CN108018372B - Molecular marker from sea island cotton Hai1 and related to verticillium wilt resistance and application thereof - Google Patents
Molecular marker from sea island cotton Hai1 and related to verticillium wilt resistance and application thereof Download PDFInfo
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Abstract
The invention discloses SSR markers linked with cotton verticillium wilt resistance QTL/major gene loci qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5, wherein qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5 are all located on chromosome C5. The SSR marker linked with the cotton verticillium wilt resistance major gene locus is used for auxiliary selection, so that the cotton verticillium wilt resistance breeding efficiency can be improved.
Description
In the notice of the first examination of the parent, the examiner indicates that there are a plurality of inventions in the parent and that there is no singleness, so the applicant decides to apply for divisional applications of inventions which are not protected in the parent. The application is a divisional application with application number 201410506198.0 (title of the invention: QTL related to cotton verticillium wilt resistance/molecular marker of major gene).
Technical Field
The invention belongs to the field of biotechnology application, and relates to a Simple Sequence Repeat (SSR) molecular marker from linkage with verticillium wilt resistance QTL/major gene of sea island cotton Hai1 with high verticillium wilt resistance, which can be used for auxiliary selection of the molecular marker for verticillium wilt resistance of cotton to improve the selection efficiency and accelerate the breeding pace.
Background
Cotton is an important commercial crop providing the majority of the world's natural fiber. China is one of the largest cotton production and consumption countries in the world, and cotton production has an important strategic position in the economy of the whole country. It is very important to breed and plant high-yield, high-quality and disease-resistant cotton varieties. The verticillium wilt is one of main diseases in cotton production, is distributed in various major cotton-producing countries in the world, is a main cause of cotton yield reduction, and becomes a disastrous disease threatening cotton production in the world. In recent years, the disease has continuous epidemic harm in 3 big cotton areas in China, and has a tendency of gradual increase, thereby causing great harm to cotton production (Jianguiliang et al, 2003). Practice proves that planting anti-verticillium wilt varieties is the most effective method for controlling the verticillium wilt. However, the resistance of the cotton varieties in China can only reach the disease resistance level (Jianguiliang, etc. 2004; Wang hong Mei, etc. 2008), so that the wilt resistance is still an important target for cotton variety breeding.
Although the yield of the sea island cotton is low, most of the sea island cotton has high verticillium wilt resistance, while the upland cotton has high yield and wide adaptability, but the sea island cotton lacks excellent disease-resistant germplasm and parent materials (Xiaosong Hua et al, 2007). Therefore, the method excavates the excellent disease-resistant gene of the sea island cotton, transfers the excellent disease-resistant gene of the sea island cotton into the background of the upland cotton, and has important significance for disease-resistant breeding of the upland cotton in China.
Because the selection in the whole process of traditional breeding is mainly phenotype selection and the phenotype character is easily affected by external conditions such as environment, the traditional breeding has long period, high cost, poor accuracy and low selection efficiency according to the phenotype selection. In recent years, the development of biotechnology has provided a new approach for crop trait improvement, and target genes can be manipulated from the molecular level by transgenic or molecular marker-assisted selection techniques to achieve improvement of target traits (durene et al, 2005). A large number of researches show that the breeding efficiency of disease-resistant varieties can be effectively improved by auxiliary selection by means of a molecular marker technology in the breeding process, and the breeding process is accelerated.
