CN110184383B - Serial molecular markers and application thereof in wheat head germination gene molecular markers - Google Patents
Serial molecular markers and application thereof in wheat head germination gene molecular markers Download PDFInfo
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Abstract
The application belongs to the technical field of wheat molecular breeding, and particularly relates to a series of molecular markers and application thereof in wheat ear germination gene molecular markers. The total number of the molecular markers is 4, and the molecular markers comprise an SSR marker and three KASP markers, wherein the SSR marker is an Xgwm383b marker; the three KASPs are labeled: a009711, A009180 and A009716. In variety cultivation, the way of transferring excellent genes in the jie wheat into the wheat is mainly to create a series of wheat/jie wheat substitution lines and introduce the jie wheat chromosomes into the wheat background by means of distant hybridization. For this breeding method, the present application takes actual F2For example, a series of molecular markers are designed for the population, so that the molecular markers are used for detecting and tracking the chromosome condition of the zygoma cruzi introduced into the wheat background, and further used for guiding the cultivation of a new wheat ear bud resistant variety with a good practical value.
Description
Technical Field
The application belongs to the technical field of wheat molecular breeding, and particularly relates to a series of molecular markers and application thereof in wheat ear germination gene molecular markers.
Background
Wheat ear sprouting (PHS) is the ear sprouting phenomenon caused by overcast and rainy or humid environment after the physiological maturity of wheat before harvest. The phenomenon of sprouting of the ear of grain not only affects the yield of wheat, but also seriously affects the processing quality, the seed storage and the sowing quality in the next year, thereby causing economic losses of different degrees.
Wheat ear germination is a climate disaster in the world, and ear germination occurs in wheat production areas all over the world, so that the loss is serious. In China, the ear sprouting mainly occurs in Yangtze river basin, southwest wheat area and northeast spring wheat area, and the large-area ear sprouting also occurs in Shaanxi Guangdong of Huang-Huai wheat area for many times. Due to the large loss, breeding researchers in various countries attach great importance to wheat ear germination, and a great deal of research is carried out on the aspects of identification methods, resistance mechanisms, genetic mechanisms, molecular markers and the like, but the wheat ear germination resistant breeding has not been developed in a breakthrough manner. Therefore, the problem of ear germination remains one of the urgent problems to be solved in wheat breeding.
In recent years, molecular markers and development work related to wheat ear germination have made great breakthroughs. For example: anderson et al (RFLP analysis of genetic regions associated with resistance to sprouting to pre-harvest spraying in Crop science.1993) found 8 genomic regions associated with ear sprouting and located 10 RFLP markers associated with resistance to ear sprouting; in addition, morphological features using gene control can also be used as markers for resistance to ear sprouting, such as: the site found at the 2B, 2D sites that controls epidermal wax is associated with resistance to pre-sprouting (King, epidermal waxes and regulation of ear wetting and pre-saline spraying in barrel and straw. Euphytoca. 2000). In the prior art, more researches are also carried out on the association degree of partial main effect QTL and pre-harvest sprouting resistance. However, in general, since the resistance to ear sprouting is controlled by a plurality of sites, intensive research on different sites and development of molecular markers related to ear sprouting aiming at different sites still have important technical value for auxiliary screening of wheat varieties resistant to ear sprouting.
Disclosure of Invention
The application aims at partially marking the primers for the molecular markers, so that the primers can mark partial pre-harvest sprouting resistance related genes and further be used for guiding the breeding of new pre-harvest sprouting resistance wheat varieties.
The technical solution adopted in the present application is detailed as follows.
