CN106676180B - Molecular marker for regulating and controlling maize tassel branch number major QTL and application thereof - Google Patents
Molecular marker for regulating and controlling maize tassel branch number major QTL and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to a molecular marker for regulating and controlling a major QTL (quantitative trait locus) of the maize tassel branch number and application thereof. The molecular marker for regulating the major QTL of the maize tassel branch number consists of two pairs of SSR markers including umc2177 and umc 1378. A method for assisting selection of maize with few tassel branches comprises the steps of extracting genomic DNA of maize to be detected, carrying out PCR amplification on the markers umc2177 and umc1378, and when amplification products with the lengths of 234bp and 331bp are obtained, determining the maize to be detected as the maize with few tassel branches candidate, and applying the maize candidate to breeding practice. The molecular marker is used for assisting in selecting the corn with few tassel branches, the number of tassel branches can be predicted only by detecting the characteristic strip of the molecular marker, and the identification method is easy to operate, simple and feasible, high in selection efficiency and has huge application potential in the field of high-yield breeding of the corn.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a molecular marker for regulating and controlling a main effect QTL of the maize tassel branch number and application thereof.
Background
Tassels serve as important reproductive organs of corn, which not only directly affect the production quality of corn seeds, but also are closely related to the yield formation of corn. A large number of researches show that the corn plants with smaller tassels have strong light transmittance at the lower parts of the groups, and the tassels grow to consume less nutrients, so that the corn yield is improved. In addition, researchers also find that the maize tassel is closely related to drought resistance, the indexes related to the maize tassel under drought stress, such as the main axis length of the tassel and the stalk length of the tassel, are obviously shortened, the tassel branch number is obviously reduced, the tassel-drawing and silking time interval (ASI) is obviously prolonged, and finally the maize is seriously reduced in yield. The tassel branch number is an important evaluation index of the tassel size, so that the genetic mechanism of the maize tassel branch character is deeply analyzed, the internal relation between the tassel branch character and the drought resistance is disclosed, and the method has important significance for maize high-yield breeding.
The difference of the tassel branch number is an important characteristic of the maize tassel, and the difference of the tassel branch number among different genetic background materials is large. Some self-bred materials have a large number of tassels, but some self-bred materials have few tassels or even no branches. In terms of genetic expression, the tassel branch number is a complex quantitative character controlled by multiple genes, the mechanism is extremely complex, and the tassel branch number is easily influenced by the environment, so that the method brings a lot of difficulties for researching the tassel mechanism of the corn. In recent years, with the intensive research of molecular biology and the continuous improvement of molecular marker technology, Quantitative Trait Loci (QTL) location can be used to roughly determine the genetic loci and positions of related quantitative traits, estimate the genetic effect of each locus, and further provide theoretical basis and technical support for future molecular breeding. At present, the research on the positioning of the maize tassel branch number QTL becomes a hot topic of attention of scholars and breeders, but the application value of the tassel branch number QTL is less, so that the mining of the main effect QTL of the tassel branch number which is stably expressed under different genetic backgrounds and different environments has great potential application value for the high-yield breeding of maize.
Disclosure of Invention
The invention aims to provide a molecular marker for regulating and controlling the major QTL of the tassel branch number of corn, also provides a method for assisting in selecting corn with fewer tassel branches, and also provides application of the molecular marker for regulating and controlling the major QTL of the tassel branch number of corn in corn breeding.
In order to solve the technical problems, the invention adopts the following three technical schemes:
firstly, a molecular marker for regulating and controlling maize tassel branch number major QTL is characterized in that: the primer is composed of two pairs of umc2177 and umc1378, wherein the sequence of the primer umc2177 is as follows:
Forward: 5’ - ACCATGCATGTCTCACGTCACT-3’
Reverse: 5’- GGGTACGTGCTGTGGAGGAC-3’
the sequence of the primer umc1378 is as follows:
Forward: 5’-GAAGTCGCTGATGAGAACGTAACC-3’
Reverse: 5’-GCTAGCTAGTGTGAGTTCTTCCGC-3’。
secondly, a method for assisting in selecting corn with less tassel branches comprises the following steps: extracting the genome DNA of the corn to be detected; PCR amplification with primers umc2177 and umc 1378; when the amplification products with the lengths of 234bp and 331bp are obtained, the corn to be detected is the corn with less tassel branches.
Thirdly, the application of the molecular marker for regulating the maize tassel branch number major QTL in maize breeding is characterized in that: the method for assisting in selecting the maize with less tassel branches provided by the invention identifies the maize with less tassel branches, and then applies the maize with less tassel branches to maize breeding.
