CN116769950A - Molecular marker closely linked with corn cob long-acting QTL and application - Google Patents

Molecular marker closely linked with corn cob long-acting QTL and application Download PDF

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CN116769950A
CN116769950A CN202310452312.5A CN202310452312A CN116769950A CN 116769950 A CN116769950 A CN 116769950A CN 202310452312 A CN202310452312 A CN 202310452312A CN 116769950 A CN116769950 A CN 116769950A
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molecular marker
corn
length
closely linked
detected
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李卫华
汤继华
张战辉
薛亚东
李冰
陈永强
张雪海
陈晓阳
郭战勇
付志远
丁冬
赵香漪
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Shennong Seed Industry Laboratory
Henan Agricultural University
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Henan Agricultural University
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Abstract

The invention belongs to the technical field of biology, and particularly relates to a molecular marker closely linked with a main effect QTL of corn ear length, and a primer and application thereof. The molecular marker is positioned on the 3 rd chromosome of the corn and comprises a molecular marker M158 and a molecular marker M0900; the nucleotide sequence of the molecular marker M158 is shown as SEQ ID NO.21, and the nucleotide sequence of the molecular marker M0900 is shown as SEQ ID NO. 24. The primer sequences of the amplified molecular marker M158 are as follows: M158-F:5'-CCGAGTGTGAGTGAGGACAA-3'; M158-R:5'-CACGTGGATTGGTTACGATG-3'; the primer sequence of the amplified molecular marker M0900 is as follows: M0900-F:5'-ACGATGCATGGTTGGAGTTG-3'; M0900-R:5'-CCATCAAACAAAGTGGCCCA-3'. The molecular marker provided by the invention is closely linked with the main effect QTL of the corn ear length, can be applied to the auxiliary breeding of the corn ear length molecular marker, is applied to the screening of corn ear length germplasm resources, and is applied to the genetic improvement of the corn ear length.

Description

Molecular marker closely linked with corn cob long-acting QTL and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular marker closely linked with a main effect QTL of corn ear length and application thereof.
Background
The corn has various functions, is not only a grain crop for human survival, but also a feed crop in animal world, and is an industrial product for promoting economic development, so that the corn yield has an extremely important influence on agricultural production in China. The length of the corn ear part is an important part of the yield forming factors, and the research is significant. Corn yield per unit depends on the number of ears and the weight of seeds, while the number of ears and rows determine the number of ears and the number of seeds, and the developmental integrity of the seeds determines the weight of the seeds. Therefore, screening and identifying the scion stock resources is an important way to cultivate new varieties of high-yield corns.
The conventional breeding method has long breeding period and low efficiency of breeding spike-length germplasm resources. However, molecular marker assisted breeding shortens the breeding period, accelerates the breeding process, improves the breeding efficiency, and overcomes the difficulties in many conventional breeding methods. The molecular marker assisted selection requires the selection of the target trait genotype by means of molecular markers by analyzing the genotype of the molecular marker closely linked to the target gene.
Linkage analysis (1 linkage analysis) is also called QTL positioning analysis, and the principle of the linkage analysis is that linkage markers associated with target traits are continuously searched according to the mutual combination of genotypes and phenotypes of the target traits, and linkage genetic maps are drawn through the integration of marker positions. A single fragment substitution refers to the identity of the remainder of the chromosome with the receptor, except for the segment of interest. The single fragment substitution (SSSL) is characterized as follows: the stability is strong; the influence caused by the environment can be eliminated; the positioning consideration factors are few, and the result accuracy is high.
Because the single-segment substitution system has high stability and small environmental impact, the single-segment substitution system is widely used for QTL positioning, but the obtained positioning interval is larger, and the molecular marker cannot be applied to production. Therefore, it is required to develop a molecular marker closely linked to the target gene for genetic improvement of corn ear length.
Disclosure of Invention
The invention aims to provide a molecular marker closely linked with a main effect QTL of corn cob length and application thereof, and the molecular marker is closely linked with the main effect QTL of corn cob length and can be applied to auxiliary breeding of the molecular marker of corn cob length.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the molecular marker is located on the 3 rd chromosome of corn and is a molecular marker M158 and a molecular marker M0900.
Preferably, the molecular marker M158 is located on chromosome 3 of corn, and the specific position chr3 is: 231733871-231734353, the molecular marker M0900 is positioned on the 3 rd chromosome of corn, and the specific position chr3 is: 232377659-232377854.
