CN117947205A

CN117947205A - Main effect QTL for regulating and controlling stem length in corn field, molecular marker and application thereof

Info

Publication number: CN117947205A
Application number: CN202410128885.7A
Authority: CN
Inventors: 赵小强; 何富强; 祁国翔; 石珍珍; 杨万忠; 李家尧
Original assignee: Gansu Longfengxiang Seed Industry Co ltd; Gansu Agricultural University
Current assignee: Gansu Longfengxiang Seed Industry Co ltd; Gansu Agricultural University
Priority date: 2024-01-29
Filing date: 2024-01-29
Publication date: 2024-04-30

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a main effect QTL for synchronously and stably regulating and controlling stem length in corn fields under various environments by comprehensively utilizing various positioning methods, and a molecular marker and application thereof. The molecular marker of the major QTL consists of three pairs of SSR molecular markers, namely umc1399, umc1659 and bnlg 1779. When the SSR molecular marker is used for auxiliary selection of the corn genotype with long and medium stem characteristics and excellent stress resistance, the characteristics of the medium stem length and the stress resistance of the corn genotype can be predicted by detecting the characteristic bands of the SSR molecular marker, the identification method is simple and easy to operate, the selection efficiency is high, the cultivation process of a new variety of excellent corn is remarkably accelerated, the influence of adverse habitats such as drought, low temperature, lodging and herbicide during the corn fertility period is remarkably improved, and the high and stable corn yield is ensured.

Description

Main effect QTL for regulating and controlling stem length in corn field, molecular marker and application thereof

Technical Field

The invention belongs to the field of corn molecular genetic breeding, and particularly relates to a main effect QTL for stably regulating and controlling stem length in corn fields under multiple environments by comprehensively utilizing multiple positioning methods and molecular markers and application thereof.

Background

The germination and emergence processes of corn (Zea mays L.) seeds are mainly comprehensively influenced by seed vitality, soil temperature, soil moisture, soil resistance, underground diseases and insect pests, sowing depth and the like. Corn varieties with the characteristics of long and medium stems (RH; referring to embryogenic tissue between seeds and the top end of coleoptile, specifically shown in figure 1) can ensure that the seeds germinate and form strong seedlings in time and normally in adverse habitats such as drought (the water content of soil in a soil layer of 15-20 cm is about 15 percent, the sucking, expanding and germinating of the seeds) and low temperature by timely deep sowing (the sowing depth of 15-20 cm is shown in the attached figure 1), and the strong seedlings are formed and closely related with early seedling vigor (EARLY SEEDLING vigor, ESV), so that later development and yield formation of plants can be obviously influenced. The Indian blue SN series excellent corn germplasm resource with long-soil medium-stem characteristics is screened by students before by utilizing the excellent characteristic of the corn, a new excellent corn variety P1213733 with long-soil medium-stem characteristics is cultivated and widely popularized and planted in the United states and the dry-farming area of Mexico, the sowing depth of the variety can reach 30cm, the normal emergence and growth of seedlings of the variety under various adverse environments can be ensured, and the stress resistance and stable yield of the variety are obviously enhanced.

The in-ground stem of corn is a typical quantitative genetic trait, is mainly influenced by the comprehensive influence of a plurality of micro/major genes and environmental factors, and brings a plurality of difficulties to systematic research on the molecular genetic mechanism of the in-ground stem of corn, the long major QTL (major quantitative trait locus)/gene positioning of the in-ground stem and the molecular marker assisted selection (marker-assisted selection, MAS) breeding of new corn varieties with excellent in-ground stem stress resistance and breeding application value in production. In recent years, with the rapid development of molecular biotechnology, particularly QTL positioning and LD (linkage disequilibrium) association analysis and BSA (bulked SEGREGANT ANALYSIS) group separation analysis methods developed by molecular marker technology, technical support is provided for positioning genetic loci of complex number genetic traits, gene mining and MAS breeding, the cultivation process of new corn varieties with excellent characteristics is remarkably accelerated, and the method has important significance in guaranteeing the safety, high yield and high-efficiency production of corn in adverse environment.

The in-ground stem is an important indicator trait for Early Seedling Vigor (ESV) of rice (Oryza sativa l.), which also significantly affects rice direct seeding. Therefore, the positioning of the in-ground stem length QTL is mainly concentrated in rice, and a plurality of main effect QTLs and candidate genes related to the in-ground stem length are positioned in the rice by a learner at present, so that a foundation is laid for explaining a molecular mechanism of regulation of in-ground stem characteristics, important regulation gene excavation and MAS breeding in the rice. However, the stem length QTL in the middle of the corn has not been reported yet. Therefore, it is very necessary to position the stem length QTL in corn fields by using various positioning methods in corn, and the positioning method will have great application potential in breeding new variety MAS of good corn.

Disclosure of Invention

The invention aims to provide a technology for comprehensively utilizing QTL positioning, LD linkage SSR association analysis, BSA group separation analysis and the like, and the main effect QTL of qRHL and molecular markers thereof for stably regulating and controlling the stem length in corn fields are detected among F ₂ extreme phenotype equivalent DNA mixed pools, F _2:3 positioning groups and natural group genotypes under the 3cm, 15cm and 20cm deep sowing environments. Furthermore, the invention provides a method for assisting in selecting the corn genotype which has long-soil mesostem characteristics and is excellent in stress resistance. In addition, the invention also provides application of the molecular marker for regulating and controlling the main effect QTL of the stem length in the corn field in corn breeding with excellent stem characteristics in the field and stress resistance.

In order to solve the technical problems, the invention adopts the following technical scheme:

1. SSR molecular marker primers for regulating and controlling the stem length major QTL in corn fields comprise three pairs of SSR molecular marker primers of umc1399, umc1659 and bnlg1779, and the sequence of the SSR molecular marker primers is as follows:

as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of the SSR molecular marker primer umc1399 is as follows:

Forward:5’-GCTCTATGTTATTCTTCAATCGGGC-3’；

Reverse:5’-GGTCGGTCGGTACTCTGCTCTA-3’；

As shown in SEQ ID NO:3 and SEQ ID NO:4, the sequence of the SSR molecular marker primer umc1659 is as follows:

Forward:5’-CAAGCTTGCTACTGTGATTTCTCG-3’；

Reverse:5’-AACTTCTCGGTGATCTTGTCCATC-3’；

as shown in SEQ ID NO:5 and SEQ ID NO:6, the sequence of the SSR molecular marker primer bnlg1779 is as follows:

Forward:5’-CCCTTTTATATCTCAAGTGTAGAACC-3’；

Reverse:5’-AGAGCACCCACCACGATAAC-3’；

2. The molecular marker for regulating and controlling the major QTL of the long-acting stem in the corn field is applied to the breeding of the corn with excellent stress resistance, and the corn genotype with the characteristic of the long-acting stem in the corn field is selected in an auxiliary way to be applied to the breeding of the corn with excellent stress resistance; the corn can be amplified by PCR by SSR primers umc1399, umc1659 and bnlg1779, and amplified products with the lengths of 121bp, 164bp and 176bp are respectively obtained, so that the corn to be detected is a corn genotype (inbred line, strain and variety) with long-soil mesostem characteristics and excellent stress resistance.

3. A method for assisting in selecting a maize genotype having long-earth mesostem characteristics and excellent stress resistance, comprising the steps of: extracting genome DNA of a corn genotype to be detected; PCR amplification is carried out by using SSR molecular marker primers umc1399, umc1659 and bnlg 1779; when amplification products with the lengths of 121bp, 164bp and 176bp are respectively obtained, the corn to be detected is a corn genotype with long-soil mesostem characteristics and excellent stress resistance.

4. A method for obtaining a molecular marker for regulating and controlling the stem length of corn field major QTL comprises the following specific steps: (1) construction of F ₂ isolated population genotype genetic map and F _2:3 positioning population genotype regulation corn field middle stem length QTL analysis, (2) construction of F ₂ middle stem length extreme phenotype equivalent DNA mixed pool and regulation corn field middle stem length BSA analysis, (3) analysis of corn inbred line natural population genotype genetic structure and regulation corn field middle stem length LD linkage SSR association analysis, and (4) regulation corn field middle stem length main effect QTL identification.