The invention is realized by two sets of F2:3The major QTL for regulating the maize tassel branch number is analyzed under the multi-moisture environment treatment, a major QTL for regulating the maize tassel branch number exists between umc2177 and umc1378 markers of a seventh chromosome Bin7.00 region of maize, and is named as qTBN-Ch.7-1 by the inventor. Under multiple moisture environment treatment, the qTBN-Ch.7-1 is in two sets of F2:3The cumulative phenotypic contribution of the pedigrees was 46.01% and 34.09%, respectively. Analysis shows that PCR amplification is carried out on the corn to be detected by utilizing the two pairs of SSR markers, and the size of the tassel of the corn to be detected can be predicted.
The invention has the beneficial effects that: the molecular marker disclosed by the invention is used for molecular marker-assisted selection, and the size of the maize tassel can be predicted only by detecting a characteristic amplification band of the molecular marker. The method can identify the corn single plant with smaller tassel in the early stage of corn growth, eliminate other single plants, has clear selection target, is not influenced by environment, and effectively improves the breeding and utilization value of candidate corn.
Drawings
FIG. 1 is a histogram of LTpop tassel branch number;
FIG. 2 is a histogram of the number of branches of the male ear of CTpop;
FIG. 3 is a schematic diagram of LTpop genetic linkage map and tassel branch number major QTL mapping;
FIG. 4 shows the CTpop genetic linkage map and the location of the tassel branch number major QTL.
Detailed Description
The present invention will be further described with reference to the following examples, wherein the test methods in the following examples are all conventional test methods unless otherwise specified, and the test reagents and consumables described in the following examples are all available from conventional biochemical reagents company.
In this embodiment, the detailed steps of obtaining the molecular marker for regulating the maize tassel branch number major QTL are as follows:
1. corn F2:3Group construction and determination of tassel branch number under different moisture environment treatment
Two sets of F are constructed by using 3 selfing lines with large difference in tassel branch number and drought resistance obtained by screening in the early stage of the subject group as parents2Segregating the population, F2Segregating the population for selfing to obtain F2:3Family members. Wherein the Luo Huang/Chang 7-2 is a strong drought-tolerant inbred line with few tassel branches as a female parent, and the TS141 is a drought-sensitive inbred line with many tassel branches as a common male parent. F constructed by corridor yellow and TS1412:3The family is abbreviated as LTpop, and comprises 202 strains; f constructed by Chang 7-2 and TS1412:3The family is abbreviated as CTpop, and comprises 218 strains. LTpop and the corresponding parents were planted in Wuwei (37.97 ℃ N, 102.63 ℃ E; 1508 m) and Zhangye (38.83 ℃ N, 106.93 ℃ E; 1536 m) in 2014. CTpop and the corresponding parents were planted in ancient waves (36.67 ℃ N, 102.85 ℃ E; 1785 m) and cloisonne (37.18 ℃ N, 104.03 ℃ E; 1640 m) in 2015. Two sets F2:3The family is respectively subjected to drought stress treatment and normal water supply treatment at four test points, and is designed according to a completely random block group by adopting a flat film drip irrigation technology, and the three times of repetition are carried out, so that the double-row area is formed, the row length is 6.0 m, the plant spacing is 0.5 m, and the row spacing is 0.6 m. The drought stress treatment is that the water is not poured before the large flare period and after the flowering period, and the water is poured once every 20 days in other periods. The normal water supply treatment is to irrigate water in time when water is deficient in the corn growth process.
Under the treatment of different moisture environments, two sets of F are respectively selected2:3The growth vigor of the family is integrally consistent with that of 10 individuals, the tassel branch number (TBN; TBN is the primary tassel branch number) of each individual is measured when the flowering period is finished, and then the average value of the number of the tassel branch numbers is taken to represent the tassel size of each line. According to the formula:
The characters in table 1 illustrate: W-W, S-W, W-Z, S-Z, W-G, S-G, W-J, S-J respectively refers to Wuwei normal water supply treatment, Wuwei drought stress treatment, stress solution normal water supply treatment, stress solution drought stress treatment, ancient wave normal water supply treatment, ancient wave stress treatment, cloisonne normal water supply treatment and cloisonne drought stress treatment; the same is as follows.
As can be seen from Table 1: under the same moisture environment treatment, the difference of the tassel branch numbers of the female parent gallery yellow/Chang 7-2 and the male parent TS141 is obvious; under drought stress treatment, the tassel branch number of 3 inbred lines is reduced, but the reduction degree of the tassel branch number of the drought-sensitive inbred line TS141 is far higher than that of the strong drought-tolerant inbred line Luo Huang/Chang 7-2.