The primer of the molecular marker closely linked with the main effect QTL of the corn ear length is amplified, and the primer sequence of the molecular marker M158 is as follows:
M158-F:5'-CCGAGTGTGAGTGAGGACAA-3' (sequence 1);
M158-R:5'-CACGTGGATTGGTTACGATG-3' (SEQ ID NO: 2);
the primer sequence for amplifying the molecular marker M0900 is as follows:
M0900-F:5'-ACGATGCATGGTTGGAGTTG-3' (SEQ ID NO: 3);
M0900-R:5'-CCATCAAACAAAGTGGCCCA-3' (SEQ ID NO: 4).
The application of the molecular marker closely linked with the main effect QTL of the corn cob length in the genetic improvement of the corn cob length.
Preferably, the method for identifying the corn ear length character in the corn ear length molecular marker assisted breeding process comprises the following steps:
extracting genome DNA of corn leaves;
taking corn leaf genome DNA as a template, and respectively carrying out PCR amplification by using the primers M158-F/M158-R, M0900-F/M0900-R;
agarose gel electrophoresis identification PCR amplification results: when the adopted primer is M158-F/M158-R, and the size of the molecular marker M158 is detected to be 321bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M158 is detected to be 502bp, the spike length of the sample to be detected is short; when the adopted primer is M0900-F/M0900-R, when the size of the molecular marker M0900 is detected to be 215bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M0900 is detected to be 205bp, the spike length of the sample to be detected is short.
Preferably, the PCR amplification system comprises 8 μl, and the components comprise: 2. Mu.LDNA, 1. Mu.L each of left and right primers, 4. Mu.L of 2 XTaq Master Mix (Norpran P112).
Preferably, the Touchdown PCR amplification procedure is used: 95 ℃ for 5min;95 ℃ for 30s and 65 ℃ for 30s, and each cycle is reduced by 1 ℃ and 72 ℃ for 45s, and 8 cycles are total; 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45s, and 28 cycles are total; and at 72℃for 10min.
Compared with the prior art, the invention has the beneficial effects that:
the molecular marker closely linked with the main effect QTL of the corn ear length is closely linked with the main effect QTL of the corn ear length, can be applied to the auxiliary breeding of the molecular marker of the corn ear length, can be applied to the screening of germplasm resources of the corn ear length and can be applied to the genetic improvement of the corn ear length.
Drawings
FIG. 1 is a photograph of a mature ear of the natural number 178 and SSSL3 of example 1, scale bar 1cm;
FIG. 2 is a mature ear phenotype analysis of accession 178 and SSSL3 of example 1;
FIG. 3 is a molecular marker polyacrylamide gel electrophoresis of example 1;
wherein A, B is an electropherogram of umc1639 and umc1136, respectively; lanes 1-4 in A and B are SSSL3, well 178, dominant pool and recessive pool, respectively;
FIG. 4 shows the qEL3 gene primary targeting of example 1;
fig. 5 is a fine positioning of qEL3 of embodiment 1.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
In the description of the present invention, unless otherwise specified, all reagents are commercially available and methods are conventional in the art.
Example 1
The embodiment provides a positioning process of a main effect QTL with the length of the corn ears, which is specifically as follows:
(1) Identification of spike length single-segment substitution line SSSL3
1.1 construction of corn Single fragment substitution line population
The experimental material is selected from more than 150 homozygous single-segment substitution lines constructed by taking a plurality of 178 as acceptor parents and taking comprehensive 3 as donor parents through multi-generation backcross selfing in the early stage of a laboratory.
1.2 field determination of the ear phenotype of the Single segment substitution population
In 2013 and 2014, 150 single-segment substitution lines and 178 acceptor materials thereof are planted in Henan Cheng county, xinxiang and Chuchang respectively. Each material was provided with 2 replicates, each 2 rows of 4 meters long, 0.65 meters row spacing, 0.25 meters row spacing. After the mature harvesting, the ear length and the ear thickness of mature ears are measured by using a seed tester (Jetion) measurement, and the ear number and the row number of the ears are counted manually.
1.3 phenotypic analysis of mature ears
Through two-year four-point field phenotype measurement, the single-segment substitution line SSSL3 is found to be obviously increased in spike length and row grain number compared with background material No. 178, and the spike thickness and the spike row number are not obviously different, and the result is shown in figure 2.
(2) Corncob long-acting main effect QTL qEL3 initial positioning
2.1 construction of a positioning population: using single-segment substitution line SSSL3 as female parent, hybridizing with Zhong 178 to obtain F1 plant, back-crossing F1 plant with Zhong 178 to obtain BC 1 F 1 A population.