The invention has the beneficial effects that: in long-term agricultural practice, the method has the advantages that drought, low temperature, soil shallow layer diseases and insect pests and herbicide influence on corn seed germination and seedling growth can be obviously improved by properly planting corn varieties with long-field middle-stem characteristics, positive effects on corn seedling morphogenesis and normal growth and development of subsequent plants are achieved, and the method is an important measure for stress resistance and high yield of corn. In order to obtain the main effect QTL for regulating and controlling the stem length in the corn field with breeding application value, the invention provides a technology for comprehensively utilizing QTL positioning, LD linkage SSR association analysis, BSA group separation analysis and the like, wherein the main effect QTL for stably regulating and controlling the stem length in the corn field is qRHL when the equivalent DNA pool of the extreme phenotype of the stem length in the F ₂ field and the genotype of the natural population of the F _2:3 positioning population are detected in the 3cm, 15cm and 20cm deep sowing environments. Further analysis shows that qRHL3 is positioned in the Bin3.07-3.08 region umc1399-umc1659-bnlg1779 of the 3 rd chromosome long arm cross-fold group ctg141-ctg145 of corn, stably regulates and controls the in-ground stem length characteristics of corn in 3cm, 15cm and 20cm deep sowing environments at the same time, and shows that the phenotype accumulated contribution rate of qRHL to the in-ground stem length is 25.90% and 33.75% in QTL positioning and LD chain SSR association analysis. Further analysis also shows that the three pairs of SSR molecular markers of qRHL are utilized to carry out PCR amplification on the corn genotype to be detected, so that the in-ground stem length characteristics of the corn genotype to be detected can be rapidly, objectively and efficiently predicted, and errors caused by environmental changes and test operation in the identification process of the in-ground stem length characteristics and stress resistance of different corn genotype materials are effectively avoided.

When the SSR molecular marker disclosed by the invention is used for carrying out molecular marker-assisted selection on corn genotypes with long and medium stem characteristics and excellent stress resistance, the characteristic amplification strips of the corresponding SSR molecular marker primers are detected, so that the medium stem phenotype and the stress resistance of the corresponding corn genotypes can be rapidly, objectively and accurately predicted. Corn genotypes with long-soil middle-stem characteristics and strong stress resistance can be rapidly and accurately identified, and other genotypes are eliminated. The method is not only free from the influence of environmental change and manual operation when the long-soil middle-stem characteristic and the excellent stress-resistant corn genotype are identified and selected, the selection target is clear, the cost of manpower and material resources excessively consumed in the phenotype identification process can be effectively reduced, the time is saved, and the method has great application potential and value in the breeding of new varieties of excellent stress-resistant corn with the long-soil middle-stem characteristic.

Drawings

FIG. 1 is a schematic representation of development of different tissue sites during germination of corn seeds; wherein Seed is Seed, rhizome (RH) is Rhizome, radicle is radicle.

FIG. 2 is a graph showing the medium stem length performance of female parent N192 and male parent Ji853 genotypes under 3cm, 15cm and 20cm deep sowing environments; wherein RHL is a stem length in the ground, 3cm is a 3cm deep-sowing environment, 15cm is a 15cm deep-sowing environment, 20cm is a 20cm deep-sowing environment, different lowercase letters "a, b and c" indicate that the stem lengths in the ground of the same parent genotypes are obviously different at the level of P <0.05 under different deep-sowing environments (3, 15 and 20 cm), and ". Times" indicate that the stem lengths in the ground of different parent genotypes (N192 and Ji 853) are obviously different at the level of P <0.05 under the same deep-sowing environment.

FIG. 3 is a medium stem length histogram of F _2:3 located population pedigree genotypes in 3cm (A), 15cm (B) and 20cm (C) deep-sowing environments; wherein RHS is the length of the stem in the ground, 3cm is the depth of sowing environment of 3cm, 15cm is the depth of sowing environment of 15cm, and 20cm is the depth of sowing environment of 20 cm.

FIG. 4 is a diagram showing the construction of F ₂ segregating population genetic maps and the positioning of the stem length QTL in the genotype of F _2:3 positioning population families in the 3cm, 15cm and 20cm deep sowing environments; the RHS is the stem length in the corn field, the black rectangle is the QTL for regulating the stem length in the corn field, the 3cm is the 3cm deep sowing environment, the 15cm is the 15cm deep sowing environment, the 20cm is the 20cm deep sowing environment, "Chr.1, chr.2, chr.3, chr.4, chr.5, chr.6, chr.7, chr.8, chr.9 and Chr.10" are 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th and 10 th chromosomes respectively.

FIG. 5 shows the medium stem growth of part F _2:3 located group pedigree genotypes under a 20cm deep sowing environment.

FIG. 6 shows the phenotype analysis of the stem length in the F ₂ region in the extreme phenotype DNA pool (35+35), the female parent pool and the male parent pool in the 20cm deep sowing environment; wherein RHI is the length of the middle ground stem, 20cm is the depth of sowing environment of 20cm, ji853 is a male parent pool, N192 is a female parent pool, L-pool is the longest F ₂ extreme phenotype DNA mixing pool of the middle ground stem, S-pool is the shortest F ₂ extreme phenotype DNA mixing pool of the middle ground stem; "x" indicates that the stem length in the corresponding pool genotype was significantly different at P <0.05 level in a 20cm deep-sowing environment.

FIG. 7 shows SNP and InDel detection in 4 pool sample resequencing (Re-Seq); wherein Ji853 is the male parent pool, N192 is the female parent pool, L-pool is the ground middle stem longest F ₂ extreme phenotype DNA pool, S-pool is the ground middle stem shortest F ₂ extreme phenotype DNA pool, hom_ref is the homozygous genotype consistent with the reference genome, HET is the heterozygous genotype, hom_alt is the homozygous genotype inconsistent with the reference genome.

FIG. 8 shows detection of BSA sites in 4 mixed pools in a 20cm deep sowing environment for regulating and controlling stem length in corn fields by using different BSA analysis methods; wherein (A) is SNP-index analysis, (B) is InDel-index analysis, (C) is ED analysis, and (D) is GPS analysis, and "1, 2, 3, 4,5, 6, 7, 8, 9, 10" is 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th, 10 th chromosomes respectively.

FIG. 9 is a medium stem length histogram of 193 parts of maize inbred natural population genotype in 3cm (A) and 20cm (B) deep-sowing environments; wherein RHS is the length of the stem in the ground, 3cm is the depth of sowing environment of 3cm, and 20cm is the depth of sowing environment of 20 cm.

FIG. 10 shows 186 pairs of distribution of SSR molecular marker primers on 10 chromosomes of corn (A), allele detection (B) and polymorphism information analysis of SSR molecular marker primers (C); wherein PIC is polymorphism information quantity of SSR molecular marker primers, and '1, 2, 3, 4, 5, 6, 7, 8, 9 and 10' respectively represent 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th and 10 th chromosomes.

FIG. 11 is a genetic map constructed by using 186 pairs of SSR molecular marker primers and shows that 193 parts of maize inbred line natural population genotypes LD are subjected to SSR linkage analysis under a 3cm and 20cm deep sowing environment; wherein 3cm is a 3cm deep sowing environment, 20cm is a 20cm deep sowing environment, GLM is a general linear model, MLM is a mixed linear model, black circles are detected SSR association sites linked with stem lengths in corn fields, and the "Chr.1, chr.2, chr.3, chr.4, chr.5, chr.6, chr.7, chr.8, chr.9 and Chr.10" are respectively 1 st, 2 nd, 3 nd, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th and 10 th chromosomes.

FIG. 12 is a genetic structural analysis of 193 maize inbred natural population genotypes using 186 pairs of SSR molecular marker primer pairs; wherein (A) delta K value is a variation curve of K value, (B) 193 parts of corn inbred line natural population genotype genetic structure analysis, (C) the genotype number/frequency statistics of the group, K is the group number, LRC is the bone group of great red, SPT is the Tang tetrad group, lan is the Lanchester group, P is the P group, and Reid is the Ruider group.