Currently, a number of genetic maps have been constructed in cotton using molecular marker technology (Shappley et al 1998; Ullloa et al 2002; Ma et al 2008; Zhang et al 2005and 2009; Lin et al 2009; Sun et al 2012; Reinsch et al 1994; Rong et al 2004; Kohel et al 2001; Guo et al 2007; Lacape et al 2009; Park et al 2005; Xiao et al 2009; Yu et al 2011; He et al 2007; Yu et al 2007; Zhang et al 2008; Yu et al 2012; Zhao et al 2012), locating QTLs for large quantities of cotton fiber quality and yield. In the aspect of positioning of the verticillium wilt resistance gene/QTL of cotton, the method has been reported in succession (Zhang et al, 2014; Zhao et al, 2014; Fang et al, 2013, 2014; Ning et al, 2013; Jianfeng et al, 2009; Yang et al, 2008; Yangyang et al, 2007; Kuihai Swallow et al, 2008; Wang Furong et al, 2007; Fangweiping et al, 2001; Gaoyuken et al, 2003; Duwei et al, 2004; Bolek et al, 2005; Wan Hongmei et al, 2005; Wan et al, Wan et al, 2007; Fangweiping et al, 2001; Gaoyun Qian et al, 2003; Duwei et alg et al, 2008; coconut et al, 2006), for a total of 100 more QTLs associated with verticillium wilt resistance. These studies provide useful information for breeding disease resistance, but the existing genetic map construction has low saturation degree, few markers available for production practice, and the most adopted for QTL positioning is F2The generation group mainly focuses on the detection of a single generation or a single environment, and has few multi-environment-stable QTL.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a molecular marker from high verticillium wilt resistant material Gossypium barbadense Hay 1 and related to verticillium wilt resistant QTL/major gene of cotton, namely: the SSR molecular markers linked with the verticillium wilt resistance major genes in the sea island cotton Hai1 from a high-verticillium wilt resistance material are screened to perform early marker auxiliary selection on the DNA level, so that the breeding efficiency is improved; and provides a method for detecting the verticillium wilt resistance of cotton and the application of the molecular marker related to the cotton verticillium wilt resistance QTL/major gene locus in cotton auxiliary breeding to improve the verticillium wilt resistance of cotton.
The technical scheme provided by the invention is as follows:
the molecular markers related to cotton verticillium wilt resistance QTL/major gene locus qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 or qDI-5-5 according to the invention are: marker linked to qDI-5-1 is DPL0247110And CIR224160(ii) a Marker linked to qDI-5-2 is PGML03048270、NAU4034220And BNL1042150(ii) a The marker linked with qDI-5-3 is DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210(ii) a Marker linked to qDI-5-4 is TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220(ii) a Marker linked to qDI-5-5 is MGHES6190、DPL0838160、NAU2494210、DPL0138240And DPL0241130Wherein, the specific primer sequence of each molecular marker and the amplified target fragment length are as follows:
according to the method for detecting the verticillium wilt resistance of cotton, the SSR marker is used for carrying out molecular marker selection on the verticillium wilt resistance in breeding groups related to the sea 1 of the gossypium barbadense and the like, so that the resistance of the gossypium hirsutum can be improved. The molecular markers used in the method are molecular markers related to verticillium wilt resistance QTL/major gene loci qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5(DI is the abbreviation of English word disease index of verticillium wilt fingers, the nomenclature of QTL is q + the name of character English abbreviation + the sequence number of chromosome + the sequence number of the QTL controlled on the same chromosome, for example, qDI-5-1 shows the 1 st QTL controlled on the 5 th chromosome), and the 5 major gene loci are all located on the chromosome C5 and are all derived from sea of island cotton 1, so that the disease fingers can be reduced and the resistance of upland cotton can be improved; the contribution rate to the cotton verticillium wilt resistance is respectively 8.10-10.91%, 9.76-13.59%, 9.80-13.52%, 10.13-16.66% and 7.72-11.63%, and the additive effect is respectively 2.7-7.0, 3.0-10.7, 3.0-10.6, 3.2-9.5 and 5.8-6.1 cN/tex.
The method is helpful for screening high-verticillium wilt-resistant materials, provides great convenience for the verticillium wilt-resistant breeding and utilization of sea-island cotton sea 1 hybridization, backcross progeny and derivative strains thereof in future, and lays a foundation for fine positioning and gene cloning of the major QTL.
According to the method for improving the verticillium wilt resistance of upland cotton by auxiliary breeding of the molecular marker related to the cotton verticillium wilt resistance QTL/major gene locus, the molecular marker is used for carrying out molecular marker selection in a breeding population related to the sea 1 of the island cotton, so that the verticillium wilt resistance of the upland cotton can be improved by reducing the disease index by 2.7-7.0, 3.0-10.7, 3.0-10.6, 3.2-9.5 and 5.8-6.1 cN/tex. The method comprises the following steps:
(1) DNA extraction and use for resisting verticillium wilt of cottonMolecular markers linked with sex QTL/major gene loci qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 or qDI-5-5, which are respectively DPL0247110And CIR224160；PGML03048270、NAU4034220And BNL1042150；DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210；TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220；MGHES6190、DPL0838160、NAU2494210、DPL0138240And DPL0241130Performing molecular detection on the genotype of the population individual plant, and taking a cotton place 36 in upland cotton and sea-island cotton sea 1 as a reference;
(2) and analyzing the detection result, and selecting the plants with the sea 1 characteristic strips of the sea island cotton to obtain the single plants with improved verticillium wilt.