2.2 identification of the ear length phenotype: BC was constructed in Hainan 2018 1 F 1 And (5) planting 45 rows of groups according to the row length of 4 meters. For BC 1 F 1 Sampling and extracting DNA from individual leaves of the population, preserving at low temperature, and carrying out self-pollination seed reserving on the individual leaves. Seed after single plant selfing in spring in 2019 in original sun (BC) 1 F 2 ) 2 replicates, each replicate 2 rows, 4 meters long, 0.65 meters row spacing, 0.25 meters row spacing. After maturation and harvesting, for BC 1 F 2 Seed test is carried out on the cluster spike length by BC 1 F 2 Determination of BC by the ear Length phenotype of the population 1 F 1 Single plant genotype.
2.3DNA extraction and molecular marker development
The corn leaf genome DNA is extracted by CTAB method and stored in a refrigerator at-20 ℃ for standby.
SSR markers: the laboratory has now overlaid 1000 pairs of SSR markers (MaizeGDB (http:// www.maizegdb.org /) database IBM) of maize whole genome.
InDel marker development: indels were synthesized using known Indel primers from the laboratory, with the sequences of the syntheses being shown in table 1.
2.4PCR procedure and amplification product genotyping
The PCR amplification system (8. Mu.L) components contained: 2. Mu.L of DNA, 2. Mu.L of primers (1. Mu.L of each of the left and right primers), 4. Mu.L of 2 XTaq Master Mix (Norwezan P112). Using the Touchdown PCR amplification procedure: 95 ℃ for 5min;95 ℃ for 30s,65 ℃ for 30s (1 ℃ drop per cycle), 72 ℃ for 45s, 8 cycles total; 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45s, and 28 cycles are total; and at 72℃for 10min. The PCR amplified products were subjected to genotyping by 6% polyacrylamide gel electrophoresis and 4% agarose gel electrophoresis.
2.5 initial positioning of major QTL for corn ear length
DNA of parent material number 178 and single-segment substitution material SSSL3 is extracted to form 2 parent pools. Selecting 10 plants of dominant trait (long ear) individuals (Aa genotype individuals in BC1F1 population) and 10 plants of recessive trait (short ear) individuals (BC 1 F 1 Aa genotype individual in the population) to form two near isogenic line pools. Genotype analysis is carried out on the parent pool by using 1000 pairs of SSR markers covering the whole genome of the corn, 2 pairs of polymorphic molecular markers, namely umc1639 and umc1136, are screened, and an electrophoresis diagram of the 2 pairs of polymorphic molecular markers is shown in figure 2. Development of markers and screening of polymorphic molecular markers was continued to the left of 3.09bin, with molecular marker primer information see table 1.
Table 1 screening of 19 pairs of linkage difference marker sequence information
Analysis of 2015 and 2016 BC Using polymorphic molecular markers of Table 1 1 F 1 Genotype of population individuals. 106 crossover individuals were classified into 8 different types (long spike individuals 4 and short spike individuals 4) of recombinant individuals in combination with the individual progeny phenotype. By cross-line mapping, the target gene was located between chromosome 3 molecular markers Chr3.09-14 and Chr3.09-176 at a physical distance of 4.5Mb, as shown in FIG. 4. The white boxes in fig. 4 represent genomic fragments from accession 178 and the black boxes represent fragments of SSSL 3.
(3) Corn cob long-acting main effect QTL qEL3 fine positioning
3.1 construction of a positioning population: at BC 1 F 1 Single plant selfing construction BC for selecting target section as heterozygous in population 1 F 2 A population.
3.2 identification of the ear length phenotype: BC was planted in three kinds of Hainan in winter in 2019 1 F 2 And (3) group, single plant selfing seed reserving. Spring 2020 and evening 2020The single plant selfing offspring seeds are planted in the original sun of the new country in summer for phenotype identification, 2 repeats are planted, each repeat is 4 m long, 2 rows are planted, the row spacing is 0.65 m, and the plant spacing is 0.25 m. Measuring the ear length of the clusters of the harvest group by using a seed tester, and judging BC by using the phenotypic result of the cluster ear length 1 F 2 Single plant genotype.
3.3DNA extraction and molecular marker development
The corn leaf genome DNA is extracted by CTAB method and stored in a refrigerator at-20 ℃.
InDel marker development: the selection of differential markers was performed with parental SSSL3 and accession 178 using Indel markers present in the Indel marker development interval on 10 chromosomes already existing in the laboratory.