FIG. 13 is a major QTL locus analysis for controlling stem length qRHL in corn fields; wherein chr.3 is chromosome 3, qRHL-1 is QTL result obtained by QTL positioning, BSA-1 is 1 BSA result obtained by SNP-index analysis, BSA-2 and BSA-3 are 2 BSA results obtained by InDel-index analysis, BSA-4, BSA-5 and BSA-6 are 3 BSA results obtained by GPS analysis, GLM and MLM are 1 correlation SSR result obtained by linkage SSR correlation analysis of General Linear Model (GLM) and MLM (mixed linear model), PVE is the contribution rate of stem length phenotype in the ground, 3cm is 3cm deep-cast environment, 15cm is 15cm deep-cast environment, 20cm is 20cm deep-cast environment, marker is molecular Marker information, mb is physical distance of molecular Marker.

Detailed description of the preferred embodiments

In order to make the objects, technical solutions and advantages of the present patent more apparent, the following detailed description of the present patent refers to the field of 'electric digital data processing'. The test methods in the following examples are all conventional test methods unless otherwise specified, and the test reagents and consumables in the following examples are all from conventional biochemical reagent companies unless otherwise specified. In order to make the objects, technical solutions and advantages of the present patent more apparent, the following detailed description of the present patent refers to the field of 'electric digital data processing'. Examples of these preferred embodiments are illustrated in the specific examples. It should be noted that, in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only the technical solutions and/or processing steps closely related to the solutions according to the present invention are shown in the embodiments, and other details having little relation are omitted.

Example 1

The invention provides a method for constructing a genotype genetic map of an F ₂ segregating population and analyzing a stem length QTL in a F _2:3 locating population genotype-regulated corn field, which comprises the following specific steps:

F ₂ segregating population genotype and F _2:3 locating population genotype construction: corn inbred line N192 genotype with excellent stress resistance (drought/cold resistance) and corn inbred line Ji853 genotype with short-field middle-stem characteristics and stress sensitivity (drought/cold sensitivity) are planted in a field at west of Gansu Province corn test points. The 2 corn inbred lines are strictly bagged before flowering, N192 is used as a female parent and Ji853 is used as a male parent, and F ₁ hybrid seeds (N192 multiplied by Ji 853) are obtained by artificial hybridization and pollination after flowering. And (3) continuously planting F ₁ hybrid seeds in a field at a west of Gansu Province corn test point in the next year, and carrying out manual self-pollination by strictly bagging to obtain the genotype of the F ₂ isolated population containing 282 kernels. 282F ₂ segregating group genotype single plants are continuously planted in a corn test point field of west of Gansu Province in the coming year, when each single plant seedling of F ₂ segregating group genotype grows to a five-leaf one-heart period, the third leaf of the corresponding seedling is sequentially numbered and cut, and the third leaf is placed into a self-sealing bag for preservation at the temperature of minus 70 ℃ and used for extracting DNA of different genotypes of the subsequent F ₂ segregating group. And (3) carrying out manual self-pollination on each single plant of F ₂ segregation population genotypes for 3 times by strictly bagging, deriving to obtain corresponding F _2:3 positioning population family genotypes, harvesting each F _2:3 positioning population family genotype after the corn is physiologically mature, and naturally air-drying and then checking.

2.2 Parts of parent inbred line genotype and 282 parts of F ₂ isolated population genotype genome DNA extraction and quality control: and (3) extracting genome DNA of the single plant five-leaf one-heart seedling samples of the genotypes of the female parent N192, the male parent Ji853 and each F ₂ in the step 1by adopting a Cetyl Trimethyl Ammonium Bromide (CTAB) method. 1% agarose gel electrophoresis is used for detecting the DNA quality of each sample, and an ultra-micro ultraviolet spectrophotometer is adopted for detecting the DNA concentration of the sample by using a American Nanodrop ^TM One/OneC (Thermo FISHER SCIENTIFIC), and the final DNA concentration is diluted to 50 ng/. Mu.L for later use.

SSR molecular marker primer is obtained: SSR molecular marker primer 1200 pairs uniformly distributed on 10 chromosomes of corn are downloaded from a corn genome database MaizeGDB (http:// www.maizegdb.org /) website and are synthesized by the division of biological engineering (Shanghai) Co. The synthesis of SSR molecular marker primers selects an HAP purification mode, and the concentration of each pair of SSR molecular marker primers (consisting of Primer 1 and Primer 2) is diluted to 1mmol/L for later use.

4.2 Parental inbred line polymorphism SSR molecular marker primer screening: and 2 parts of the genomic DNA of the parent genotype sample extracted in the step 2 are used as templates, the SSR molecular markers obtained in the step 3 are used as primers, and a Biometra-T1 PCR instrument produced in Germany is used for PCR amplification. Specifically, a 20 mu L reaction system is adopted, namely ：Primer 1(1mmol/L)0.6μL,Primer 2(1mmol/L)0.6μL,DNA(50ng/μL)1.4μL,2×Power Taq PCR Master Mix 10.0μL,UPH₂O 7.4μL.PCR reaction procedures are specifically as follows: pre-denaturation at 95℃for 5min,1 cycle; denaturation at 94℃for 0.5min, annealing at 49.8-61.5℃for 0.5min and extension at 72℃for 0.5min for 35 cycles; finally, the extension is carried out at 72 ℃ for 1min, and the preservation is finished at the temperature of 4 ℃ for 60min.2 PCR amplified products of the parental genotype genome DNA are subjected to gel running and silver staining by 8% non-denaturing polyacrylamide gel electrophoresis (PAGE), and finally SSR molecular markers with obvious polymorphism and clear bands between N192 and Ji853 genotypes are screened out for standby.

5.282F ₂ segregating population genotype whole genome SSR scanning and genetic map construction: and (3) selecting 282 parts of F ₂ segregating group genotypes and genomic DNAs of parent N192 and Ji853 genotypes extracted in the step (2), carrying out full genome SSR scanning on 282 parts of F ₂ segregating group genotypes by using polymorphic SSR molecular markers screened among the parent genotypes according to the method in the step (4), and detecting the genotypes of each F ₂ segregating group genotype on each pair of SSR molecular markers. The distribution condition of each pair of SSR molecular markers in the genotype of the F ₂ separation population is analyzed by the card square test, so that the genotype of the female parent is obtained: f ₁ heterozygous genotype: the male parent genotype separation ratio accords with 1:2:1 SSR molecular marker matrix information. Genetic maps of F ₂ segregating populations were constructed using JoinMap4.0 software, and distances (cM) between map markers were calculated by the Kosambi function.

F _2:3 positioning group family genotype corn field stem length phenotype measurement and data statistical analysis under 6.3cm, 15cm and 20cm deep sowing environment: each F _2:3 positioning group family obtained in the step 1 and 20 seeds of the parental N192 and Ji853 genotypes are sequentially weighed, then 70% ethanol (v/v) is used as a disinfectant, the seeds are disinfected for 10min according to the proportion of dry seed mass to disinfectant volume of 1g to 25mL, ddH ₂ O water is used for washing the seeds for 5 times, and then ddH ₂ O water is used as seed soaking liquid for each corresponding disinfected genotype seed, and seed soaking seeds are obtained according to the proportion of dry seed mass to seed soaking liquid volume of 1g to 25mL, and the seeds are soaked for 24h at normal temperature and in a dark place at the temperature of 25 ℃. 20 seeds of each parent genotype and F _2:3 family genotype seed with good seed soaking are uniformly sown in a seed deep sowing test device (authorized bulletin number: CN209768182U; specification of the deep sowing test device: height 50cm and inner diameter 17 cm) which is provided with sterilized vermiculite (the sterilized vermiculite for sowing is mixed and stirred uniformly in a proportion of 5g:1mL of water volume of ddH ₂ O according to the mass of the sterilized vermiculite in advance), and 3 deep sowing environments are sequentially arranged, wherein the 3 deep sowing environments are respectively 3cm, 15cm and 20cm. The deep seeding test device is placed in a climatic chamber for culture after seeding, the environment is set to be illumination intensity of 600 mu mol/s.m ² during culture, illumination is carried out for 12 hours per day, the relative humidity is 65%, and the culture is carried out at a constant temperature of 22+/-0.5 ℃. The corresponding parental genotypes and F _2:3 positioning group genotypes cultured in each deep-sowing environment are supplemented with 20mL ddH ₂ O water every 2d, and 3 biological repeats are set. After 10d of culture, the vermiculite adhered to the seedlings is washed off, 10 seedlings with the same growth vigor are selected, and the genotype of each parent is measured by a centimeter scale, and the length of the middle soil stem (RHL) of the genotype of the colony family is positioned by F _2:3.