The series of molecular markers for marking the pre-harvest sprouting related genes comprise 4 markers in total, wherein the series of molecular markers comprises a SSR marker and three KASP markers (the SSR marker is a pair of primers, each KASP marker is a set of primers), the SSR marker is named as Xgwm383b marker, and the three KASP markers are respectively named as: mark A009711, A009180 and A009716; the base sequence is shown in SEQ ID NO. 1-11, and the specific sequence is as follows:
Xgwm383b：
Xgwm383b -F：5'- ACGCCAGTTGATCCGTAAAC-3'，
Xgwm383b -R：5'- GACATCAATAACCGTGGATGG-3'；
A009711：
A009711-1：5'-TGGTGATTAGCATCATCGGAATGG-3'，
A009711-2：5'-TTGGTGATTAGCATCATCGGAATGT-3'，
A009711-3：5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180：
A009180-1：5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3'，
A009180-2：5'-AGTGAATTTTTAAAGTTCGCATACCCC-3'，
A009180-3：5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716：
A009716-1：5'-GTTGCTATGTAACGGAATAAGAACG-3'，
A009716-2：5'-CGTTGCTATGTAACGGAATAAGAACT-3'，
A009716-3：5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'。
the application of the series of molecular markers in the pre-harvest sprouting resistance gene marker is matched with the conventional Xcfd223 to carry out the molecular marker on the pre-harvest sprouting resistance gene, and when the molecular markers are specifically applied:
xcfd223 was labeled in conjunction with A009711;
a009711 and A009180 are matched for marking;
a009716 marking in combination with Xgwm383 b;
the Xcfd223 has the following specific sequence:
Xcfd 223：
5'-AAGAGCTACAATGACCAGCAGA-3'，
5' -GCAGTGTATGTCAGGAGAAGCA-3'。
statistics have shown that white wheat is generally more prone to ear sprouting than red wheat, and analysis suggests that this is due to the fact that the red R gene is a transcriptional regulator that affects grain dormancy by regulating the expression of several genes that control flavonoid synthesis, thereby affecting ear sprouting resistance. However, the development of the anti-pre-germination genes of white-grain wheat and the cultivation of the anti-pre-germination varieties of white-grain wheat are still the main research focus because white-grain wheat has the advantages of high flour yield, high purchase price and the like compared with red-grain wheat.
In the existing research, the related species jiegeng wheat of wheat has many excellent traits, such as stress resistance, disease resistance and other excellent traits, so it is usually used as an important gene source of the genetic variation of wheat variety and as a donor species of ordinary wheat D group chromosome to carry out genetic improvement on wheat. In the actual variety cultivation, the way of transferring excellent genes in the jie wheat into the wheat is mainly to create a series of wheat/jie wheat substitution lines and introduce the chromosome of the jie wheat into the wheat background by means of distant hybridization. For this breeding method, the present application takes actual F2For example, a series of molecular markers are designed for population, so that the molecular markers are used for detecting and tracking the chromosome condition of the arthrocele wheat introduced into the wheat background, and further used for guiding the cultivation of a new wheat pre-harvest sprouting resistant variety; by combining with the evidence of practical research results, the related molecular marker provided by the application has better practical value for guiding the cultivation of new wheat pre-harvest sprouting resistant varieties.
Drawings
FIG. 1 shows strain 1884F2A 7 day germination index determination histogram for the population;
FIG. 2 shows the SSR primer Xcfd223-3D pair F2Population partial material and parental week 18 and T093 amplification comparison results, in the figure: 1-36 represent the moiety F2A population; m represents Marker, T represents festival wheat T093; z represents Zhouma 18;
FIG. 3 shows wheat F2Positioning a 3D chromosome ear sprouting QTL in a colony; in the left graph, the right side of the chromosome is provided with 4 positioned SSR markers and 10 KASP markers, the left side of the chromosome is the genetic distance of each marker in the genetic map, and the unit of the genetic distance is the Remo; the red mark under the color image is the QTL position for resisting pre-harvest sprouting; the right panel shows the LOD (logarithm of the odds) values for the markers on the chromosome, with the LOD values for the three QTLs being 119, 70 and 20, respectively.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific examples, a brief description of the background of some of the experimental materials in the following examples is provided below.
Experimental materials:
the recurrent background parent week wheat 18 (which is a material easy to germinate), and the nodal wheat T093 (resistant to germination due to a longer dormancy period) are common germplasm resources which can be publicly obtained;
1884 parts of F referred to in the examples2The material is created by hybridizing diploid jiejie T093 and Zhoumai 18, and the creation process comprises the following steps: cross week 18 with T093, hybrid F1Obtaining artificial octaploid synthetic wheat (AABBDDDD,2n =8x =56) by young embryo culture and colchicine doubling; backcrossing and selfing with week 18, selecting the single plant resisting pre-germination, hybridizing with week 18, and selfing to form F2A population; the specific operation can be performed by referring to the prior art, and is not repeated;
during the experiment, parental derivative population F2And the parent Zhongmai 18 and Jiejiao wheat T093 are planted in the school test field of Henan university, and when the Zhongmai T093 is planted: randomly selecting 10 grains according to each family system in a colony, uniformly sowing in a single row, setting the plant spacing to be 10cm, the row length to be 1.0m and the row spacing to be 25cm, and conventionally managing cultivation;
the 4 SSR primers related in the following examples are synthesized and provided by Beijing Sanbo Polygala tenuifolia Biotech, Inc.;
the KASP (Kompetitive Allele Specific PCR) marker is provided by Zhongyujin marking (Beijing) Biotechnology GmbH.