Through the selection of the molecular markers, upland cotton varieties with improved fiber strength can be obtained, and the breeding process of the cotton fiber quality is accelerated.
The invention relates to a molecular marker linked with cotton verticillium wilt resistance QTL/major gene locus, which is obtained by the following method:
1) the excellent early-maturing upland cotton variety with the cotton institute 36 as recurrent parent and the sea-island cotton sea 1 as donor is first hybridized and backcrossed to obtain BC1F1、BC1S1、BC2F1A population;
2) examination BC2F1Verticillium wilt disease finger and BC in different periods of field and disease nursery1S1The absolute value of skewness of the verticillium wilt disease of the field is less than 1, and the verticillium wilt disease accords with normal distribution;
3) extracting parent and F by CTAB method1And BC1F1DNA of single plant leaves of 135 cotton in a population;
4) and selecting 23569 from different sources to perform polymorphism screening on the parent by the SSR primer. These primers included 14 sets of 12504 pairs of genomic SSRs (NAU6124-NAU6701, 578 pairs; BNL113-BNL4108, 689 pairs; CIR001-CIR418, 392 pairs)(ii) a CM003-209, 49 pairs; COT001-COT165, 70 pairs; DC20001-DC40441, 465 pairs; pairs of CGR5001-CGR7005, 1244; pairs C20001-C20139, 93; DPL0009-DPL0922, 849 pairs; GH001-GH700, 700 pairs; JESPR1-JESPR311, 309 pairs; MUSB0001-MUSB1316, 1316 pairs; TMB0010-TMB2963, 750 pairs; PGML00001-PGML05000, 5000 pairs), and 10 series of 11065 pairs of EST-SSRs (CICR0001-CICR1000, 1000 pairs; CER0001-CER0170, 121 pairs; pairs of HAU0001-3407, 3407; MGHES1-MGHES78, 82 pairs; pairs MUCS001-MUCS624, 624; pairs MUSS001-MUSS607, 554; pairs of NAU0747-NAU5513, 3198; SHIN0011-SHIN1640, 295 pairs; pairs STV001-STV192, 192; SWU0001-SWU1592, 1592 pairs); the preliminary screening of the molecular markers of the parents shows that 2173 pairs of SSR primers have difference among the parents, and 135 BC are paired by using the polymorphic primers1F1The population was expanded and the BC constructed using the JoinMap 4.0 software (Van Ooijen 2006)1F1A molecular linkage map of the population;
5) using 2 generations (BC)2F1And BC1S1) Verticillium wilt data of populations in different disease stages of field and disease nursery combined with BC1F1The genetic linkage map of colony is obtained by screening verticillium wilt resistance major QTL (quantitative trait locus) with composite interval mapping method of Windows QTL Cartographer 2.5 software, wherein 5 QTL can be screened in 2 generations (BC)2F1And BC1S1) Or 2 environments (disease nursery and field) or stable detection in different disease stages, and the verticillium wilt resistance synergistic genes are all from the sea island cotton sea 1, namely: qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5, wherein the 5 major gene loci are all located on chromosome C5, and the marker linked with qDI-5-1 is DPL0247110And CIR224160(ii) a Marker linked to qDI-5-2 is PGML03048270、NAU4034220And BNL1042150(ii) a The marker linked with qDI-5-3 is DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210(ii) a Marker linked to qDI-5-4 is TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220(ii) a Marker linked to qDI-5-5 is MGHES6190、DPL0838160、NAU2494210、DPL0138240And DPL0241130。
Step 4) above selects 23569 to carry out inter-parent polymorphism screening on SSR primers, the PCR reaction system is 10 mu 1, wherein the ultrapure water is 6.40 mu 1, 10 xBuffer is 1.0 mu 1, 10mM dNTPs is 0.50 mu 1, 10 mu M forward primer is 0.50 mu 1, 10 mu M reverse primer is 0.50 mu 1, 30 ng/mu 1 template DNA is 1.0 mu 1, 5U/mu 1Taq DNA polymerase is 0.10 mu 1, and the PCR reaction program is: pre-denaturation at 94 ℃ for 45 s; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 1min, 29 cycles; denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 2min, PCR reaction at TGRADIENT and PTC-200, electrophoresis of the amplified product in 8% polypropylene gel, and recording the result.