As can be seen from table 1, fig. 1 and fig. 2: under the condition of different moisture environmental treatments, two sets of F are constructed2:3The number of tassel branches of the family is typically normally distributed, and the generalized heritability of the tassel branches is 78.69% and 84.26%, which indicates that the tassel branches are quantitative under the control of multiple genes and greatly influenced by genetic nature, and QTL analysis is feasible.
2. SSR marker design and polymorphism screening thereof
SSR marker 872 pairs evenly distributed in 10 chromosomes of maize were selected at maize genomic database MaizeGDB website (http:// www.maizegdb.org /), synthesized by Sangon in Shanghai. Extracting genome DNA of parent Luo Huang, Chang 7-2 and TS141 by CTAB method, detecting DNA quality by 1% agarose gel electrophoresis, and detecting DNA concentration by German IMPLEN microspectrophotometer. The PCR reaction system is shown in Table 2, the PCR amplification reaction program is shown in Table 3, and the amplification product is subjected to 8% non-denaturing polyacrylamide gel electrophoresis and silver staining.
Through analysis, 213 pairs of SSR markers with clear bands and good polymorphism are screened out between the parent Luo Huang and the parent TS141, and 217 pairs of SSR markers with clear bands and good polymorphism are screened out between the parent Chang 7-2 and the parent TS 141. These polymorphic SSR markers will be used in two sets F2Separating the genotype analysis of the population, and constructing a corresponding genetic linkage map in step 3.
3. Construction of genetic linkage map
Two sets F constructed using step 12Segregating the population and the corresponding polymorphic SSR markers screened in step 2 for two sets of corresponding F2The population was isolated for genotyping and genetic linkage maps were constructed using the JoinMap4.0 software (http:// www.kyazma.nl/index. php/mc. JoinMap/sc. Evaluate /), and genetic distance (cM) was calculated using the Kosambi function. The genetic linkage map of the obtained LTpop is shown in FIG. 3; the resulting genetic linkage map of CTpop is shown in FIG. 4.
As can be seen from fig. 2 and 3: the two sets of constructed genetic linkage maps respectively comprise 199 and 205 pairs of SSR markers, cover 10 linkage groups of corn, the total genetic distance is 1542.5 cM and 1648.8 cM, and the average genetic distance between molecular markers is 7.8 cM and 8.0 cM. The relative order of the markers on the reference map was highly consistent for both sets of genetic maps compared to IBM 22008 Neighbors (http:// www.maizegdb.org/data _ center/map).
4. Tassel branch number major QTL location
According to two sets F2:3Family systemDetecting corresponding F by adopting a Composite Interval Mapping (CIM) in Windows QTLCartographer version 2.5 software (http:// statgen. ncsu. edu/qtlrat/WQTLctart. htm) under the treatment of different moisture environments2:3The tassel branch number QTL of the family. For CIM, using the Zmapqtl program module Model 6 with a window size of 10.0 cM, the tassel branch number was genotyped every 0.5 cM and the LOD threshold (LOD) was determined by 1000 samplings>3.0). QTL mode of action was estimated from the absolute value of the ratio of the dominant (D) and additive (a) effects (stubbeet al 1987): D/A | = 0.00-0.20 is additive (A), | D/A | = 0.21-0.80 is Partially Dominant (PD), | D/A | = 0.81-1.20 is dominant (D), | D/A | = hair cut>1.20 is hyperdominant (OD). QTL nomenclature reference is made to the nomenclature of mccuch et al, (1997), i.e. q + trait name abbreviation + chromosome name + QTL numbering on the chromosome. Adopting Patentin version 3.5 software to generate an SSR marker sequence table at two ends of a main effect tassel branch number QTL; the SSR marker sequence table at two ends of the major tassel branch number QTL is shown in the nucleotide and amino acid sequence table of the specification. The schematic diagram of the location of the major QTL of the LTpop tassel branch number is shown in figure 3; the schematic diagram of the obtained CTpop tassel branch number major QTL location is shown in FIG. 4. Two sets F2:3The main effect QTL detection result of the branch number of the tassel of the family is shown in the table 4.
