CN117904357A - Main effect QTL for regulating photosynthetic characteristics of corn seedlings under low-temperature stress and molecular marker and application thereof - Google Patents
Main effect QTL for regulating photosynthetic characteristics of corn seedlings under low-temperature stress and molecular marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to a multi-effect main effect QTL for simultaneously regulating and controlling the activity, pore conductance and transpiration rate of ribulose-1, 5-bisphosphate carboxylase of corn seedlings under low-temperature stress, and a molecular marker and application thereof. The molecular marker of the main effect QTL consists of two pairs of SSR molecular markers, namely bnlg1443 and umc 1020. When the SSR molecular marker is used for auxiliary selection of the corn genotype with high photosynthetic performance and cold resistance, the photosynthetic performance and the cold resistance of the corn genotype can be predicted by only detecting the characteristic bands of the SSR molecular marker, the identification method is simple and easy to operate, the selection efficiency is high, the application potential is great in the field of high-efficiency utilization of corn light and cold resistance breeding, and the safe production of corn in northern high-altitude areas is served.
Description
Technical Field
The invention belongs to the field of corn molecular genetic breeding, and particularly relates to a main effect QTL for regulating and controlling photosynthetic characteristics of corn seedlings under low-temperature stress, namely a multi-effect main effect QTL for simultaneously regulating and controlling activity, stomatal conductance (stomatal conductance, gs) and transpiration rate (transpiration rate, tr) of ribulose-1, 5-bisphosphate carboxylase (ribulose, 5-biphosphate carboxylase, rubisco) of the corn seedlings under low-temperature stress, and molecular markers and application thereof.
Background
The low-temperature cold damage is one of the most important adversity stress factors causing grain yield fluctuation in China, and has large scale, comprehensiveness and geographic region difference. As a typical C 4 short-day crop with a preference for temperature, corn (Zea mays l.) is early in fertility, particularly from sowing to seedling morphogenesis, and is vulnerable to cold damage at low temperatures, when corn encounters cold damage at low temperatures, the vigor of the seed will be reduced, the time for emergence of the seed will be prolonged, resulting in mildewing and rot of the seed or sprout; seedlings will stop growing and even the seedling cells and tissues will be irreversibly damaged, resulting in serious seedling-missing and ridge-breaking phenomena. The occurrence frequency of low-temperature cold injury in northern corn production areas in China is high, and the corn yield can be generally reduced by 5% -15%. Therefore, the prevention of the low-temperature cold damage of the corns in the northern high-altitude areas of China is very important to ensure the production safety of grains.
The growth and development of corn and the accumulation of organic matters in the body mainly depend on photosynthesis, and the low-temperature cold injury can directly damage photosynthetic organs such as corn leaves, and the photosynthesis performance of the corn is influenced by inhibiting activities of various photosynthetic pigments (such as chlorophyll; chlorophyll, chl a) and photosynthetic enzymes (such as ribulose-1, 5-bisphosphate carboxylase; ribulose, 5-biphosphate carboxylase, rubisco is a key enzyme determining carbon assimilation rate in photosynthesis), so that the net photosynthetic rate (net photosynthetic rate, pn) of the corn is reduced, the absorption function is reduced, the biochemical change is changed and the like. The photosynthetic characteristics of the corn are extremely obviously positively correlated with the cold resistance, the photosynthetic characteristics can be used as a rapid, effective and noninvasive tool for exploring the influence of low-temperature cold injury on photosynthetic organs of the corn, and the deep research of the photosynthetic characteristics has become an important cutting point and direction for revealing the cold resistance mechanism of the corn. The photosynthetic characteristics of the corn belong to typical quantitative inheritance traits, are commonly regulated and controlled by a plurality of micro/major genes, locate gene loci (quantitative trait loci, QTL) for regulating and controlling the photosynthetic characteristics of the corn under the low-temperature condition, can lay a solid theoretical basis for systematically exploring the molecular genetic mechanism of the cold resistance of the corn, and provide references for marker-assisted selection (MAS) assisted selection of the cold resistance molecular markers of the corn and CRISPR gene editing application, so that the cultivation process of new varieties of the excellent cold resistance corn is accelerated.