After measuring the stem length in each F _2:3 positioning group genotype, female parent N192 and male parent Ji853 genotype under 3 seed depth environments of 3cm, 15cm and 20cm, the stem length is sorted by Excel 2016 software, and the Mean value (Mean), standard deviation (Standard deviation, SD), kurtosis (Kurtosis) and bias (Skewness), amplitude (Range), genotype variance (Genotypic variance, V _G), environment variance (Environmental variance, V _E), genotype-environment interaction variance (Variance of genotype × Environment interaction, V _GEI), error of machinery (Error of variance, V epsilon), generalized force transmission (Broad-sense heritability, H _B).H_B) of F _2:3 positioning group genotypes in 3 seed depth environments are calculated by the following formula H _B＝V_G/(V_G+V_GEI/n+V epsilon/nr) ×100% (1). Wherein, H _B is generalized genetic force, V _G is genotype variance, V _GEI is genotype and environment interaction variance, V epsilon is machine error, n (n=3) is deep sowing environment, r (r=10) is the number of genotypes of each F _2:3 positioning group measured in each deep sowing environment. The medium stem length of the F _2:3 located population genotype was plotted using IBM-SPSS19.0 (SPSS Inc., chicago, IL, USA) software under 3 seed depth environments.

F _2:3 location group under 7.3cm, 15cm and 20cm deep sowing environment: combining the above-mentioned in-field stem length phenotype of 282 parts of F _2:3 positioning group genotypes under the condition of 3 sowing depths (3 cm, 15cm and 20 cm) of step 6 and the genotype genetic map information of F ₂ separating group genotypes constructed in step 5, adopting a composite interval mapping method (CIM) in Windows QTL Cartographer version 2.5.5 software to carry out QTL positioning analysis on the in-field stem length of F _2:3 positioning group genotypes under the condition of single sowing depth. For CIM, genotype scans were performed on stem length in corn at intervals of 0.5cM using Zmapqtl program module Model 6, window size 10.0cM, and the likelihood function ratio log value (logarithm of odds, LOD; LOD > 3.0) threshold was determined by 1000 samples. Estimating the QTL genetic effect of stem length in corn land from the absolute value of the ratio of dominant effect (dominance effect, d) to additive effect (ADDITIVE EFFECT, a), namely: additive effects (ADDITIVE EFFECT, a; |d/a |=0.00-0.20), partial dominant effects (partial-dominance effect, PD; |d/a |=0.21-0.80), dominant effects (dominance effect, D; |d/a |=0.81-1.20), overdominant effects (over-dominance effect, OD |d/a| > 1.20). The physical reference genome of maize B73 RefGen_v3 (https:// www.maizegdb.org/genome /) is searched for the physical positions of SSR molecular markers at both sides of the stem length QTL locus interval in the regulatory maize field.

Example 2

The invention provides a method for constructing a DNA mixed pool with equal quantity of extreme phenotype of stem length in F ₂ land and regulating BSA analysis of stem length in corn land, which comprises the following specific steps:

Construction of a DNA pool with the medium stem length extreme phenotype of F ₂: 282 parts of F _2:3 positioning colony genotype seeds obtained by N192 XJi 853 manual self-pollination are used for weighing 20 seeds in sequence, 70% ethanol (v/v) is used as a disinfectant, seeds are disinfected for 10min according to the ratio of dry seed mass to disinfectant volume of 1g to 25mL, ddH ₂ O water is used for washing the seeds for 5 times, and each genotype seed correspondingly disinfected is used for soaking the seed with ddH ₂ O water as a seed soaking solution according to the ratio of dry seed mass to seed soaking solution volume of 1g to 25mL, and the seed soaking seeds are obtained at normal temperature and in the dark for 24 h. Each F _2:3 positioning group genotype seed with good seed soaking is uniformly sown in a seed deep sowing test device (authorized bulletin number: CN209768182U; specification of the deep sowing test device: height 50cm, inner diameter 17 cm) which is provided with sterilized vermiculite (the sterilized vermiculite for sowing is mixed and stirred uniformly in advance according to the mass of the sterilized vermiculite and the water volume of ddH ₂ O of 5g:1 mL), and the sowing depth is set to 20cm. The deep sowing test device is placed in a key laboratory artificial climate chamber of a crop science country of arid habitat of provincial department for cultivation after sowing, the environment is set to be illumination intensity of 600 mu mol/s.m ² during cultivation, 12h of illumination is carried out each day, the relative humidity is 65%, and the temperature is constant for cultivation at 22+/-0.5 ℃. 282 parts of F _2:3 -localized population genotypes cultured in a 20cm deep-sown environment were supplemented with 20mL ddH ₂ O water every 2d and 3 biological replicates were made. After 10d of cultivation, the vermiculite adhered to the seedlings is washed off, 10 seedlings with the same overall growth vigor are selected, and the in-ground stem length (RHI) of each F _2:3 positioning group genotype is measured by a centimeter scale. According to the phenotype of the stem length of the 282F _2:3 positioning population genotypes in the 20cm deep sowing environment, respectively screening out 35 parts of the extreme genotypes of the stem length and the extreme genotypes of the stem length in the ground, finding out the corresponding F ₂ genotypes, uniformly mixing 0.1g of equal quantity of seedling leaves to respectively form a stem length extreme mixing pool (L-pool) in the ground and a stem length extreme mixing pool (S-pool) in the ground, wherein the sizes of the stem length phenotype extreme mixing pools in the 2 fields are 35+35. And selecting 0.5g of each of female parent N192 and male parent Ji853 genotype seedling leaves as a female parent pool and a male parent pool respectively. These 4 equal-amount DNA pools were prepared for subsequent DNA Re-Sequencing (Re-Seq) analysis.

DNA resequencing analysis of 2.4 equal amount DNA pools: taking 4 equal amount DNA mixed pool samples constructed in the step 1, extracting a genome DNA library by using a Cetyl Trimethyl Ammonium Bromide (CTAB) method, sending the genome DNA library to a Person biotechnology Co., ltd (Shanghai) for DNA re-sequencing analysis (project number: BS202309181155 MKVT), generating Raw Data of FASTQ, and filtering low quality Reads. FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc) software. The high quality data obtained after filtration was aligned to the reference genome (Zea_mays. B73_RefGen_v4.dna.toplevel. Fa) using bwa (0.7.17-r 1188) mem mode. The Duplicates is removed using markduplics in the Picard package. The detection of SNP (single nucleotide polymorphisms) mutation sites was performed using GATK software. The unifiedgenotyrer program in GenomeAnalysisTK v 3.8.8 software package was used to extract InDel (insertion and deletions) mutation site information.

3. Regulation of stem length BSA site detection in maize fields: in order to accurately screen candidate sites associated with stem length genes in corn, the stem length BSA sites in F ₂ in equivalent DNA pools of the extreme phenotype of stem length in corn were detected using a SNP-index (Takag et al, 2013) assay, inDel-index (Takag et al, 2013) assay, ED (Euclidean distance; jonathon et al, 2013) assay, GPS (GradePool-Seq; wang et al, 2019) assay, mutMap (Abe et al, 2012) assay, and their physical locations were determined.

Example 3

The invention provides a method for analyzing and regulating the genetic structure of natural corn colony genotypes and SSR linkage analysis of a stem length LD in corn fields, which comprises the following specific steps:

1.193 natural population genotypes of maize inbred lines are collected and bred: 193 natural population genotype seeds of corn inbred lines from different sources are collected, all natural population genotype seeds of the corn inbred lines are sown at west of Gansu Province corn test points in the same year by adopting a random block design, and are planted in double-row areas, wherein the row length is 1.5m. And (3) uniformly fertilizing and covering films according to field management before sowing, bagging and artificial self-pollination in a strict flowering period, harvesting after physiological maturity of corn, naturally airing, and examining seeds to obtain new seeds of natural population genotype corn of different corn self-bred lines with consistent seed vigor.