The invention has the following beneficial effects:
the invention relates to 5 loci (qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5) related to cotton verticillium wilt resistance QTL/major gene, and synergistic genes are all from high verticillium wilt resistance material sea island cotton sea 1 and can be stably detected in different environments or different morbidity periods or different generations. qDI-5-1 at BC2F12 different disease periods in the two environments of the disease nursery of 9-month 17-day and 7-month 19-day of the population explain 10.91% and 8.10% of the phenotypic variation respectively, and the additive effects are 7.0 and 2.7 respectively; qDI-5-2 at BC2F19-month 17-day field, 7-month 19-day disease nursery, 8-month 15-day disease nursery and BC of the population1S1Population 9 months and 17 days in the field (two generations, two environments and 3 disease periods) accounted for 12.31%, 9.76%, 11.74%% and 13.59% of phenotypic variation, respectively, with additive effects of 7.5, 3.0, 6.0 and 10.7, respectively; qDI-5-3 at BC2F19-month 17-day field, 7-month 19-day disease nursery, 8-month 15-day disease nursery and BC of the colony1S1The population at 9 months and 17 days (two generations, two environments and 3 disease periods) accounted for 13.46%, 9.80%, 13.52% and 13.13% of phenotypic variation, respectively, with additive effects of 7.9, 3.0, 6.5 and 10.6, respectively; qDI-5-4 at BC2F19-month 17-day field, 7-month 19-day disease nursery, 8-month 15-day disease nursery and BC of the population1S1Group 9 month and 17 day field (two)Generation, two environments and 3 disease stages) account for 10.13%, 12.01%, 16.66% and 10.83% of phenotypic variation, respectively, with additive effects of 6.8, 3.2, 7.3 and 9.5, respectively; qDI-5-5 at BC2F1The two 2 disease stages in the population, the 9-month, 17-day field and the 8-month, 15-day nursery, accounted for 7.72% and 11.63% of the phenotypic variation, respectively, with additive effects of 5.8 and 6.1, respectively. The located QTL has stable and reliable performance in multiple environments, multiple disease stages and different generations, and can be used for early auxiliary selection on the level of cotton verticillium wilt resistance DNA and improve the breeding efficiency.
Drawings
FIG. 1 shows that the QTL of the present invention related to verticillium wilt resistance is at BC1F1The position of the molecular marker linkage map.
Wherein qDI-5-1 is located at marker interval DPL0247 of chromosome C5110–CIR224160Internal, the label linked with it is DPL0247110And CIR224160The specific positions are 25.6cM and 30.5 cM; qDI-5-2 marker interval PGML03048 at chromosome C5270–BNL1042150In that, the label linked with it is PGML03048270、NAU4034220And BNL1042150The specific positions are 31.3cM, 32.4cM and 32.6 cM; qDI-5-3 marker interval DPL0063 located on chromosome C5130–DPL0724210The label of internal, linked with it is DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210The specific positions are 35.4cM, 38.2cM, 38.6cM and 39.0 cM; qDI-5-4 is located at marker interval TMB1120 of chromosome C5390–HAU1712220In, with it is linked with the label TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220The specific positions are 39.1cM, 39.4cM, 39.8cM, 40.2cM and 43.1 cM; qDI-5-5 marker interval MGHES6 located on chromosome C5190–DPL0241130In, the label linked with it is MGHES6190、DPL0838160、NAU2494210、DPL0138240And DPL0241130The specific position is 43.9cM, 44.6cM, 44.8cM, 45.0cM and 47.4 cM.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.