Although QTL/genes for multiple cold-resistance related traits have been mapped in rice (oryza sativa l.) and arabidopsis thaliana (Arabidopsis thaliana), only a few QTL/genes have been shown to play a role in rice and arabidopsis thaliana response to low temperature stress. Recently, the domestic Cookara team localizes cold-resistant QTL main gene in japonica rice as COG3 (chilling-tolerance in geng/japonica rice 3), and domestically selected high-expression protein COG3 plays a role in key components (OsFtsH 2-D1) of rice chloroplast photosystem II (Photosystem II, PSII) under low-temperature induction. The professor Mark g.m.aarts, netherlands, warz Ning Genda, measured the steady state quantum yield of PSII electron transport (phitsii) in the natural population of arabidopsis using chlorophyll fluorescence imaging autophenotype platform, localized to multiple QTL sites associated with phitsii changes under low temperature stress, one of which was confirmed to be a candidate gene involved in PSII response to low temperature, encoding PSII-associated protein PSB27. These studies demonstrate that the major QTL/genes involved in photosynthetic property correlation can significantly affect cold resistance of different plants, providing technical support for different crop cold resistance molecular breeding applications. But no QTL/gene has been located on maize to low temperature related photosynthetic properties. Therefore, by adopting the QTL analysis technology, the main effect QTL/gene related to photosynthetic property is positioned and regulated on the corn under low-temperature stress, which is the key of corn cold-resistant MAS breeding and is one of effective measures for solving the problem of low-temperature stress resistance of the corn.
Disclosure of Invention
The invention aims to provide a 'one-factor multiple-effect' main effect QTL and an SSR molecular marker thereof, which can simultaneously regulate and control the activity of ribulose-1, 5-biphosphate carboxylase (ribulose, 5-biphosphate carboxylase, rubisco), stomatal conductance (stomatal conductance, gs) and transpiration rate (transpiration rate, tr) of corn seedlings under low-temperature stress. Furthermore, the invention provides a method for assisting in selecting the genotype of the cold-resistant corn with high photosynthetic performance. In addition, the invention also provides application of the molecular marker for simultaneously regulating and controlling the activity of ribulose-1, 5-biphosphate carboxylase, air pore conductivity and transpiration rate of the multi-effect main effect QTL of the maize seedling under low-temperature stress in maize light efficient utilization and cold-resistant maize breeding.
In order to solve the technical problems, the invention adopts the following technical scheme:
1. SSR molecular marker primers for simultaneously regulating and controlling the activity, stomatal conductance and transpiration rate of ribulose-1, 5-bisphosphate carboxylase of corn seedlings under low-temperature stress, wherein the SSR molecular marker primers comprise two pairs of SSR molecular marker primers of bnlg1443 and umc1020, 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 bnlg1443 is as follows:
Forward:5’-TACCGGAATCCTCTTTGGTG-3’；
Reverse:5’-TTTGACAACCTCTTCCAGGG-3’；
As shown in SEQ ID NO:3 and SEQ ID NO:4, the sequence of the SSR molecular marker primer umc1020 is as follows:
Forward:5’-CCTGGAGAGCCACTACAAGGAA-3’；
Reverse:5’-TCAGCCTGAGCTCACATCATCT-3’；
2. The molecular marker of the multi-effect main effect QTL is used for simultaneously regulating and controlling the activity of ribulose-1, 5-bisphosphate carboxylase, the air pore conductivity and the transpiration rate of corn seedlings under low-temperature stress in the breeding of the cold-resistant corn with high efficient utilization of corn light, and the genotype of the cold-resistant corn with high photosynthetic performance is selected in an auxiliary way to be applied to the breeding of the cold-resistant corn; can be amplified by SSR primers bnlg1443 and umc1020 by PCR; and obtaining amplification products with lengths of 183bp and 216bp, the corn to be detected is a corn genotype (germplasm, strain and variety) with high photosynthetic performance and cold resistance.
