CN105063201B - The molecular labeling of corn Chromosome 9 tassel row number main effect QTL and its application - Google Patents

Abstract

The invention discloses a molecular marker and an application of a major QTL for the ear row number of the No. 9 chromosome of maize. The present invention also provides a pair of primers for identifying or assisting in the identification of corn ear row number traits. In order to be able to amplify and obtain corn genomic DNA as a template, the primer pair shown in sequence 1 and sequence 2 is used for PCR amplification. The primer pair for the DNA fragment is specifically a primer pair composed of two single-stranded DNAs shown in Sequence 1 and Sequence 2. Using the maize genome as a template, the PCR amplification product obtained by using the primers is the molecular marker closely linked to the major QTL for ear row number on chromosome 9 of maize. Experiments have shown that the additive effect of the main QTL for corn ear row number is 0.9-1.3 rows, and the phenotypic variation that can be explained is 3.72%-10.78%. This QTL is closely linked to the SSR marker jsr21051, and jsr21051 can be effectively used in seedling The selection of ear row number can also be used for molecular marker breeding of corn ear row number, providing a marker basis for the fine mapping of corn ear row number QTL, and accelerating the process of high-yielding maize breeding.

Description

Molecular markers and their application of the major QTL for ear row number on chromosome 9 in maize

technical field

The invention belongs to the field of biotechnology, and relates to a molecular marker and an application of a major QTL for the ear row number of the No. 9 chromosome of maize.

Background technique

Corn is an important grain, feed and economic crop. According to the figures released by the National Grain and Oils Information Center, the sown area of corn in China reached 36.2 million hectares in 2014, becoming the largest grain crop in my country, and it occupies more than 100% of my country's agricultural production and national economic development. increasingly important position. However, with the continuous increase of population and the continuous decrease of cultivated land resources, it is still a more and more urgent task in my country's corn production to increase the grain yield per unit area of corn.

Under a specific planting density, the yield per unit area is composed of corn single ear grain yield and ear number, and single ear grain yield is composed of ear row number, row kernel number and 100-kernel weight. It can be seen that yield is a very complex trait, and direct genetic analysis of yield traits is a very complex and difficult research topic, and it is undoubtedly a better choice to decompose yield traits into each yield component factor and study them separately. Ear row number is an important yield component factor, which is significantly positively correlated with yield. Exploring the genetic mechanism of ear row number will help to elucidate the genetic mechanism of maize yield traits. The discovery of new genes related to ear row number is the key to study its formation mechanism, realize efficient conventional and molecular selection, and improve breeding efficiency and effect.

Classical quantitative genetics research shows that corn ear row number is a quantitative trait, and there is a complex interaction with other yield components; and the trait is controlled by multiple genes, and there is a complex interaction between genes, and the performance of the trait is easily affected by the environment. influences. Anederon emphasized as early as 1944: "The number of panicle rows is easy to measure accurately, and it is a good model for studying quantitative traits." The characteristics of the number of panicle rows are easy to count, so many early scholars used it as a model to study the law of inheritance. In the 1840s, Emerson selected a set of maize inbred lines with 12 ear rows, planted them for many years, and carried out single cross and double cross among the materials, and found that the trait of ear row number was both stable and variable. Afterwards, the researchers used different generations, diallell crosses and other methods to conduct quantitative genetic analysis on the trait of maize ear row number.

Contents of the invention

The first object of the present invention is to provide a pair of primers for identifying or assisting in identifying the trait of corn ear row number.

The pair of primers provided by the present invention for identifying or assisting in the identification of the number of rows of corn ears is a pair of primers capable of amplifying the following DNA fragment: the DNA fragment uses the genomic DNA of corn as a template, and uses the sequence 1 in the sequence table The DNA fragment obtained by performing PCR amplification with the primer pair shown in Sequence 2.

In the present invention, the primer pair is specifically composed of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence listing.

The preparation method of the primer pair also belongs to the protection scope of the present invention.

The preparation method of the primer pair includes the step of separately packaging the two single-stranded DNAs.