2.193 Maize inbred natural population genotypes medium stem length phenotype identification and data statistical analysis: 193 parts of corn inbred line natural population genotype new seeds obtained in the step 1 are sequentially weighed, 70% ethanol (v/v) is used as a disinfectant, seeds are disinfected for 10min according to the proportion of dry seed mass and disinfectant volume of 1g to 25mL, ddH ₂ O water is used for washing the seeds for 5 times, and each genotype seed correspondingly disinfected is used as seed soaking liquid according to the proportion of dry seed mass to seed soaking liquid volume of 1g to 25mL, and seed soaking seeds are soaked at normal temperature and at 25 ℃ in a dark state for 24h, so that seed soaking seeds are obtained. 20 seeds of natural population genotype seeds of each inbred line with good seed soaking are uniformly sown in a seed deep sowing test device (authorized bulletin number: CN209768182U; specification of the deep sowing test device: height 50cm and inner diameter 17 cm) which is provided with sterilized vermiculite (the sterilized vermiculite for sowing is mixed and stirred uniformly in advance according to the mass of the sterilized vermiculite and the water volume of ddH ₂ O to be 5g:1 mL), and 2 sowing deep environments are respectively 3cm and 20cm in sequence. The deep sowing test device is placed in a key laboratory artificial climate chamber of a crop science country of arid habitat of provincial department for cultivation after sowing, the environment is set to be illumination intensity of 600 mu mol/s.m ² during cultivation, 12h of illumination is carried out each day, the relative humidity is 65%, and the temperature is constant for cultivation at 22+/-0.5 ℃. The genotypes of the natural populations of the corresponding inbred lines cultured in each deep-sowing environment were supplemented with 20mL ddH ₂ O water every 2d, and 3 biological replicates were made. After 10d of culture, washing off vermiculite adhered to seedlings, selecting 10 seedlings with the same growth vigor, measuring the length of the middle soil stem (RHL) of the natural population genotype of each inbred line by using a centimeter scale, taking corresponding seedling tissues, filling the tissues into a self-sealing bag for preservation at the temperature of minus 70 ℃ for subsequent 193 parts of corn inbred line natural population genotype DNA extraction.

The stem length in the genotype land of each maize inbred natural population is measured under 2 deep-sowing environments of 3cm and 20cm, and is finished by Excel2016 software, the Mean value (Mean), standard deviation (Standard deviation, SD), kurtosis (Kurtosis) and bias (Skewness) of the stem length in the genotype land of 193 parts of the maize inbred natural population under each deep-sowing environment is calculated by statistical analysis software IBM-SPSS19.0 (SPSS Inc., chicago, IL, USA), the variation coefficient (Genetic variation coefficient, CV _g).CV_g) is calculated by the following formula: CV _g =SD/mean×100% (2) in the formula, CV _g is the genetic variation coefficient of the stem length in the genotype land of 193 parts of the maize inbred natural population, SD is the standard deviation of the stem length in the genotype land of 193 parts of the maize inbred natural population, and the Histogram of the stem length in the genotype land of 193 parts of the maize inbred natural population is calculated by IBM-SPSS19.0 (SPSS Inc., chicago, IL, hi 2).

Extracting and quality testing of 3.193 corn inbred line natural population genotype genome DNA: the 193 parts of genomic DNA of the maize inbred line natural population genotype seedling sample collected in the step 2 is extracted by adopting a Cetyl Trimethyl Ammonium Bromide (CTAB) method. 1% agarose gel electrophoresis is used for detecting the DNA quality of each sample, and an ultra-micro ultraviolet spectrophotometer is adopted for detecting the DNA concentration of the sample by using a American Nanodrop ^TM One/OneC (Thermo FISHER SCIENTIFIC), and the final DNA concentration is diluted to 50 ng/. Mu.L for later use.

4. The polymorphic SSR molecular marker primer is obtained: the maize MaizeGDB (http:// www.maizegdb.org /) database website downloads polymorphic SSR molecular marker primer 186 pairs uniformly distributed on 10 chromosomes of maize, which are synthesized by the division of biological engineering (Shanghai). The synthesis of SSR molecular marker primers selects an HAP purification mode, and the concentration of each pair of SSR molecular marker primers (consisting of Primer 1 and Primer 2) is diluted to 1mmol/L for later use.

Detection of genotype genetic diversity and analysis of genetic structure of natural population of 5.193 maize inbred lines: taking 193 corn inbred line natural population genotype sample genome DNA extracted in the step 3 as a template, taking 86 pairs of polymorphic SSR molecular markers obtained in the step 4 as primers, and carrying out PCR amplification by using a Biometra-T1 PCR instrument produced by Germany. Specifically, a 20 mu L reaction system is adopted, namely ：Primer 1(1mmol/L)0.6μL,Primer 2(1mmol/L)0.6μL,DNA(50ng/μL)1.4μL,2×Power Taq PCR Master Mix 10.0μL,UPH₂O 7.4μL.PCR reaction procedures are specifically as follows: pre-denaturation at 95℃for 5min,1 cycle; denaturation at 94℃for 0.5min, annealing at 49.8-61.5℃for 0.5min and extension at 72℃for 0.5min for 35 cycles; finally, the extension is carried out at 72 ℃ for 1min, and the preservation is finished at the temperature of 4 ℃ for 60min.193 PCR amplified products of natural population genotype genomic DNA of the maize inbred line are subjected to gel running and silver staining by 8% non-denaturing polyacrylamide gel electrophoresis (PAGE), a '0, 1' allele matrix of the PCR amplified products is constructed, and polymorphism information (Polymorphism of information content, PIC) of each pair of SSR loci is calculated. PIC is calculated using the following formula: pic=1- Σpi ² (3). Wherein PIC is polymorphism information of SSR molecular markers, and Pi is gene frequency of an ith site. And carrying out population genetic Structure analysis on 193 parts of natural population genotypes of the maize inbred line by adopting Structure 2.3.1 software, estimating the optimal population group number K (K is set to be 1-12) according to the maximum likelihood principle, and calculating the covariate Q parameter.

6.186 Construction of SSR molecular marker genetic map: searching the genetic distance (cM) of 186 pairs of polymorphic SSR molecular marker primers obtained in the step 4 in the corn IBM2 2008 Neighbors Map Frame map, and constructing the 186 pairs of SSR molecular marker genetic map by adopting BioMercator v.4.2 software.

In-soil stem length LD linkage SSR association analysis of 193 corn inbred line natural population genotypes under 7.3cm and 20cm deep sowing environments: SSR site information associated with significant stem length (P < 0.01) in 193 parts of maize inbred natural population genotypes in 3cm and 20cm deep-sowing environments was calculated using a general linear model (GENERAL LINEAR model, GLM) and a mixed linear model (Mixed linear model, MLM) in Tassel 3.0.0 software. The physical location of the LD-linked SSR molecular markers regulating the stem length in corn fields is found in the corn B73 RefGen_v3 physical reference genome (https:// www.maizegdb.org/genome).

Example 4

The invention provides a method for obtaining and regulating main effect QTL identification of stem length in corn fields, which comprises the following specific steps:

1. Regulating and controlling the identification of major QTL locus of stem length in corn land: according to the result of the positioning of the stem length QTL in the corn field by F _2:3 positioning group genotypes under the 3cm, 15cm and 20cm deep sowing environments, the result of the equivalent phenotype DNA mixed pool of F ₂ under the 20cm deep sowing environments for regulating the stem length BSA locus in the corn field, and the result of the analysis of the linkage SSR associated locus of the stem length LD in the natural group genotypes of 193 parts of corn inbred lines under the 3cm and 20cm deep sowing environments, if 2 or more positioning methods detect the genetic locus (QTL locus, BSA locus or SSR associated locus) for regulating the stem length in the corn field, the physical positions of the genetic locus overlap, and the genetic locus can be stably detected under 2 or more deep sowing environments, and if the phenotype contribution rate (phenotypic variation explained, PVE) for explaining the stem length in the corn field under a single deep sowing environment is more than or equal to 10 percent, the genetic locus is the main effect QTL locus for regulating the stem length in the corn field. According to the physical distance (Mb) of molecular marker information on two sides of a main QTL interval of the middle-stem length of the regulated corn field, bioMercator v.4.2 software is adopted to construct a main QTL molecular marker physical map of the regulated corn field.