3.4PCR procedure and amplification product genotyping
The PCR amplification system (8. Mu.L) components contained: 2. Mu.L of DNA, 2. Mu.L of primers (1. Mu.L of each of the left and right primers), 4. Mu.L of 2 XTaq Master Mix (Norwezan P112). Using the Touchdown PCR amplification procedure: 95 ℃ for 5min;95℃30s,65℃30s (1℃drop per cycle), 72℃45s, 8 cycles total; 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45s, and 28 cycles are total; and at 72℃for 10min. And carrying out genotype analysis on the PCR amplification product by agarose gel electrophoresis.
3.5 Fine positioning of major QTL for corn ear length
The polymorphic molecular marker primer information obtained by differential marker screening was shown in Table 2 using the Indel markers present in the Indel marker development interval on 10 chromosomes existing in the laboratory using parental SSSL3 and accession number 178.
TABLE 2 molecular marker primer information
Extracting DNA of individual leaves of a colony planted in Hainan in 2019, screening by using molecular markers at two ends to obtain 123 recombinant individual plants, identifying the ear length phenotype of two-point two-by-two repetition in two years, performing genotype analysis on the individual plants by using markers in a section, and fixing a target gene between markers M158 and M0900 by using a cross-fold diagram, wherein the distance is 643Kb. White boxes represent genomic fragments from accession 178, black boxes represent SSSL3 genomic fragments, and grey boxes represent heterozygous fragments of accession 178 and SSSL 3. The right bar graph indicates the average number of the ear lengths of the selfing progeny of the recombinant single plant, and the asterisks are obtained by t-test with the number 178. * Represents P <0.01 and P <0.001. The molecular marker M158 is positioned on the 3 rd chromosome of corn, and the specific position chr3:231733871-231734353, the molecular marker M0900 is positioned on the 3 rd chromosome of corn, and the specific position chr3 is: 232377659-232377854.
In many 178, the size of the M158 amplified sequence is 502bp, and the amplified sequence is SEQ ID NO26:
CCGAGTGTGAGTGAGGACAAAGTGTATAGAGGTGAACTGTGACTTCATAGGCCAATGTTTAGAGGGTGTTTGGTTTCTAAGGACTAATTTTTAGTCCCTATATTTTATTCTATTTTAGTTCAAAATTGTCAAATATAGAAACTAAAATTCTATTTTAATTTCTATATTTGGCAATTTATAGACTAAAATGAATAAAAATAAAGGGACTAAACATTAGTCCCTATAAACCAAACACCCCCTTAATATGATTCATATAATACCAATGCCATCAAGTGCACCTTATTTTTGGGAAGTTTATCTAAAAACAATCTGGATTAAATGTGTTTAGCTTAGATTTTTTTTTTTGGGGATGTGACAAAAATTCCCCTCGAGAGTAATCACTGCTGATCGTACTGTCCGTGAGAGACTGAAGTGTTACAAATTAACTTGAAGTTCTGTTACGAATTAACTTAAAAGTATAACTTCGAGTAGCAACCTTTCTACATCGTAACCAATCCACGTG;
the SSSL 3M 158 has an amplified sequence size of 321bp, and the amplified sequence is SEQ ID NO27:
CCGAGTGTGAGTGAGGACAAAGTGTATAGAGGTGAACTGTGACTTCATAGGCCAATGCTTAATATGATTCATATAATACCAATGCCATCAAGTGCACCTTATTTTTGGGAAGTTTATCTAAAAACAATCTGGATTAAATGTGTTTAGCTTAGATTTTTTTTTTTGGGGATGTGACAAAAATTCCCCTCGAGAGTAATCACTGCTGATCGTACTGTCCGTGAGAGACTGAAGTGTTACAAATTAACTTGAAGTTCTGTTACGAATTAACTTAAAAGTATAACTTCGAGTAGCAACCTTTCTACATCGTAACCAATCCACGTG;
in many 178, the size of the M0900 amplified sequence is 205bp, and the amplified sequence is SEQ ID NO28:
ACGATGCATGGTTGGAGTTGGAGGGCTGAACGGTTCGGGCCTGGGTCAAATTCAACTACGTACCACGGAGTAGCAACACATCAGTTGCAATTTGCAAATACGCAGCCGACACGTTACCCCTTTTTTTTTGCACCCTTTTTGGTATCGCCTTAAACCGCCGCTTGAAGCATAAACGGAAACCATATGGGCCACTTTGTTTGATGG;
the size of the M0900 amplified sequence in SSSL3 is 215bp, and the amplified sequence is SEQ ID NO29:
ACGATGCATGGTTGGAGTTGGAGGGCTGAACGGTTCGGGCCTGGGTCAAATTCAACTACGTACCACGGAGTAGCAACACATCAGTTGCAATTTGCAAATACGCAGCCTCTGGGCAGCCGACACGTTACCCCTTTTTTTTTGCACCCTTTTTGGTATCGCCTTAAACCGCCGCTTGAAGCATAAACGGAAACCATATGGGCCACTTTGTTTGATGG。
example 2
The molecular marker closely linked with the main QTL of the corn ear length and the application of the primer thereof in the corn ear length genetic improvement, which are obtained in the embodiment 1, the method for identifying the corn ear length character in the corn ear length molecular marker assisted breeding process comprises the following steps:
extracting genome DNA of corn leaves;
taking corn leaf genome DNA as a template, and respectively carrying out PCR amplification by using the primers M158-F/M158-R, M0900-F/M0900-R;
agarose gel electrophoresis identification PCR amplification results: when the adopted primer is M158-F/M158-R, and the size of the molecular marker M158 is detected to be 321bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M158 is detected to be 502bp, the spike length of the sample to be detected is short; when the adopted primer is M0900-F/M0900-R, when the size of the molecular marker M0900 is detected to be 215bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M0900 is detected to be 205bp, the spike length of the sample to be detected is short.