Examples
It should be noted that the main technical idea of the present application is: by the phenotype judgment of whether the related gene segments in the Jiejiemai T093 exert a reduction effect on the wheat germination or not after being introduced into the wheat, and further combining the technologies such as SSR (simple sequence repeat) markers, QTL (quantitative trait locus) analysis and the like, a series of molecular markers are provided by specific design, so that a foundation can be laid for further wheat pre-harvest germination resistant variety cultivation. Therefore, the present embodiment is briefly described below with respect to the relevant experimental conditions.
(ii) identification of anti-pre-sprouting phenotype
Each wheat plant is marked with a label at the flowering stage of 4 months, the wheat plants enter a wax ripeness stage after about 35 days of flowering, after the wheat ears are completely green, 5 mature and consistent main stem ears of each material are taken out and dried outdoors for 7 days, and then the wheat ears of each material are subjected to a seed germination experiment. The specific experimental method is described below.
The germination grain number is determined statistically by the method of Imtiaz et al (Characterization of QTL controlling genetic variation for pre-harven germination in synthetic background minerals lines, Genetics 2008), with specific reference to the following:
manually threshing wheat ears, randomly taking 50 full and healthy grains from each variety, placing the 50 full and healthy grains with the abdominal furrows downwards and neatly in a culture dish padded with double-layer filter paper, adding a proper amount of distilled water into the culture dish, covering the culture dish, placing the culture dish in a culture room for culture at the temperature of 22 ℃, and keeping the filter paper moist during the experiment;
taking the broken and white seed coat as germination, counting the number of the germinated seeds every day, removing the seeds, counting the number until 7 days, and calculating the germination index for 7 days according to the following formula;
to 1884 Strain F2The germination index of (2) was counted, and the results are shown in FIG. 1. Analysis can see that: at F2 The germination indexes in the population are distributed in the range of 0-100%, and have no obvious proportional relation, so the population belongs to quantitative trait inheritance.
(II) SSR marker analysis
By utilizing a PCR technology, 4 SSR markers are adopted for further marker analysis, and the adopted SSR markers (each SSR marker is a primer pair) specifically comprise:
Xcfd152：
Xcfd152-F：5'-TGGAAGTCTGGAACCACTCC -3'，
Xcfd152-R：5' -GCAACCAGACCACACTCTCA -3；
Xgwm383b：
Xgwm383b -F：5'- ACGCCAGTTGATCCGTAAAC- 3'，
Xgwm383b -R：5' -GACATCAATAACCGTGGATGG- 3'；
Xcfd 223：
Xcfd 223-F：5' -AAGAGCTACAATGACCAGCAGA- 3'，
Xcfd 223-R：5'- GCAGTGTATGTCAGGAGAAGCA- 3'；
Xgpw 5094：
Xgpw 5094-F：5'-GACGATCAACAGCGAGTCAA-3'，
Xgpw 5094-R：5'-TTACAATCTCACCCTGGCAA-3'。
before PCR amplification, the Oncorhynchus japonicus T093, Zhoumai 18 and F collected from the field in 3 months2The genomic DNA of the young leaf sample of the colony strain is extracted and used as a template for PCR amplification. Extraction of DNA was carried out by the CTAB method (Rogers S O. Extraction of DNA from Plant tissues. Plant molecular biology manual 1989).