2. And (3) regulating and controlling molecular marker information retrieval of the stem length major QTL in the corn field: searching SSR molecular marker information on two sides of a main effect QTL interval of the stem length in the regulated corn field identified in the step 1 in a corn genome database MaizeGDB (http:// www.maizegdb.org /) website, wherein the SSR molecular marker information comprises chromosome where an SSR molecular marker is located, cross-fold group (mail B73 Genome sequencing Project contig 2005), bin position, chromosome arm and repeated sequence information.

Example 5

The invention provides a result of F ₂ segregating population genotype genetic map construction and F _2:3 positioning population genotype control corn field stem length QTL analysis, which comprises the following specific results:

1.3 medium stem length phenotype analysis of parent genotype and F _2:3 positioning group family genotype under deep sowing environment: for 2 parental genotypes, the in-ground stem length of 2 parental genotypes increased significantly (P < 0.01) with increasing depth of sowing from 3cm to 20cm (fig. 2). Compared with 3cm depth, the stem length of female parent N192 genotype in 15cm and 20cm deep sowing environment is increased by 103.41% and 196.93%, respectively, while the stem length of male parent Ji853 genotype in 15cm and 20cm deep sowing environment is increased by 35.92% and 90.14%, respectively (figure 2). The stem length in the ground of the female parent N192 genotype was 1.03 times, 1.54 times and 1.61 times that of the male parent Ji853 genotype in 3cm, 15cm and 20cm deep-sowing environments, respectively (figure 2). The result shows that the length of the middle-ground stem of the drought/cold resistant female parent N192 genotype is obviously larger than that of the drought/cold sensitive male parent Ji853 genotype, and the length of the middle-ground stem of the N192 genotype is obviously increased along with the increase of deep sowing. In addition, in the case of 282F _2:3 -located colony pedigree genotypes, the stem lengths in the 3cm, 15cm and 20cm deep-sowing environments were 4.16cm, 9.50cm and 15.86cm, respectively, and the luffing amplitudes were 2.11-6.91 cm, 2.86-16.76 cm and 3.50-26.77 cm, respectively (FIG. 3). It is shown that the sowing depth can obviously influence the medium stem lengths of different corn genotypes, and the medium stem length of the locating group family genotypes is obviously increased along with the increase of the sowing depth F _2:3.

F _2:3 positioning group pedigree genotype medium stem length normal distribution detection, variance analysis and generalized genetic force analysis under 2.3 seed sowing deep environments: f _2:3 positioning group genetype is characterized by that its stem length deflection and kurtosis are respectively-1.0 (Table 1) under 3 kinds of deep sowing environments of 3cm, 15cm and 20cm, etc., and they are represented by typical normal distribution characteristics (figure 3), and are characterized by quantitative genetic characteristics. In addition, the genotype variance (V _G), the environment variance (V _E), and the genotype and environment interaction variance (V _GEI) of the middle stem length of the genotype of the F _2:3 localization population families under the 3 deep-sowing environments all differed significantly at the P <0.01 level (table 1), indicating that the middle stem length of corn is simultaneously co-regulated by the genotype, the environment and the genotype and environment interaction effect. Analysis of the generalized genetic transmission (H _B) showed that the generalized genetic transmission (H _B) of the stem length in the F _2:3 -localized family genotype was 87.130% (Table 1), and therefore QTL localization of the stem length in the F _2:3 -localized family genotype was feasible.

Table 13 Normal distribution of stem length in the genotype of the positioning population families of 282F _2:3 under the deep sowing environment (3 cm, 15cm and 20 cm), analysis of variance and generalized genetic statistics

Note that: 3cm is a 3cm deep-sowing environment, 15cm is a 15cm deep-sowing environment, 20cm is a 20cm deep-sowing environment, the values in the analysis of variance are F-values, "x" represents that the analysis of variance is significantly different at a level of P < 0.01.

3. Parent genotype inbred line polymorphism SSR molecular marker primer screening and F ₂ segregation population genetic map construction: the SSR molecular marker primer 1200 pairs are downloaded from a corn MaizeGDB database, polymorphic SSR molecular marker primer screening is carried out between female parent N192 and male parent Ji853 genotypes, finally, SSR molecular marker primers with clear strips and obvious polymorphism are screened out and used for carrying out whole genome SSR scanning on 282 parts of F ₂ segregation population genotypes, and finally, 1 set of F ₂ segregation population genotype genetic map with the total length of 1668.0cM (shown in figure 4) is constructed, and compared with an IBM2 2008 Neighbors Map Frame reference map, the sequence of SSR molecular markers is highly consistent with the reference map, so that the constructed F ₂ segregation population genotype genetic map is accurate and can be used for positioning stem long QTL in 282 parts of F _2:3 positioning population genotypes in the subsequent 3cM, 15cM and 20cM sowing depth environments.

F _2:3 positioning group pedigree genotype in-ground stem length QTL positioning under 4.3 deep sowing environments: the method comprises the steps of carrying out QTL positioning on the stem length in the genotype of 282F _2:3 positioning groups under the deep sowing environments of 3cm, 15cm and 20cm by adopting a composite interval mapping method (CIM), wherein the total of the positioning to 4 stem length QTLs in the ground are respectively positioned on chromosome 1 (2 QTLs, in Bin 1.08 and Bin 1.09-1.10), chromosome 2 (1 QTL, in Bin 2.05), chromosome 3 (1 QTL, in Bin 3.07) and chromosome 6 (1 QTL, in Bin 6.04), all the QTLs are detected under the deep sowing environments of 2 or more, and the phenotype contribution rate (PVE) of single QTL is between 3.03% and 12.43% (Table 2, figure 4). Furthermore, the modes of gene action of these QTLs were mainly additive effect (a) and dominant effect (D), which account for 76.9% and 23.1%, respectively (table 2), indicating that stem length in corn is mainly additive inheritance. Further analysis also found that the alleles in these QTLs that increased stem length in corn land were all from the maternal N192 genotype (table 2). Thus, genetically improving stem length in corn fields should not only focus on the accumulation of additive effects but also on the effects of dominant effects, while focusing particularly on the selection of maternal genotype materials with stem characteristics in the field and on the effects of heterosis.

Table 23 positioning of the Medium stem length QTL of the genotype of the family of 282F _2:3 positioning groups under the deep sowing environment (3 cm, 15cm and 20 cm)

Note that: RHS is the in-ground stem length, 3cm is the 3cm deep-sowing environment, 15cm is the 15cm deep-sowing environment, 20cm is the 20cm deep-sowing environment, bin is the chromosome Bin position, LOD is the likelihood function ratio logarithmic value, PVE is the phenotype contribution rate of the in-ground stem length QTL. Additive (a) positive values indicate that alleles from the male parent Ji853 genotype are potentiating, whereas negative values indicate that alleles from the female parent N192 genotype are potentiating.

Example 6

The invention provides a result of F ₂ in-soil stem length extreme phenotype equivalent DNA mixed pool construction and corn field stem length BSA regulation analysis, which comprises the following specific results:

DNA pool mixing analysis of the medium stem length extreme phenotype of F ₂ in a 1.20cm deep sowing environment: phenotype identification was performed on the medium stem length of F _2:3 -located colony genotypes under a 20cm deep-sowing environment (FIG. 5), 35 parts of the extreme genotypes of the longest and the shortest medium stem in the ground were screened out according to the medium stem length phenotype of F _2:3 -located colony genotypes, and the corresponding F ₂ genotypes were found as DNA pools (L-pool and S-pool, 35+35) of medium stem length extreme phenotypes of F ₂, and 4 resequencing pools were composed with 2 parental pools (Ji 853 and N192). The in-ground stem length phenotype analysis showed that the difference between the in-ground stem lengths of the female parent N192 pool and the male parent Ji853 pool under the 20cm deep sowing environment was significant (P < 0.05), the lengths thereof were 17.4cm and 10.8cm, respectively, and the difference between the in-ground stem lengths of the L-pool and the S-pool were significant (P < 0.05), the lengths thereof were 22.5cm and 8.8cm, respectively (FIG. 6), the in-ground stem lengths of the L-pool were 1.3 times and 2.1 times that of the 2 parent N192 and Ji853 pools, and the in-ground stem lengths of the S-pool were 51.0% and 82.1% that of the 2 parent N192 and Ji853 pools, respectively (FIG. 6). Thus, the 4 pools can be used to resequence BSA analysis for stem length in corn fields.