In this example, the PCR amplification system was 8. Mu.L, and the components contained: 2. Mu.L of DNA, 1. Mu.L of each of the left and right primers, 4. Mu.L of 2 XTaq Master Mix (Norvirginia P112). Using the Touchdown PCR amplification procedure: 95 ℃ for 5min;95 ℃ for 30s and 65 ℃ for 30s, and each cycle is reduced by 1 ℃ and 72 ℃ for 45s, and 8 cycles are total; 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45s, and 28 cycles are total; and at 72℃for 10min.
It should be noted that, since the steps and methods adopted are the same as those of the embodiments, the preferred embodiments are described in order to prevent redundancy. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (7)

1. The molecular marker closely linked with the corn cob long-acting QTL is characterized in that the molecular marker is positioned on a 3 rd chromosome of corn and is a molecular marker M158 and a molecular marker M0900.
2. The molecular marker closely linked to the major QTL for corn ear length according to claim 1, wherein the molecular marker M158 is located on chromosome 3 of corn at position chr3:231733871-231734353, the molecular marker M0900 is positioned on the 3 rd chromosome of corn, and the specific position chr3 is: 232377659-232377854.
3. The molecular marker closely linked to the major QTL for corn cob length of claim 1, wherein the primer of the molecular marker is:
the primer sequence for amplifying the molecular marker M158 is as follows:
M158-F:5’- CCGAGTGTGAGTGAGGACAA -3’;
M158-R:5’- CACGTGGATTGGTTACGATG -3’;
the primer sequence for amplifying the molecular marker M0900 is as follows:
M0900-F:5’- ACGATGCATGGTTGGAGTTG -3’;
M0900-R:5’- CCATCAAACAAAGTGGCCCA -3’。
4. the application of the molecular marker closely linked with the main effect QTL of the corn ear length is characterized in that the molecular marker is used for the genetic improvement of the corn ear length.
5. The use of molecular markers closely linked to major QTLs for corn ear length according to claim 4, wherein the method for identifying the coarse trait of corn ear in the genetic improvement of corn ear length comprises the steps of:
extracting genome DNA of corn leaves;
taking corn leaf genome DNA as a template, and respectively carrying out PCR amplification by using the primers M158-F/M158-R, M0900-F/M0900-R;
agarose gel electrophoresis identification PCR amplification results: when the adopted primer is M158-F/M158-R, and the size of the molecular marker M158 is detected to be 321bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M158 is detected to be 502bp, the spike length of the sample to be detected is short; when the adopted primer is M0900-F/M0900-R, when the size of the molecular marker M0900 is detected to be 215bp, the spike length of the sample to be detected is longer; when the size of the molecular marker M0900 is detected to be 205bp, the spike length of the sample to be detected is short.
6. The use of molecular markers closely linked to the major QTL for maize cob length of claim 5, characterized in that said PCR amplification system comprises 8 μl of the components: 2. mu.L of DNA, 1. Mu.L of each of left and right primers, 4. Mu.L of 2 XTaq Master Mix.
7. The use of molecular markers closely linked to the major QTL for corn cob length of claim 5, characterized by using the Touchdown PCR amplification procedure: 95 ℃ for 5min;95 ℃ for 30s and 65 ℃ for 30s, and each cycle is reduced by 1 ℃ and 72 ℃ for 45s, and 8 cycles are total; 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 45s, and 28 cycles are total; and at 72℃for 10min.
CN202310452312.5A 2023-04-25 2023-04-25 Molecular marker closely linked with corn cob long-acting QTL and application Pending CN116769950A (en)

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