The second object of the present invention is to provide a kit for identifying or assisting in identifying the trait of corn ear row number.

The kit for identifying or assisting in identifying the trait of corn ear row number provided by the present invention contains the primer pair and at least one of the following: dNTP, DNA polymerase and PCR amplification buffer.

The preparation method of the kit also belongs to the protection scope of the present invention.

The preparation method of the kit includes the step of separately packaging the primer pair and at least one of the following: dNTP, DNA polymerase and PCR amplification buffer.

The third object of the present invention is to provide a method for identifying or assisting in identifying the number of ear rows of maize hybrid progeny.

The method for identifying or assisting in identifying the ear row number traits of corn hybrid progeny provided by the present invention may specifically include the following steps:

(a1) Using the genomic DNA of each individual in corn A, corn B, and the corn hybrid offspring population crossed from the corn A and the corn B as templates, using the primer pair (sequence 1 and sequence 2) performing PCR amplification respectively to obtain the PCR product of the corn A, the PCR product of the corn B and the PCR product of each individual in the corn hybrid offspring population;

The ear row number of the corn A is more than the ear row number of the corn B;

(a2) Perform electrophoresis on the PCR product of the corn A, the PCR product of the corn B, and the PCR product of each individual in the corn hybrid offspring population, and determine the corn hybrid offspring according to the following electrophoresis results Ear row number traits: the number of ear rows of the corn hybrid progeny with the same band type as the electrophoresis band of the PCR product of the corn A is more or more candidate than that of the electrophoresis band of the PCR product of the corn B The offspring of said maize hybrids with the same belt pattern.

The fourth object of the present invention is to provide a method for obtaining individuals with a relatively high ear row number from the maize hybrid progeny population.

The method for obtaining individuals with a relatively high ear row number from the corn hybrid progeny population provided by the present invention may specifically include the following steps:

(b1) Using the genomic DNA of each individual in corn A, corn B, and the corn hybrid offspring population crossed from the corn A and the corn B as templates, using the primer pair (sequence 1 and sequence 2) performing PCR amplification respectively to obtain the PCR product of the corn A, the PCR product of the corn B and the PCR product of each individual in the corn hybrid offspring population;

The ear row number of the corn A is more than the ear row number of the corn B;

(b2) Perform electrophoresis on the PCR product of the corn A, the PCR product of the corn B, and the PCR products of each individual in the corn hybrid offspring population, and select the Individuals with the same electrophoresis band pattern of the PCR product of A are or are candidates for individuals with a relatively high ear row number in the maize hybrid progeny population.

For the above two methods, in the present invention, the corn A is specifically the corn inbred line B73; the corn B is specifically the corn inbred line Xiwu 211. Correspondingly, the corn hybrid progeny population is specifically a corn hybrid progeny population obtained by crossing the corn inbred line B73 and the corn inbred line Xiwu 211.

Of course, the corn A can also be any variety of corn that meets the condition A except the corn inbred line B73; the condition A is: using genomic DNA as a template and using the primer pair (sequence 1 and sequence 2) Carry out PCR amplification, gained PCR product is carried out electrophoresis, and the band type of electrophoresis band is identical with reference to band type A; Described reference band type A is with the genomic DNA of maize inbred line B73 as template, adopts described primer pair ( Sequence 1 and Sequence 2) were amplified by PCR, and the obtained PCR products were subjected to electrophoresis to obtain band patterns. For example, the target segment (the amplified product of PCR amplification using the primer pair (sequence 1 and sequence 2) using genomic DNA as a template) is the same as the RIL line of the maize inbred line B73.

Correspondingly, the corn B can also be any variety of corn that meets the condition B except the corn inbred line Xiwu 211; the condition B is: using genomic DNA as a template, using the primer pair (sequence 1 and Sequence 2) PCR amplification was performed, and the obtained PCR product was subjected to electrophoresis, and the band type of the electrophoresis band was the same as that of the reference band type B; the reference band type B was based on the genomic DNA of the corn inbred line Xiwu 211 as a template, using The primer pair (sequence 1 and sequence 2) is amplified by PCR, and the band pattern obtained by electrophoresis of the PCR product is obtained. For example, the target segment (the amplified product of PCR amplification using the primer pair (sequence 1 and sequence 2) using genomic DNA as a template) is the same RIL line as the corn inbred line Xiwu 211.