During PCR amplification, a 10. mu.L reaction system was designed as follows:
DNA sample, 2. mu.l (50 ng/ul);
10 PCR buffer(Mg2+)，1μl；
d NTP(2.5mmol/L)，1μl；
Primer F (10umol/L)，0.5μl；
Primer R (10umol/L)，0.5μl；
TaqE (5U/ul) ，0.1μl；
ddH2O，4.9 ul。
after the reaction system was mixed well, PCR amplification was performed according to the following procedure:
pre-denaturation at 95 ℃ for 3 min;
denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 45 s, and extension at 72 ℃ for 1 min for 35 cycles;
extension for 10 min at 72 ℃.
The PCR amplification product was directly analyzed by electrophoresis (or stored at 16 ℃ for further use). When the PCR amplification product is subjected to electrophoresis, 2 mu L of non-denatured 6 Xloading buffer is added into the PCR amplification product, and 8% polyacrylamide electrophoresis detection is carried out after the mixture is uniformly mixed.
The electrophoresis results were photographed and the band patterns were recorded, and the same band pattern as the parent jiejing wheat T093 was designated as "2", the same band pattern as the parent Zhouyi wheat 18 was designated as "0", the heterozygous band pattern was designated as "1", and the band pattern which was missing or not detected was designated as "-1".
The results of some of the experiments are shown in FIG. 2.
(III) BSA (bulked Segregant analysis) analysis
Using segregating population grouping analysis (BSA), at F2In the population, 60 strains of each of the extreme resistant pool and the extreme sensitive pool were selected. SNP detection is carried out on 18 weeks of parents, Jijiezhi wheat T093, a polar resistance pool and a polar induction pool by using a wheat 660k chip developed by the existing Jia.
And (5) combining the statistical results of the step (II), and comprehensively analyzing to consider that:
after comparison with wheat IWGSC _ RefSeq _ v1.0 version, for F2The 4 SSR markers can position a 3D chromosome target section in a 538M-565M interval, and further combine the SNP detection results of a wheat 660k chip, and in the interval, 52 different SNPs are found in an anti-pool and a sensing pool. Further designing and developing KASP primer groups according to flanking sequences at two sides of the 52 SNP sites, finally successfully obtaining 10 KASP molecular markers, and further carrying out F18, T093 and 1884 strains on the parents2Typing was performed.
QTL localization analysis
And (5) carrying out QTL analysis by using QTL IciMapping v 4.0 based on the typing result of the KASP and the electrophoresis results of the 4 SSR primers in the step (III).
Although the number of KASP tags is designed to be plural, there are typically 3 tags, specifically:
A009711：
A009711-1：5'-TGGTGATTAGCATCATCGGAATGG-3'，
A009711-2：5'-TTGGTGATTAGCATCATCGGAATGT-3'，
A009711-3：5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180：
A009180-1：5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3'，
A009180-2：5'-AGTGAATTTTTAAAGTTCGCATACCCC-3'，
A009180-3：5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716：
A009716-1：5'-GTTGCTATGTAACGGAATAAGAACG-3'，
A009716-2：5'-CGTTGCTATGTAACGGAATAAGAACT-3'，
A009716-3：5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'。
in specific application, the existing Xcfd223 and the SSR marker Xgwm383b are matched to carry out molecular marking on the pre-harvest sprouting resistant gene, and the specific combined application mode is as follows:
xcfd223 was labeled in conjunction with A009711;
a009711 and A009180 are matched for marking;
a009716 was labeled in combination with Xgwm383 b;
the Xcfd223 has the following specific sequence:
Xcfd 223：
5'-AAGAGCTACAATGACCAGCAGA-3'，
5' –GCAGTGTATGTCAGGAGAAGCA-3'。
it should be noted that the Xcfd223 marker belongs to a sequence disclosed in the prior art, and specifically, see: an Advanced backs marketing approach noise Synthetic ideal Wheat as a "Bridge": Development and QTL Detection for Seed Dormacy (Dale Zhang et al, Frontiers in Plant Science, 2017).
Using IciMapping4.0 software, performing 1000 times of arrangement detection, and determining the critical value of the pre-harvest sprouting resistant LOD as2.3604. Method of plotting complete intervals for F2QTL analysis was performed on the population as shown in the following table, with 3 QTL sites detected in total.
The specific analysis results are shown in the following table and fig. 3.