CIM analysis showed that the two sets F are shown in Table 4, FIG. 3 and FIG. 42:3The major QTL for regulating the tassel branch number is named as qTBN-Ch.7-1 and is located at the seventh chromosome Bin7.00 and between markers umc2177 and umc1378, the action mode of the gene is mainly the super dominant effect, and alleles for reducing the tassel branch number are all from parent corridor yellow/Chang 7-2. The QTL was detected in LTpop under 2 drought stress treatments and 1 normal water supply treatment simultaneously with a cumulative phenotype contribution of 46.01% and a genetic distance of 19.7 cM. The QTL was detected simultaneously under 2 drought stress treatments and 1 normal water supply treatment in CTpop, which was a test for the presence of the QTL in the plantThe cumulative phenotype contribution rate was 34.19% and the genetic distance was 0.6 cM. The QTL for reducing the tassel branch number is derived from parent corridor yellow/Chang 7-2, so that the QTL can be used for predicting the size of the tassel of the corn, but the QTL has a gene action mode of super-dominance, so that the influence of the heterosis on the size of the tassel is particularly paid attention to when the hybrid is assembled.
The molecular marker for regulating the maize tassel branch number major QTL consists of two pairs of SSR markers umc2177 and umc1378, wherein the sequence of the marker umc2177 is as follows:
Forward: 5’ - ACCATGCATGTCTCACGTCACT-3’
Reverse: 5’- GGGTACGTGCTGTGGAGGAC-3’
the sequence of the primer umc1378 is as follows:
Forward: 5’-GAAGTCGCTGATGAGAACGTAACC-3’
Reverse: 5’-GCTAGCTAGTGTGAGTTCTTCCGC-3’
the method for selecting the maize with fewer tassel branches by utilizing the molecular marker for regulating the major QTL of the tassel branch number of the maize comprises the following steps: extracting the genome DNA of the corn to be detected, carrying out PCR amplification by using markers umc2177 and umc1378, and obtaining the amplification products with the lengths of 234bp and 331bp, wherein the corn to be detected is a corn single plant with smaller candidate tassel branch number, other single plants are eliminated, purposeful combination and hybridization combination are matched, and a new corn variety with moderate tassel size and high yield is bred, and further the new corn variety is applied to production practice.
Sequence listing
SEQUENCE LISTING
<110> university of agriculture in Gansu province
<120> molecular marker for regulating and controlling maize tassel branch number major QTL and application thereof
<130>2016
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<170>PatentIn version 3.5
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<211>22
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1
Claims (2)
1. The method for selecting the maize with less tassel branches by using the molecular marker for regulating the major QTL of the tassel branch number of the maize is characterized by comprising the following steps of: extracting the genome DNA of the corn to be detected; PCR amplification of the molecular markers umc2177 and umc1378 with the primer sequence of the amplification molecular marker-; when the amplification products with the lengths of 234bp and 331bp are obtained, the corn to be detected is the corn with less tassel branches; wherein the primer sequence of the amplification molecular marker umc2177 is as follows:
Forward: 5’ - ACCATGCATGTCTCACGTCACT-3’；
Reverse: 5’- GGGTACGTGCTGTGGAGGAC-3’；
the primer sequence of the amplification primer umc1378 is as follows:
Forward: 5’-GAAGTCGCTGATGAGAACGTAACC-3’；
Reverse: 5’-GCTAGCTAGTGTGAGTTCTTCCGC-3’。
2. the application of the molecular marker for regulating the major QTL of the maize tassel branch number in maize breeding is characterized in that: the method for selecting maize with fewer tassel branches as provided in claim 1 identifies maize with fewer tassel branches as candidate and applies maize with fewer tassel branches as candidate for maize breeding.
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| CN112029897B (en) * | 2020-10-14 | 2022-07-22 | 江苏省农业科学院 | SNP marker closely linked with continuous multi-leaf-position leaf width main effect QTL under corn tassel and application thereof |
| CN112626261B (en) * | 2021-01-18 | 2022-05-17 | 吉林大学 | SNP molecular marker related to maize tassel branch number character and application |
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Non-Patent Citations (3)
| Title |
|---|
| Association analysis of important agronomical traits of maize inbred lines with SSRs;Qianqian Zhang et al.;《Australian Journal of Crop Science》;20121231;第6卷(第7期);Supplementary Data Table S4 * |
| Genetic and QTL analysis of maize tassel and ear inflorescence architecture;N.Upadyayula et al.;《Theor Appl Genet》;20060105;第112卷(第4期);摘要部分、图3和表4 * |
| Missouri maize project releases new SSR marker data;Mary Polcco et al.;《http://www.bio.net/bionet/mm/maize/2000-March/000746.html》;20000331;第8页 * |
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