RIL is RIL (Recombinant Inbred Lines), a type of population used for genetic analysis and mapping in biology. It is produced from hybridized materials through multiple generations of selfing. Each strain in the population is pure. suitable.

Among the corn A and the corn B, either is the female parent, and the other is the male parent.

In the above two methods, the corn hybrid progeny can specifically be homozygous corn hybrid progeny, such as RIL line, DH line, or homozygous F2 generation, homozygous BC generation, etc.

The fifth object of the present invention is to provide a method for cultivating corn varieties with increased ear rows.

The method for cultivating corn varieties with increased ear rows provided by the present invention is to select corns that meet the following conditions as parents for breeding: use genomic DNA as a template, and use the primer pair (sequence 1 and sequence 2) to perform PCR amplification , the resulting PCR product is electrophoresed, and the band type of the electrophoresis band is the same as the reference band type A;

The reference band pattern A is the band pattern obtained by using the genomic DNA of the corn inbred line B73 as a template, using the primer pair (sequence 1 and sequence 2) for PCR amplification, and electrophoresis of the obtained PCR products.

In the aforementioned three methods, the annealing temperature used in the PCR amplification may specifically be 58°C.

Further, the specific reaction conditions for the PCR amplification are: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 40s; 72°C for 10 minutes; and storage at 12°C.

In the aforementioned three methods, the electrophoresis may specifically be polyacrylamide gel electrophoresis, and in the polyacrylamide gel electrophoresis, the concentration of the polyacrylamide gel is 8% (mass percentage). The polyacrylamide gel electrophoresis is non-denaturing polyacrylamide gel electrophoresis.

The sixth object of the present invention is to provide a molecular marker related to the corn ear row number trait.

The molecular markers related to corn ear row number traits provided by the present invention are specifically the DNA fragments obtained by PCR amplification using the primer pair (Sequence 1 and Sequence 2) using corn genomic DNA as a template.

Wherein, the annealing temperature adopted during the PCR amplification may specifically be 58°C. Further, the specific reaction conditions for the PCR amplification are: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 40s; 72°C for 10 minutes; and storage at 12°C.

The application of the primer pair (Sequence 1 and Sequence 2) or the kit or the molecular marker in any of the following also falls within the protection scope of the present invention:

(1) Identification or auxiliary identification of corn ear row number traits;

(2) Maize breeding.

The present invention first detects a QTL qKRN9 for the number of ear rows on the No. 9 chromosome of maize through the initial positioning of the QTL. The SSR marker jsr21051 is closely linked. jsr21051 can be effectively used for the selection of ear row number at seedling stage, and at the same time, it can be used for molecular marker breeding of corn ear row number. .

Description of drawings

Figure 1 is the genetic linkage map of chromosome 9 and the genetic linkage map on the target interval of maize. Among them, A is the genetic linkage map of chromosome 9; B is the genetic linkage map on the target interval.

Figure 2 is a part of the segregation population produced by selfing of the individual plants heterozygous for the target segment (umc1191-umc2343) selected in the F ₆ generation of B73 (male parent) × Xiwu 211 (female parent) using jsr21051 The genotype detection results of a single plant. Among them, A indicates the same genotype as the male parent B73 (same electrophoresis band pattern); B indicates the same genotype as the female parent Xiwu 211 (same electrophoresis band pattern); H indicates heterozygous band type; - indicates deletion.

Figure 3 is the number of panicle rows of the segregated population produced after selfing of a single plant heterozygous on the target segment (umc1191-umc2343) selected from the F ₆ generation of B73 (male parent) × Xiwu 211 (female parent) Distribution.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Corn inbred line B73: described in the article "Zhao Yongping. Corn inbred line B73 low nitrate response miRNA and its target gene identification. Chinese Academy of Agricultural Sciences, 2013 master's thesis", the public can obtain from the applicant to repeat the present invention experiment.