2. Mixed pool high throughput resequencing (Re-Seq) analysis: we sent L-pool, S-pool, female N192, male Ji853 pool samples to Peseno Biotechnology Co., ltd (Shanghai) for high throughput Re-sequencing (Re-Seq) analysis (project number: BS202309181155 MKVT), removed low quality Reads, and obtained 53.1Gb, 30.2Gb, and CLEAN READS of 30.2Gb in L-pool, S-pool, female N192, male Ji853 pools, respectively (Table 3); the GC percentages were 49.62%, 49.39%, 47.98% and 48.97%, respectively (table 3). The CLEAN READS of the 4 pools were further aligned to the reference genome (zea_mays. B73_refgen_v4.Dna. Toplevel. Fa) at alignment rates of 98.74%, 98.73%, 98.68% and 98.79%, respectively (table 3). In addition, the average sequencing depth of these 4 pools was 32.18×, 32.38 ×, 18.49×, and 18.54× (table 3). Thus, the resequencing data may be used for subsequent BSA analysis.

Table 34 high throughput resequencing (Re-Seq) data quality control for pool samples

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Note that: n192 is a female parent pool, ji853 is a male parent pool, L-pool is an extreme phenotype DNA mixing pool of the longest F ₂ of the middle ground stem, and S-pool is an extreme phenotype DNA mixing pool of the shortest F ₂ of the middle ground stem.

3. Pool SNP and InDel mutation site analysis: the SNPs and InDels detected in the L-pool, S-pool, female parent N192, and male parent Ji853 pools were predominantly homozygous genotype (HOM_REF) and heterozygous genotype (HET) consistent with the reference genome (FIG. 7). The total number of SNPs detected in the 4 pools was 16492287, with the number of non-synonymous mutations of SNP (Nonsynonymous SNV) being 195013, accounting for 1.18% (Table 4). The total amount of InDel detected in the 4 pools was 1556827, with a frame shift deletion resulting in a 6890 number of frame changes (FRAMESHIFT DELETION) in the protein encoding gene accounting for 0.44% of InDel type; frame shift insertion resulted in a frame change (FRAMESHIFT INSERTION) in the protein encoding gene of 5844 in number, accounting for 0.38% of InDel type (table 5).

TABLE 44 high throughput resequencing (Re-Seq) post SNP site annotation analysis of pool samples

TABLE 54 InDel site annotation analysis after high-throughput resequencing (Re-Seq) of pool samples

4.20Cm deep sowing environment, BSA analysis of the stem length in the corn field: the length of stem BSA sites in 193 parts of maize inbred line natural population genotype regulated maize fields under 20cm deep sowing environment are analyzed by adopting different BSA analysis methods. Specifically, 41 regulatory maize fields were detected for stem length BSA sites on chromosomes 2,3, 4, 6, 7, and 8 using SNP-index analysis (FIG. 8A); 12 regulatory maize plant stem length BSA sites were detected using InDel-index analysis and located on chromosomes 2,3, 8, and 10 (FIG. 8B); 1 site of BSA, which was located on chromosome 6 (FIG. 8C), was detected in the regulatory maize field using the ED assay; 5 regulatory maize fields were detected for stem length BSA sites on chromosome 3, 6 using GPS analysis (FIG. 8D); no control of the long BSA sites in corn was detected using MutMap assay.

Example 7

The invention provides a result of genetic structure analysis of natural population genotypes of maize inbred lines and SSR linkage analysis of LD linkage of the length of stems in a regulatory maize field, which comprises the following specific results:

Identification of the medium stem length phenotype of the genotype of the natural population of 193 maize inbred lines in 1.3cm and 20cm deep-sowing environments: the genotype of 193 maize inbred natural population was identified for the medium stem length phenotype in 3cm and 20cm deep-sowing environments. The results showed that as the depth of sowing increased from 3cm to 20cm, the in-ground stem length of the natural population genotype of 193 parts of maize inbred lines increased significantly (P < 0.05), the genetic variation coefficient (CV _g) increased from 13.79% to 25.67%, and kurtosis and skewness were both between-1.0 and 1.0 (table 7), with a typical normal distribution trend (fig. 9) and exhibited several genetic characteristics.

TABLE 7 statistical analysis of the medium stem length phenotype of the natural population genotypes of 193 maize inbred lines in 3cm and 20cm deep-sowing environments

Note that: 3cm is a 3cm deep sowing environment, 20cm is a 20cm deep sowing environment, and different lowercase letters "a and b" indicate that the stem length in 193 parts of corn inbred line natural population genotypes is obviously different at the P <0.05 level under the 3cm and 20cm deep sowing environments.

Genotypic genetic diversity analysis of natural populations of 2.193 maize inbred lines: the genotype of 193 maize inbred natural population was analyzed for genetic diversity on chromosome 1,2, 3, 4, 5,6, 7, 8, 9, 10 of maize with the polymorphic SSR molecular marker primers 29, 14, 15, 18, 17, 18, 27, 17, 14 selected respectively (FIG. 10A). The result shows that 186 pairs of SSR molecular marker primers detect 587 alleles in total among 193 corn inbred natural population genotypes, and each pair of SSR molecular marker primers detects 2-7 alleles (shown in figure 10B); the Polymorphism Information (PIC) value of each pair of SSR molecular marker primers is 0.138-0.734, and the average value is 0.445 (figure 10C), which shows that the 193 corn inbred line natural population genotypes have rich genetic background and higher genetic diversity.

SSR molecular marker genetic map construction: the genetic distance (cM) of the 186 pairs of polymorphic SSR molecular marker primers is searched in a corn IBM2 2008 Neighbors Map Frame map, and the 186 pairs of SSR molecular marker genetic maps are constructed by BioMercator v.4.2 software, wherein the total length of the maps is 6187.8cM, and the total lengths of chromosomes 1,2, 3, 4, 5, 6, 7, 8, 9 and 10 are 1116.8cM, 632.3cM, 804.9cM, 728.7cM, 602.5cM, 614.1cM, 585.1cM, 492.3cM, 611.1cM and 496.6 respectively (shown in figure 11).

Genotypic genetic structural analysis of 4.193 maize inbred lines natural populations: the full genome scanning is carried out on 193 corn inbred natural population genotypes by 186 pairs of SSR molecular marker primers, and the genetic Structure of the 193 corn inbred natural population genotypes is analyzed by adopting Structure 2.3.1 software. The results indicate that the 193 maize inbred natural population genotypes can be divided into 5 populations (fig. 12A). Further, 193 parts of natural group genotypes of the maize inbred line are classified into 5 groups according to the group attribute ratio of more than or equal to 0.5, and the other 9 parts of genotypes (4.7%) have no definite group attribution characteristic, so that a Mixed group (fig. 12B and 12C) is formed. These 5 clusters are respectively the hotel red bone cluster (LRC), comprising 30 genotypes, accounting for 15.5%; a tetrad group (SPT) of tangs comprising 47 genotypes, 24.4%; the lanchester group (Lan) comprises 23 genotypes, 11.9%; group P (P) contained 33 genotypes, accounting for 17.1%; the Reed population (Reid), which contains 51 genotypes, accounts for 26.4% (FIGS. 12B and 12C).

In-soil stem length LD linkage SSR association analysis of 193 corn inbred line natural population genotypes under 5.3cm and 20cm deep sowing environments: carrying out LD linkage SSR association analysis on the middle stem length of 193 corn inbred natural population genotypes under 3cm and 20cm deep sowing environments by adopting Tassel 3.0.0 software, wherein a total of 7 SSR markers and the middle stem length of the corn in 3cm or 20cm deep sowing environments are detected in a General Linear Model (GLM) (P < 0.01), which are respectively positioned on chromosome 2 (1 obvious association SSR locus), chromosome 3 (2 obvious association SSR locus), chromosome 6 (2 obvious association SSR locus), chromosome 8 (1 obvious association SSR locus) and chromosome 10 (1 obvious association SSR locus), and the phenotype contribution rate of the single association SSR locus is between 2.57 and 14.08 percent (table 8); a total of 5 SSR markers were detected in the Mixed Linear Model (MLM) as being significantly associated with stem length in corn plots under 3cm or 20cm deep-sowing environments (P < 0.01), which were located on chromosome 1 (1 significantly associated SSR site), chromosome 3 (1 significantly associated SSR site), chromosome 6 (2 significantly associated SSR site), chromosome 8 (1 significantly associated SSR site), respectively, with a phenotypic contribution rate of a single associated SSR site between 2.84% and 7.95% (table 8).

