants or strains of target groups; and analyzing the detection result, and selecting plants with characteristic bands to obtain cotton varieties with remarkably improved bell weight.
8. The method of claim 7, wherein the characteristic band is 167 bp or/and 242bp band.
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