Maize inbred line Xiwu 211: recorded in the article "Li Yan, Wang Jiangmin. Breeding and Utilization of Maize Inbred Line "Xiwu 211". Yunnan Agricultural Science and Technology, 1998 Issue 01", the public can obtain it from the applicant To repeat the experiment of the present invention.

Embodiment 1, the localization of the main effect QTL qKRN9 of maize ear row number

1. Experimental materials

1. DNA was extracted from young leaves of fine maize inbred lines B73 and Xiwu 211 and used to screen for differential primers.

_2. The F2 population of B73 (male parent)×Xiwu 211 (female parent), with a total of 148 individual plants, was used for the construction of the genetic map and the initial mapping of the QTL for the number of panicle rows.

3. Continuously select F ₂ , F ₃ , F ₄ , and F ₅ lines of B73 (male parent)×Xiwu 211 (female parent) that are heterozygous for the target segment to self-cross to generate segregated populations, and perform panicle rows Detection of several main QTLs.

2. Mapping of the main effect QTL qKRN9 for corn row number

1. Massive extraction of maize genomic DNA (improved CTAB method)

(1) Take 10-20 mg of young leaves of corn plants, put them in a 1.5 ml Eppendorf tube, add liquid nitrogen, and grind them into fine powder.

(2) Add 600 μl of 1×CTAB extraction buffer preheated at 65°C to the centrifuge tube, and shake gently to mix.

(3) Incubate in a 65°C water bath for 30 minutes, and shake the centrifuge tube carefully every 10 minutes.

(4) Take out the centrifuge tube after 30 minutes, add an equal volume of chloroform: isoamyl alcohol (volume ratio 24:1) in the fume hood, and shake the centrifuge tube carefully and fully for 2-3 minutes, then let it stand until the organic phase changes from colorless to green →Dark green.

(5) Centrifuge at 10,000 rpm for 10 min at room temperature, and then pipette 500 μl of the supernatant into a new centrifuge tube.

(6) Add an equal volume of pre-cooled isopropanol (-20°C) to the supernatant, mix carefully and place at -20°C for 15 minutes.

(7) Centrifuge at 10,000 rpm for 5 min at room temperature, pour off the supernatant carefully, add 600 μl of 70% ethanol for rinsing, and shake the centrifuge tube slightly to suspend the DNA.

(8) Centrifuge at 10,000 rpm for 3 minutes at room temperature, then pour off the supernatant carefully and place it to dry at room temperature.

(9) After the DNA is dried, add an appropriate amount of sterilized ddH ₂ O to fully dissolve the DNA.

(10) Use an ultraviolet spectrophotometer to detect the concentration and purity of the DNA, and store it in a -20°C refrigerator for later use.

2. Amplification of the target fragment

The PCR reaction system (10 μl system) was as follows: 40ngμL ^-1 template DNA 2.0μL; 10×PCR buffer 1.0μL; dNTPs 0.2μL; primer 1.0μL; rTaq DNA polymerase 0.1μL; ddH ₂ O 5.7μL.

The PCR reaction program (10 μl system) was as follows: 94° C. for 5 min; 94° C. for 30 s, 55° C. for 30 s, 72° C. for 30 s, 36 cycles; 72° C. for 10 min; 12° C. for incubation.

3. Polyacrylamide gel electrophoresis detection

A. Glue making

(1) Tighten the clean two glass plates and spacers with clips, mix 8% glue (ml): 20% APS (μl): TEMED (μl) in a ratio of 1:10:1 and mix well quickly. back cover;

(2) Prepare a clean comb, wait for the glue on the back cover to solidify, mix the glue with 35ml of 8% glue per plate according to the same ratio, and fill the glue immediately; if there are air bubbles, gently tap the glass plate to drive out the air bubbles, and insert them in parallel clean comb;

(3) In order to prevent residual glue, carefully pull out the comb in time after the gel is solidified, fix the gel plate on the electrophoresis tank, and add 1×TBE buffer.

40% PAGE solution (5L): 2000ml of deionized water; 50g of N,N'-methylenebisacrylamide; 1950g of acrylamide; stir with a stirrer, fully dissolve, and make up to 5L.