Example 1: screening molecular markers
The molecular marker linked with the cotton verticillium wilt resistance QTL/major gene is screened by the following method:
(1) the cotton seed place 36 in upland cotton is used as recurrent parent, sea island cotton sea 1 is used as donor parent, and the hybridized backcross combination is prepared. The Zhongmiao cottage 36 (Zhong36) is an excellent early-maturing upland cotton cultivar (national cotton 990007), and the sea 1(Hai1) is a highly verticillium wilt-resistant gossypium barbadense cultivar (Jing deep-desertification et al, 1990, Chinese agricultural science).
In summer of 2003, the two parents are hybridized to obtain 36 XHai of the Zhongmian institute1F1Seeds, additional backcrossing of 2003 winter in Hainan China cotton institute wild cotton plantation test base, and obtaining BC by using recurrent parent China cotton institute 36 as female parent1F1And (4) seeds. Planting parents and F in Anyang test field in 4 months in 20041And BC1F1Generation, and extracting gland plants in seedling stage. Uses the cotton seed 36 in the recurrent parent as the female parent and BC1F1Backcrossing with 135 individual plants as male parents while crossing under the condition of BC1F1And (5) selfing the single plants. And (4) hybrid bolls are harvested according to the male parent source in a mixed mode, and the male parent single-plant selfing bolls are harvested separately. Planting parents and F in Anyang experimental field 4 months in 20051、BC2F1(133 lines) and BC1S1(120 lines), parent and F12 rows each, 1 row each for each line of the two generations, 5m row length, 25cm plant spacing, and also pulled out glandular plants at seedling stage. Two generations BC combined on Mitsubishi 36 planted in test field on 17.9.20052F1And BC1S1All the plants were subjected to cotton verticillium wilt grading investigation. Meanwhile, in the appraisal of disease nursery of Anyang cotton institute, 36 combination BC of cotton institute2F1Verticillium wilt resistance of 133 generations of families, taking Ji 11 and Yu 2067 asDisease-sensitive and disease-resistant control materials, wherein each disease nursery is identified to be an artificially infected cement pond with the length of 20 meters and the width of 2.5 meters, and pathogenic bacteria are of an Anthriscus system and have medium pathogenicity. The verticillium wilt is respectively researched in four different periods (6 months, 23 days, 7 months, 19 days, 8 months, 15 days and 8 months, 25 days) in a single-row planting mode, wherein the row length is 2.5 meters, three random repetitions are arranged in total.
The verticillium wilt investigation adopts a standard grade 5 system, and the disease incidence index (DI) is calculated (refer to the Chinese cotton genetic breeding science published in 2003 by the Cotton research institute of Chinese academy of agricultural sciences).
The disease index calculation formula is as follows:
the statistical description and the normality distribution test of the disease fingers are shown in Table 2. The absolute value of skewness is less than 1, and the normal distribution is conformed.
(2) Extraction of parent, F, by CTAB method (Paterson, 1993)1And BC1F1135 cotton single plant leaf DNA.
(3) The primers 23569 from different sources are selected for polymorphism screening of the parents, and the primers comprise 24 series of SSR primers. Among them, 14 series 12504 pairs of genome SSRs (NAU6124-NAU6701, 578 pairs; BNL113-BNL4108, 689 pairs; CIR001-CIR418, 392 pairs; CM003-209, 49 pairs; COT001-COT165, 70 pairs; DC20001-DC40441, 465 pairs; CGR5001-CGR7005, 1244 pairs; C20001-C20139, 93 pairs; DPL0009-DPL0922, 849 pairs; GH001-GH700, 700 pairs; JESPR 86SPR 24-SPR JE311, 309 pairs; SB0001-MUSB1316, 1316 pairs; TMB0010-TMB2963, 750 pairs; PGML00001-PGML05000, 5000 pairs) and 10 series 11065 pairs of EST-SSCICR 624, 1000 pairs; CER0001-CER0170 pairs; MUL 00001-PGML05000, 5000 pairs; MUSS 3407-NAML 7 pairs; MUSS 3402-SHS 15951 pairs; SHU 3402-SHSU pairs; SHSU 2-SPR 6347 pairs; SHSU pairs; SHSB 0001-SHSU pairs). Wherein the CICR series is SSR primer autonomously developed by doctor by Cotton research institute of Chinese academy of agricultural sciencesPrimers for the PGML and SWU series are SSR primers developed by professor