TABLE 8 SSR correlation analysis of the middle stem length of 193 maize inbred line natural population genotypes under 3cm and 20cm deep-sowing environments

Note that: 3cm is a 3cm deep sowing environment, 20cm is a 20cm deep sowing environment, bin is a chromosome Bin position, and PVE is a phenotype contribution rate of stem length associated SSR sites in corn fields.

Example 8

The invention provides a method for obtaining a result of regulating and controlling the identification of a main effect QTL of a stem length in corn fields, which comprises the following specific results:

1. Regulating and controlling the identification of major QTL locus of stem length in corn land: we analyzed the physical location of the F _2:3 positioning group in 3cm, 15cm and 20cm deep sowing environments to regulate the stem length QTL locus in corn, the physical location of the F ₂ extreme phenotype equivalent DNA pool in 20cm deep sowing environments to regulate the stem length BSA locus in corn, and the physical location of the stem length SSR association locus in 193 parts of natural group genotypes of maize inbred lines in 3cm and 20cm deep sowing environments. 1 in-field stem length QTL of corn is detected to be qRHL-1 in 3 rd chromosome Bin 3.07-3.08 region under the conditions of 3cm, 15cm and 20cm sowing depth, the Quantitative Trait Locus (QTL) is positioned between umc1399-bnlg1779, and the accumulated contribution rate of the phenotype of the in-field stem length is 25.90%; the SNP-index, inDel-index and GPS analysis method are also used for detecting that the BSA loci of the stem length in 6 regulatory corn lands are BSA-1, BSA-2, BSA-3, BSA-4, BSA-5 and BSA-6; 1 SSR site correlated with the stem length in the corn field, which is detected by a General Linear Model (GLM) and a Mixed Linear Model (MLM) under the 3cm and 20cm deep sowing environments, is also stably regulated and controlled to be umc1659, and the phenotype accumulated contribution rate of the stem length in the corn field is 33.75%. Further analysis found that these 8 genetic loci were highly overlapping, designated qRHL (FIG. 13) for regulating the stem length major QTL in corn fields. Further analysis also found that the main QTL regulating stem length qRHL in corn fields was able to stably regulate stem length in corn fields under 3cm, 15cm and 20cm deep-sowing environments, which was regulated by gene addition effects, and that the allele with increased stem length in corn fields was derived from the maternal genotype (table 2).

2. And (3) regulating and controlling stem length qRHL in corn field, and searching molecular marker information of a main effect QTL: 1 main effect QTL for simultaneously and stably regulating and controlling stem length qRHL in corn fields in 3cm, 15cm and 20cm deep sowing environments is detected by adopting 3 positioning methods such as QTL positioning, BSA analysis and SSR association analysis. The major QTL is located in the umc1399-umc1659-bnlg1779 region of the 3 rd chromosome long arm Bin 3.07-3.08 region, the ctg141-ctg145 span the contig, the 198458408-209429475bp physical region (Table 9).

TABLE 8 qRHL major QTL molecular marker information for controlling stem length in corn fields

Note that: bin is the chromosomal Bin position.

In conclusion, the research combines 3 positioning methods such as QTL positioning, BSA analysis and SSR association analysis to comprehensively detect 1 main effect QTL for simultaneously and stably regulating and controlling the stem length qRHL in the corn field in the 3cm, 15cm and 20cm deep sowing environments, and provides reliable SSR marker information for MAS breeding of a new stress-resistant corn variety with excellent stem characteristics and stress-resistant main effect genes of the corn field, and has huge application potential.

Example 9

The invention provides an application of a molecular marker for regulating and controlling a major QTL of a long stem in a corn field in corn breeding with excellent stress resistance, which comprises the following specific steps:

1. The application of the molecular marker for regulating and controlling the main effect QTL of the stem length in the corn field in the corn breeding with excellent stem characteristics in the soil and stress resistance adopts a plurality of positioning methods, the main effect QTL of the stem length in the corn field is qRHL3 simultaneously and stably under the conditions of 3cm, 15cm and 20cm sowing depth, the molecular marker of qRHL3 consists of three pairs of SSR molecular markers of umc1399, umc1659 and bnlg1779, wherein the sequence of the SSR molecular marker primer umc1399 is as follows:

Forward:5’-GCTCTATGTTATTCTTCAATCGGGC-3’；

Reverse:5’-GGTCGGTCGGTACTCTGCTCTA-3’；

The sequence of the SSR molecular marker primer umc1659 is as follows:

Forward:5’-CAAGCTTGCTACTGTGATTTCTCG-3’；

Reverse:5’-AACTTCTCGGTGATCTTGTCCATC-3’；

The sequence of the SSR molecular marker primer bnlg1779 is as follows:

Forward:5’-CCCTTTTATATCTCAAGTGTAGAACC-3’；

Reverse:5’-AGAGCACCCACCACGATAAC-3’；

As can be seen from the above examples, the method for assisted selection of maize genotypes (inbred lines, varieties) with excellent stress resistance and long-medium stem characteristics by using the above molecular markers for controlling the main QTL of medium stem length in maize fields comprises: extracting genome DNA of a corn genotype to be detected; PCR amplification is carried out by using SSR molecular marker primers umc1399, umc1659 and bnlg 1779; when the amplification products with the lengths of 121bp, 164bp and 176bp are obtained, the corn genotype to be detected is a corn genotype with long-soil middle-stem characteristics and excellent stress resistance, other genotypes are eliminated, the corn new variety or new line with long-soil middle-stem characteristics and excellent stress resistance is bred through purposeful combination hybridization combination, and the corn new variety or new line is popularized and applied to corn field production in combination with deep sowing cultivation measures, so that adverse effects of drought, low temperature, lodging, herbicide and the like suffered in the corn growth and development process can be remarkably improved, positive effects are generated for corn production safety, and economic, ecological and social benefits are remarkable.

Claims

1. The molecular marker for regulating and controlling the stem length major QTL in the corn field is characterized in that the molecular marker is identified by primers umc1399, umc1659 and bnlg 1779; umc1399 is shown in sequence listing SEQ ID NO:1 and SEQ ID NO:2, umc1659 is shown in the sequence table SEQ ID NO:3 and SEQ ID NO:4, bnlg1779 is shown in a sequence table SEQ ID NO:5 and SEQ ID NO: shown at 6.

2. The molecular marker for regulating and controlling the main effect QTL of the stem length in the corn field according to claim 1, wherein the molecular marker is used for simultaneously and stably regulating and controlling the stem length characteristic in the corn field in a 3cm, 15cm and 20cm deep sowing environment.

3. The application of the molecular marker for regulating and controlling the major QTL of the middle-length stem of the corn field in screening the corn genotypes with excellent stress resistance, which is characterized in that the primers umc1399, umc1659 and bnlg1779 are adopted for PCR amplification, and when amplification products with the lengths of 121bp, 164bp and 176bp are respectively obtained, corn to be tested is the corn genotypes with excellent stress resistance and middle-length stem characteristics.

4. A method for obtaining a molecular marker for regulating and controlling a main effect QTL of a stem length in a corn field is characterized by comprising the following steps: (1) construction of F ₂ isolated population genotype genetic map and F _2:3 positioning population genotype regulation corn field middle stem length QTL analysis, (2) construction of F ₂ middle stem length extreme phenotype equivalent DNA mixed pool and regulation corn field middle stem length BSA analysis, (3) analysis of corn inbred line natural population genotype genetic structure and regulation corn field middle stem length LD linkage SSR association analysis, and (4) regulation corn field middle stem length main effect QTL identification.