8% PAGE solution: 40% PAGE solution is diluted 5 times and diluted according to the ratio of 40% PAGE:5×TBE buffer:deionized water=1:1:3.

5×TBE buffer (5L): Tris 270.00g; boric acid 137.50g; EDTA-Na ₂ 18.61g; deionized water to 1L.

B. Electrophoresis

(1) Add 2.0 μl of 6×Loading Buffer to the amplified PCR product, after centrifugation, spot 4 μl per well and spot 4 μl of 100bp DNA Ladder in 1 well of each plate;

(2) At room temperature, at GT nucleic acid electrophoresis system (Bio-Rad, USA) pre-electrophoresis at 200V for 2min, and then electrophoresis at a constant voltage of 120V for about 5h.

6×Loading Buffer (100ml): 2ml of 0.5M EDTA (pH8.0); 98ml of deionized formamide; 0.05g of bromophenol blue; 0.05g of xylene cyanide.

1 × TBE electrophoresis buffer: 5 × TBE buffer is diluted 5 times and then diluted according to the ratio of 5 × TBE buffer: deionized water = 1:4.

C. Silver staining

(1) 0.1% staining solution: after the electrophoresis is finished, take a clean silver staining basin, weigh 0.50g of AgNO ₃ , 500ml of deionized water, and mix well;

(2) Staining: remove the rubber sheet, peel the glue and put it in 0.1% staining solution, stain 4 sheets of glue per pot with silver, then shake gently on the shaker, and dye for 15 minutes;

(3) Chromogenic solution: NaOH 10.00g, anhydrous Na ₂ CO ₃ 0.20g-0.30g, deionized water 500ml, formaldehyde 750μl;

(4) Color development: after dyeing for 15 minutes, carefully pour off the color development solution and quickly rinse with deionized water for 30 seconds, add 500ml of color development solution to each pot, continue to place on the shaker, and shake gently for about 10 minutes;

(5) Illumination of the gel: When the gel turns light yellow and the DNA bands are completely visible, pour off the chromogenic solution, rinse with water, and use a camera to record the bands of each plate of gel, so as to read the band pattern.

4. Screening of maize chromosome 9 SSR markers and construction of genetic map

The SSR primer sequences used in the PCR reaction were from the Maize Database (www.maizegdb.org), and were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. After PCR amplification between the parents, 8% non-denaturing polyacrylamide gel electrophoresis detection and small group verification, the SSR marker with obvious polymorphism and clear band pattern was finally obtained.

The SSR markers are co-dominant markers. The band pattern of the marker locus in the F ₂ individual plant is the same as that of the male parent B73, which is marked as A, and the same as that of the female parent Xiwu 211 is marked as B, and the heterozygous band pattern is marked as H , Deletion was recorded as "-", and the JoinMap 4.0 software was used to construct the genetic linkage map.

Using the genotype of the polymorphic SSR marker on chromosome 9 screened from the maize database ( _{www.maizegdb.org} ) in the F2 population of B73 (paternal parent) × Xiwu 211 (female parent), the A maize molecular marker linkage map (A in Figure 1) containing 7 co-dominant SSR markers, the map is 36.6cM in total, the average distance between markers is 5.23cM, and the chromosomal loci of most primers are the same as the genetic linkage map of IBM (www.maizegdb.org).

5. QTL mapping for panicle row number

According to the genotype of each SSR marker and the phenotype of each individual plant in the population, the genetic linkage map on the target segment was constructed with the help of the composite interval mapping method (CIM) of JoinMap4.0 (Zeng, 1994). Among them, the Kosambi function was selected to convert the recombination value into the genetic map distance unit (cM). Then run WinQTLcartgrapher2.0 software, and conduct preliminary analysis and positioning of QTL by composite interval mapping (Composite Interval Mapping, CIM) method (Zeng Z B. Precision mapping of quantitative trait loci. Genetics, 1994, 136: 1457-1468).