Table 1: f2Additive QTL position, effect and contribution rate of population pre-harvest sprouting resistance character
The QTL analysis of the table above shows that there are 3 QTLs on the 3D chromosome that are associated with ear sprouting:
the first QTL is between xcfd223 and A009711, the physical distance between the two markers is 2.1M, the genetic distance is 5.25 c M, the LOD value of the QTL is 119, the phenotype contribution rate is 24.16%, and the additive effect is-16.61;
the second QTL was between a009711 and a009180, with a physical distance of 6.9M between the two markers and a genetic distance of 17.44 c M. The LOD value of the QTL is 70, the phenotype contribution rate is 15.49 percent, and the additive effect is-12.59;
the third QTL is between A009716 and xgwm383b, and the genetic distance between the two markers is 43.56 c M. The LOD value of this QTL was 22 with a phenotypic contribution of 4.74% and an additive effect of-6.03.
The additive effects are negative, which indicates that the introduced segment from the Jiejiemai T093 plays a role in reducing the germination of grains, namely the germination rate of wheat grain germination is reduced, and the method is used for molecular marker assisted breeding and research and breeding work in the aspect of wheat pre-harvest germination resistance.
It should be explained that 201 BC are constructed in the published article "An Advanced backs Power consumption through structural outline As a" Bridge ": Development and QTL Detection for Seed management3F4And the population carries out primary positioning on the pre-harvest sprouting QTL, and the positioning mark is Xcfd 223. On the basis of preliminary positioning of the pre-germination QTL, the population quantity is enlarged, and 1884F plants are subjected to quantitative analysis2660k chip analysis and KASP marker development are carried out on the colony by using BSA, and the SSR marker is combined to further narrow the germination QTL locating interval of the ear of grainThe QTL interval for germination is 9M, wherein 3 QTLs relevant to the ear germination are identified, and meanwhile, the preliminarily positioned QTLs are verified to provide reference for further determining the ear germination genes.
SEQUENCE LISTING
<110> university of Henan
<120> series molecular markers and application thereof in wheat ear germination gene molecular markers
<130> none
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial design
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<210> 2
<211> 21
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<213> Artificial design
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gacatcaata accgtggatg g 21
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tggtgattag catcatcgga atgg 24
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ttggtgatta gcatcatcgg aatgt 25
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ccaaatagaa gtatcacttg aacaatgctt 30
Claims (3)
1. The series of molecular markers are used for marking the pre-harvest sprouting related genes, and are characterized in that the number of the series of molecular markers is 5 in total, and the series of molecular markers comprises two SSR molecular markers and three KASP molecular markers; the two SSR molecular markers are respectively: xgwm383b, Xcfd223, three KASP molecular markers are respectively: a009711, a009180, a 009716; the specific sequences of the series of molecular markers are respectively as follows:
Xgwm383b：
Xgwm383b -F：5'- ACGCCAGTTGATCCGTAAAC-3'，
Xgwm383b -R：5'- GACATCAATAACCGTGGATGG-3'；
A009711：
A009711-1：5'-TGGTGATTAGCATCATCGGAATGG-3'，
A009711-2：5'-TTGGTGATTAGCATCATCGGAATGT-3'，
A009711-3：5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180：
A009180-1：5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3'，
A009180-2：5'-AGTGAATTTTTAAAGTTCGCATACCCC-3'，
A009180-3：5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716：
A009716-1：5'-GTTGCTATGTAACGGAATAAGAACG-3'，
A009716-2：5'-CGTTGCTATGTAACGGAATAAGAACT-3'，
A009716-3：5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'；
Xcfd 223：
5'-AAGAGCTACAATGACCAGCAGA-3'，
5'-GCAGTGTATGTCAGGAGAAGCA-3'。
2. the use of the series of molecular markers of claim 1 in the marking of pre-harvest sprouting resistance genes, which is used for carrying out the molecular marking of the pre-harvest sprouting resistance genes transferred into wheat genomes in the genome of the segmental wheat in genetic breeding.
3. The molecular marking method of the pre-harvest sprouting resistance gene using the series of molecular markers according to claim 1 is characterized in that, in the specific application:
xcfd223 was labeled in conjunction with A009711;
a009711 and A009180 are matched for marking;
a009716 was labeled in conjunction with Xgwm383 b.
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| WO2001073091A2 (en) * | 2000-03-24 | 2001-10-04 | Pioneer Hi-Bred International, Inc. | Methods of selection and development of plants having improved root quality and root lodging resistance |
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