Zhangzheng, university of southwest. Primer sequences are published in the CMD (Cotton Marker database) database (http:// www.cotton-Marker. org /) for free download or in the literature. Primer synthesis was performed by shanghai handsome biotechnology limited. The SSR amplification reaction system is 10 mu 1, wherein ultrapure water is 6.40 mu 1, 10 xBuffer is 1.0 mu 1, 10mM dNTPs is 0.50 mu 1, a forward primer (10 mu M) is 0.50 mu 1, a reverse primer (10 mu M) is 0.50 mu 1, a template DNA (30 ng/. mu.1) is 1.0 mu 1, and Taq DNA polymerase (5U/. mu.1) is 0.10 mu 1. SSR amplification reaction program: pre-denaturation at 94 ℃ for 45 s; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 1min, 29 cycles. Denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 2 min. The amplification reactions were carried out on BIOMETRA TGRADIENT and BIO-RAD PTC-200, and the amplification products were electrophoresed on 8% polypropylene gel, silver stained on the gel according to Zhang et al (2002), and the results were recorded. The result of molecular marker screening shows that 2173 pairs of SSR primers show polymorphism between parents, and BC is further analyzed1F1The DNA polymorphism of 135 individuals in the population obtains 2365 polymorphic sites in total.
(4) Construction of BC Using JoinMap 4.0 software (Van Ooijen 2006)1F1And (3) constructing a linkage group by adopting a Kosambi function and setting a LOD value to be more than or equal to 4.0 in the mapping of the group genetic linkage map. Consensus markers among several molecular linkage maps containing SSR markers based on published data on the web (Guo et al 2007; Yu et al 2011; Yu et al 2012; Lacape et al 2005) and other linkage group localization studies (David Fang, 2007,http://www.cottonmarker.org/ projects/dpl/) The linkage group constructed is mapped to a specific chromosome using conventional nomenclature for chromosomes or linkage groups (Guo et al 2007). A molecular linkage map containing 2292 loci, 26 linkage groups (corresponding to 26 chromosomes, respectively), covering 5115.16cM genome was constructed (see FIG. 1 for chromosome 5).
(5) The genetic linkage map is combined with BC2F1Four different disease peak periods and BC in disease nursery1S1And BC2F1Mean disease index data of respective verticillium wilt in field by using Windows QTL CarQTL mapping and positioning are carried out by a Tographer2.5 composite interval mapping method, an LOD value is set to be 2.5, 1000 times of sequencing test is carried out, multiple environment multi-generation multiple different disease onset periods show stable verticillium wilt resistance QTL, 5 verticillium wilt resistance QTL showing stable expression are screened in total, are qDI-5-1, qDI-5-2, qDI-5-3, qDI-5-4 and qDI-5-5 and are positioned on a chromosome C5. The 5 QTLs are all derived from sea island cotton Hai1, can reduce greensickness index of upland cotton and improve greensickness resistance. qDI-5-1 marker interval DPL0247 located on chromosome C5110–CIR224160The new label of inner, connected with it is DPL0247110And CIR224160The specific positions are 25.6cM and 30.5 cM; qDI-5-2 marker interval PGML03048 at chromosome C5270–BNL1042150The new label linked with it is PGML03048270、NAU4034220And BNL1042150Specific positions are 31.3cM, 32.4cM and 32.6cM, and other nearby marks CIR102230And DC20067120Reported in the tonic talent (2006) and Ning et al (2013) studies, respectively; qDI-5-3 marker interval DPL0063 located on chromosome C5130–DPL0724210The new label of internal, connected with it is DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210The specific positions are 35.4cM, 38.2cM, 38.6cM and 39.0 cM; qDI-5-4 is located at marker interval TMB1120 of chromosome C5390–HAU1712220In, the new label linked with it is TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220The specific positions are 39.1cM, 39.4cM, 39.8cM, 40.2cM and 43.1cM, and the nearby marker is DPL0022250Reported in the Ning et al (2013) study; qDI-5-5 marker interval MGHES6 located on chromosome C5190–DPL0241130The new label of MGHES6 associated with it190、DPL0838160、NAU2494210、DPL0138240And DPL0241130The specific positions were 43.9cM, 44.6cM, 44.8cM, 45.0cM and 47.4cM (fig. 1 and table 3). The base sequences of these primers are shown in Table 1.