The results showed that a main QTL for corn ear row number was detected in the interval between molecular markers umc1191 and umc2343, named qKRN9, its LOD value was 3.0488, the additive effect was 0.644 lines, and the genetic variation for ear row number was 3.72%. And the synergistic gene comes from the male parent B73.

Primer pairs used to amplify molecular marker umc1191:

Upstream primer: 5'-AAGTCATTGCCCAAAAGTGTTGC-3' (SEQ ID NO: 3);

Downstream primer: 5'-ACTCATCACCCTCCAGAGTGTC-3' (SEQ ID NO: 4).

Primer pairs used to amplify the molecular marker umc2343:

Upstream primer: 5'-TCATCTTCCCCACAAATTTTCATT-3' (SEQ ID NO: 5);

Downstream primer: 5'-GACTGACAACTCAGATTTTCACCCA-3' (SEQ ID NO: 6).

Example 2, the acquisition of the main QTL qKRN9 closely linked SSR marker jsr21051 for the number of corn ear rows

1. Development of target interval SSR markers

For the target interval of the main QTL qKRN9 on chromosome 9 in maize, according to the genome sequence information of the published maize inbred line B73, different segments in the target interval were retrieved from the maize database MaizeGDB (http://www.maizegdb.org) The sequence above was combined with primer 3.0 to design corresponding primers. Then, after PCR amplification and 8% non-denaturing polyacrylamide gel electrophoresis detection and small group verification, SSR primers with obvious polymorphism and clear band pattern between B73 and Xiwu 211 were selected for marker analysis, and finally A total of 9 pairs of SSR markers with obvious polymorphism and clear band pattern in the QTL interval were obtained, which were recorded as polymorphic molecular markers jsr16931, jsr20512, jsr21671, jsr19851, jsr21311, jsr21051, jsr18135, jsr22023 and jsr22381.

2. Construction of the genetic map of the target segment

For the target interval umc1191-umc2343, referring to the genome sequence of the maize inbred line B73, through design and screening, a total of 9 pairs of polymorphic molecular markers with obvious differences and clear band patterns between B73 and Xiwu 211 were developed. Using newly developed 9 pairs of SSR markers and 3 known SSR markers (umc1191, _umc1078 and umc2343) on the target interval, target the F2 population of B73 (father) × Xiwu 211 (female) in this interval Genotype heterozygous individual plants self-crossed offspring segregated populations, identified and counted the genotypes of each individual plant. With the help of JoinMap4.0 software, "A" is recorded as 2, "B" is recorded as 0, "H" is recorded as 1, and "-" is recorded as -1 (the one with the same band pattern as the parent B73 is recorded as A, and the 211 is the same as B, the heterozygous pattern is marked as H, and the deletion is marked as '-'). Using the Kosambi function, the optimal order of the 12 molecular markers on the target segment and the new genetic distance between the markers, that is, the genetic linkage map in the target segment (B in Figure 1) were obtained.

3. Acquisition of closely linked marker jsr21051

According to the genotype data of the encrypted genetic map constructed in each generation, combined with the data of ear row number of the corresponding generation, the main QTL qKRN9 of ear row number was located by composite interval mapping method. The results showed that in the segregated populations produced by selfing of the lines heterozygous for the target segment in F ₂ , F ₃ , F ₄ , and F ₅ of B73 (male parent)×Xiwu 211 (female parent), all of them could The QTLs controlling the number of panicle rows were detected near the marker jsr21051, and their additive effects were 1.3 rows, 2.2 rows, 0.94 rows, and 0.9 rows, and the explainable phenotypic variation was 3.72%-10.78%. From the male parent B73 (Table 1).

Table 1 Detection results of maize chromosome 9 QTL qKRN9 in each generation

Note: "F ₂ , F ₃ , F ₄ , F ₅ " in the table represent the individual plants heterozygous for the target segment (umc1191-umc2343) selected from the F ₂ , F ₃ , F ₄ , and F ₅ generations, respectively Segregating populations produced after selfing.

Primer pairs used to amplify the SSR marker jsr21051:

Upstream primer: 5'-tgaagagaagcttttgagacg-3' (SEQ ID NO: 1);

Downstream primer: 5'-ggctgcagcagagagataat-3' (SEQ ID NO: 2).