qDI-5-1 in the population BC2F12 different disease periods in the two environments of the disease nursery of 9-month 17-day and 7-month 19-day of the population explain 10.91% and 8.10% of the phenotypic variation respectively, and the additive effects are 7.0 and 2.7 respectively; qDI-5-2 in the population BC2F19-month 17-day field of colony, 7-month 19-day disease nursery, 8-month 15-day disease nursery and colony BC1S1Population 9 months and 17 days in the field (two generations, two environments and 3 disease periods) accounted for 12.31%, 9.76%, 11.74%% and 13.59% of phenotypic variation, respectively, with additive effects of 7.5, 3.0, 6.0 and 10.7, respectively; qDI-5-3 in the population BC2F19-month and 17-day field of colony, 7-month and 19-day disease nursery, 8-month and 15-day disease nursery and colony BC1S1The 9 month 17 day field (two generations, two environments and 3 disease stages) accounted for 13.46%, 9.80%, 13.52% and 13.13% of phenotypic variation, respectively, with additive effects of 7.9, 3.0, 6.5 and 10.6, respectively; qDI-5-4 in the population BC2F19-month 17-day field of colony, 7-month 19-day disease nursery, 8-month 15-day disease nursery and colony BC1S1The population at 9 months and 17 days (two generations, two environments and 3 disease periods) accounted for 10.13%, 12.01%, 16.66% and 10.83% of phenotypic variation, respectively, with additive effects of 6.8, 3.2, 7.3 and 9.5, respectively; qDI-5-5 at BC2F1The two environmental 2 disease stages of the population, the 9-month 17-day field and the 8-month 15-day nursery, accounted for 7.72% and 11.63% of the phenotypic variation, respectively, with additive effects of 5.8 and 6.1, respectively (table 3). The molecular markers linked with the stable QTL can be used for molecular marker-assisted selection of cotton verticillium wilt resistance, and the disease-resistant breeding efficiency is improved.
Example 2: molecular marker selection method for improving verticillium wilt resistance of gossypium hirsutum
Using the molecular marker obtained in example 1, the marker linked to qDI-5-1 was DPL0247110And CIR224160(ii) a Marker linked to qDI-5-2 is PGML03048270、NAU4034220And BNL1042150(ii) a The marker linked with qDI-5-3 is DPL0063130、HAU0746210、TMB1296180、CGR6708110And DPL0724210(ii) a Marker linked to qDI-5-4 is TMB1120390、CGR5590160、PGML02063210、CGR5025160And HAU1712220(ii) a Marker linked to qDI-5-5 is MGHES6190、DPL0838160、NAU2494210、DPL0138240And DPL0241130The molecular marker selection is carried out in breeding populations related to the sea island cotton Hai1 and the like, and comprises the following steps:
(1) DNA extraction: taking the sea island cotton sea 1 as a donor parent, taking the upland cotton variety or strain as a receptor parent, carrying out hybridization and backcross to obtain a separation population, or taking the sea island cotton sea 1 as the donor parent, taking the upland cotton variety as the receptor parent, carrying out hybridization high-generation backcross to obtain a introgression line and a derivative line thereof, or a progeny population of hybridization and backcross of the introgression line and the upland cotton variety, and extracting single plant DNA of the separation population at a seedling stage by adopting a CTAB method;
(2) performing molecular marker detection on the genotype of the individual plant of the population (1) by using the molecular marker, and taking the gossypium hirsutum 36 in upland cotton and the gossypium barbadense sea 1 as a reference;
(3) analyzing the detection result;
(4) plants with sea-island cotton sea 1 characteristic bands are selected, and the verticillium wilt resistance of selected individual plants can be improved to different degrees.
408 line groups of the Chinese cotton institute 36 and sea 1 backcrossed for 5 generations and selfed for 6 generations are respectively provided with 2 rows of areas 2 for repeated tests, the test places are planted in Xinjiang rock river Chinese cotton institute in 2014, the test places are heavily sick with cotton verticillium in agricultural science institute in rock river city, and the two test places are respectively subjected to verticillium wilt disease index investigation in 9 months and 8 days. Using randomly selected SSR markers TMB1120 linked to qDI-5-1 and qDI-5-2, respectively390And CIR224160Molecular detection is carried out in 408 strain groups to obtain strains with improved resistance and lower average verticillium wilt index. Table 4 shows the average disease index of verticillium wilt of the obtained partial strains.
TABLE 1 SSR molecular markers of forward and reverse primer sequences
TABLE 2 basic statistics and Normal test for verticillium wilt resistance trait data of three-generation segregating population
Remarking: FD denotes the field test
TABLE 3 Stable 5 QTLs identified by composite Interval
Remarking: 7.19, 8.15, 8.25, 9.17 represent respectively 7 months 19 days, 8 months 15 days, 8 months 25 days and 9 months 17
TABLE 4 expression of the mean disease index of verticillium wilt of 9 lines of 5-generation selfing 6-generation backcross of medium cotton institute 36 and sea 1 selected by molecular marker
| Material numbering | TMB1120 | CIR224 | Disease finger (trial floor one) | Disease finger (trial Di) |
| MBC093 | + | 15.68 | 26.18 | |
| MBC153 | + | 14.81 | 27.17 | |
| MBC162 | + | 16.36 | 26.46 | |
| MBC178 | + | 15.11 | 24.98 | |
| MBC222 | + | 13.96 | 24.22 | |
| MBC238 | + | 16.93 | 31.72 | |
| MBC528 | + | 14.96 | 25.58 | |
| MBC639 | + | 19.10 | 24.93 | |
| MBC095 | + | + | 17.74 | 23.41 |
| Middle cotton institute 36 | 22.66 | 36.36 |
"+" indicates a characteristic band in which the mark can be detected; the test site is a test site for cotton in the stone river of Xinjiang and a test site II for severe verticillium wilt of cotton of agricultural academy of sciences of stone river city.
Claims (2)
1. A method for detecting verticillium wilt resistance of cotton is characterized by comprising the following steps: the method comprises the following steps:
(1) DNA extraction, 5 molecular markers linked with cotton verticillium wilt resistance QTL/major gene locus qDI-5-4 are used, and TMB112 is used respectively0390、CGR5590160、PGML02063210、CGR5025160And HAU1712220(ii) a Performing molecular detection on the genotype of the population individual plant, and taking a cotton place 36 in upland cotton and sea-island cotton sea 1 as a reference;
(2) analyzing the detection result, selecting the plants with the sea 1 characteristic bands of the sea island cotton with the 5 molecular markers simultaneously, and obtaining the single plants with improved verticillium wilt resistance, wherein the specific primer sequences of the molecular markers linked with the cotton verticillium wilt resistance QTL/major gene locus qDI-5-4 and the lengths of the amplified target segments are as follows:
。
2. The application of a molecular marker in cotton assisted breeding to improve the cotton verticillium wilt resistance of cotton is characterized in that the specific amplification primer sequence of the molecular marker is as follows:
。
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| CN106048033B (en) * | 2016-06-30 | 2019-12-20 | 中国农业科学院棉花研究所 | Evaluation method of cotton land-sea introgression line |
| CN108130381B (en) * | 2017-11-29 | 2020-08-04 | 中国农业科学院棉花研究所 | Molecular marker from sea island cotton Hai 1 and related to verticillium wilt resistance and application thereof |
| CN108130380B (en) * | 2017-11-29 | 2020-08-04 | 中国农业科学院棉花研究所 | Molecular breeding method for synchronously improving verticillium wilt resistance and fiber quality of cotton |
| CN108184652B (en) * | 2018-01-24 | 2020-03-24 | 山东棉花研究中心 | Molecular breeding method for improving cotton fiber length by using chr.19 single QTL fragment replacement line |
| CN111825751B (en) * | 2019-03-27 | 2022-02-11 | 中国农业科学院棉花研究所 | Clone of land cotton bud yellow gene vsp and its function |
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| 棉花抗黄萎病基因的分子标记辅助选择研究;李志坤等;《河北农业大学学报》;20111130;第34卷(第6期);第1-4页 * |
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