Example 3, the application of the tightly linked SSR marker jsr21051 in the selection of the number of corn ear rows

1. Experimental materials

Identification of the tightly linked SSR marker jsr21051 using a segregating population generated after selfing of individual plants heterozygous for the target segment (umc1191-umc2343) selected in the F ₆ generation of B73 (male) × Xiwu 211 (female) Application in ear row number selection.

2. Experimental method

Aiming at the segregation population produced by the self-crossing of individual plants heterozygous on the target segment (umc1191-umc2343) selected in the F _6th generation of B73 (male parent)×Xiwu 211 (female parent), the single plant was listed and sampled at the seedling stage Finally, extract its genomic DNA and use it as a template, carry out PCR amplification with the amplification primers (sequence 1 and sequence 2) of the tightly linked marker jsr21051 of the main effect QTL qKRN9 of the number of panicle rows obtained, detect and count the number of individual plants genotype. At the same time, after the maize matured, the number of main panicle rows of each individual plant in the F ₆ population was examined and counted.

PCR reaction system (10 μL): Mix 5.0 μL; ddH ₂ O 2.5 μL; upstream and downstream primers 1.5 μL (50 ng); DNA template with a concentration of 20 ng/μL 1.0 μL. Among them, Mix is a product of Beijing Kangrun Chengye Biotechnology Co., Ltd.

The PCR reaction was carried out on the Gene Amp PCR System 9700 PCR reaction instrument, and the PCR reaction program was as follows: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 40s; 72°C for 10 minutes; and storage at 12°C.

After the reaction, 8% non-denaturing polyacrylamide gel electrophoresis was performed according to the method of Example 1.

Figure 2 shows the genotype detection results of some individual plants in the isolated population using the SSR marker jsr21051. At the same time, after the corn matured, the number of main panicle rows of each individual plant in the isolated population was examined and counted.

Statistical analysis was carried out on the genotype and number of ear rows of each individual plant in the segregation population (Table 2, Figure 3), and it was found that using the SSR marker jsr21051, 27 genotypes of the parent B73 were screened out of 119 individual plants in the segregation population. The genotype is the same (that is, the band type of the electrophoresis band is the same, which is A), and the average number of ear rows is 11.55; the genotype of 45 strains is the same as that of the parent Xiwu 211 (that is, the band type of the electrophoresis band is the same, which is B), the average number of panicle rows is 10.53, which is 1.02 rows less than the average number of panicle rows of 27 plants with the same genotype as B73, which has a statistically significant difference (P<0.05). It can be seen from this that using the newly developed SSR marker jsr21051 can effectively screen out corn with a large number of ear rows at the seedling stage, saving experimental costs and improving selection efficiency, thereby quickly screening out lines with a large number of ear rows for use in Maize breeding for high yield.

Table 2 Statistical results of panicle row number of F ₂ population of ZC16×C7-2

Based on the above results, the present invention used the excellent inbred lines B73 and Xiwu 211 as basic materials to construct the F2 population, and detected a major QTL _qKRN9 for the number of panicle rows in the interval between the SSR markers umc1191 and umc2343 of chromosome 9, which The LOD value was 3.0488, the additive effect was 0.644, and the explainable genetic variation in panicle row number was 3.72%. On this basis, a continuous self-crossing strategy was used to construct segregated populations, and molecular markers were developed for target segments. Segregating populations derived from each generation were genotyped using the developed markers and a genetic linkage map of the segment of interest was constructed. The QTL detection of segregation populations derived from F ₂ , F ₃ , F ₄ and F ₅ generations showed that the QTL controlling the number of panicle rows could be detected near the marker jsr21051, and the additive effects were 1.3 lines, 2.2 lines, and 0.94 lines, respectively. row, 0.9 row, the explainable phenotypic variation is 3.72%-10.78%, and the synergistic loci are all from the male parent B73, which shows that the panicle row number QTL qKRN9 on chromosome 9 exists stably, and is related to SSR Marker jsr21051 is tightly linked. In addition, using this tightly linked marker to test the segregation population derived from the F ₆ generation, it was found that the average number of panicle rows in plants with the same genotype as B73 was 1.02 more than the average number of rows in plants with the same genotype as the parent Xiwu 211, indicating that jsr21051 can be effectively used for the selection of ear row number at the seedling stage, and can be used for molecular marker breeding of corn ear row number, providing a marker basis for the fine mapping of corn ear row number QTL, and accelerating the process of high-yielding maize breeding.

Claims (10)

1. The pair of primers used to identify or assist the identification of corn ear row number traits is a primer pair capable of amplifying the following DNA fragments: the DNA fragment is based on the corn genomic DNA as a template, using sequence 1 and sequence 2 in the sequence table The DNA fragments obtained by PCR amplification with the indicated primer pairs. 2. The primer pair according to claim 1, characterized in that: the primer pair is a primer pair composed of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence listing. 3. The method for preparing the primer pair according to claim 1 or 2, comprising the step of separately packaging the two single-stranded DNAs according to claim 1 or 2. 4. The kit for identifying or assisting in identifying the character of corn ear row number, containing the primer pair described in claim 1 or 2 and at least one of the following: dNTP, DNA polymerase and PCR amplification buffer. 5. A method for identifying or assisting in identifying the ear row number traits of corn hybrid progeny, comprising the steps of: (a1) Using the genomic DNA of each individual in corn A, corn B, and the corn hybrid progeny population crossed from the corn A and the corn B respectively as a template, using the primers described in claim 1 or 2 Carrying out PCR amplification respectively to obtain the PCR product of the corn A, the PCR product of the corn B and the PCR product of each individual in the corn hybrid progeny population; The ear row number of the corn A is more than the ear row number of the corn B; (a2) Perform electrophoresis on the PCR product of the corn A, the PCR product of the corn B, and the PCR product of each individual in the corn hybrid offspring population, and determine the corn hybrid offspring according to the following electrophoresis results Ear row number traits: the number of ear rows of the corn hybrid progeny with the same band type as the electrophoresis band of the PCR product of the corn A is more or more candidate than that of the electrophoresis band of the PCR product of the corn B The offspring of said maize hybrids with the same belt pattern. 6. A method of obtaining a relatively high individual with the number of ear rows from the maize hybrid progeny population, comprising the steps of: (b1) Using the genomic DNA of each individual in corn A, corn B, and the corn hybrid progeny population crossed from the corn A and the corn B as templates, using the primers described in claim 1 or 2 Carrying out PCR amplification respectively to obtain the PCR product of the corn A, the PCR product of the corn B and the PCR product of each individual in the corn hybrid progeny population; The ear row number of the corn A is more than the ear row number of the corn B; (b2) Perform electrophoresis on the PCR product of the corn A, the PCR product of the corn B, and the PCR products of each individual in the corn hybrid offspring population, and select the Individuals with the same electrophoresis band pattern of the PCR product of A are or are candidates for individuals with a relatively high ear row number in the maize hybrid progeny population. 7. A method for cultivating a corn variety that increases the number of ear rows is to select the corn that meets the following conditions to breed as a parent: using genomic DNA as a template, using the primers described in claim 1 or 2 to carry out PCR amplification, and The obtained PCR product was subjected to electrophoresis, and the band type of the electrophoresis band was the same as that of the reference band type A; The reference band pattern A is the band pattern obtained by using the genomic DNA of the corn inbred line B73 as a template, using the primer pair described in claim 1 or 2 for PCR amplification, and electrophoresis of the obtained PCR product. 8. The method according to any one of claims 5-7, characterized in that: the annealing temperature used in the PCR amplification is 58°C. 9. Molecular markers related to corn ear row number traits, which are DNA fragments obtained by PCR amplification using the primers described in claim 1 as templates using corn genome DNA. 10. The primer pair described in claim 1 or 2 or the test kit described in claim 4 or the application of the molecular marker described in claim 9 in any of the following: (1) Identification or auxiliary identification of corn ear row number traits; (2) Maize breeding.