3. A method for assisting in selecting a maize genotype with high photosynthetic performance and cold resistance comprises the following steps: extracting genome DNA of a corn genotype to be detected; PCR amplification is carried out by using SSR molecular marker primers bnlg1443 and umc 1020; when amplification products with lengths of 183bp and 216bp are obtained, the corn to be detected is a corn genotype with high photosynthetic performance and cold resistance.
4. A method for evaluating cold-resistant genotypes of corn seedlings comprises the following specific steps: (1) collecting seeds of maize inbred lines with different genotypes, (2) pre-treating the seeds, (3) testing different temperature gradients of maize seedlings, (4) measuring cold resistance traits, and (5) carrying out data statistical analysis and cold resistance evaluation.
5. A method for obtaining a multi-effect main QTL molecular marker for simultaneously regulating and controlling the activity, stomatal conductance and transpiration rate of ribulose-1, 5-bisphosphate carboxylase of maize seedlings under low-temperature stress comprises the following specific detailed steps: (1) F 1 hybrid, F 2 segregating group and F 2:3 locating group construction, (2) parental genotype inbred line and F 2 segregating group genome DNA extraction and quality inspection, (3) SSR molecular marker primer acquisition, (4) parental genotype inbred line polymorphism SSR molecular marker primer screening, (5) F 2 segregating group whole genome scanning and genetic linkage map drawing based on SSR molecular markers, (6) corn F 2:3 locating group seedling photosynthetic property determination and statistical analysis under different temperature treatment, (7) corn F 2:3 locating group seedling photosynthetic property QTL location under different temperature treatment, (8) simultaneous regulation of multiple photosynthetic property 'one-factor multiple-effect' main effect QTL identification of corn seedlings under low temperature stress, (9) simultaneous regulation of multiple photosynthetic property 'one-factor multiple-effect' main effect QTL molecular marker information retrieval of corn seedlings under low temperature stress.
The invention has the beneficial effects that: the invention uses a moderate cold-resistant corn inbred line N192 as a female parent and a cold-sensitive corn inbred line Ji853 as a male parent to hybridize to construct 1 set of F 2:3 positioning groups of 282 families, the F 2:3 positioning groups and parents of the seedling three-leaf stage carry out photosynthetic property identification under the environment of normal temperature contrast 25 ℃ and low temperature stress 15 ℃, a composite interval mapping method (composition INTERVAL MAPPING, CIM) is adopted to carry out QTL positioning research on photosynthetic property of corn seedlings, and finally, the cumulative phenotype contribution of 1 maize seedling ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductivity and seedling transpiration rate under 2 environments is stably detected in a Bin 6.05 area bnlg1443-umc1020 interval of 6 chromosome cross-overlapped group ctg404-ctg373, and simultaneously regulated and controlled under the environment of 15 ℃ and 25 ℃, and the one-factor multi-effect QTL is respectively 24.16%, 9.43% and 15.54% of the cumulative phenotype contribution of the maize ribulose-1, 5-bisphosphate carboxylase activity, the stomatal conductivity and the seedling transpiration rate under 2 environments. Further analysis shows that the corn genotype to be detected is subjected to PCR amplification by utilizing the two pairs of SSR molecular markers of the multi-effect main effect QTL, so that the photosynthetic performance and cold resistance of the corn genotype to be detected can be rapidly, objectively and efficiently predicted, and the error caused by temperature environment change in the process of identifying and selecting the corn genotype with cold resistance and high photosynthetic performance is effectively avoided.
When the SSR molecular marker disclosed by the invention is used for carrying out molecular marker-assisted selection on the corn genotype with high photosynthetic performance and cold resistance, the photosynthetic performance and the cold resistance of the corn genotype can be rapidly, objectively and accurately predicted by detecting the characteristic amplification strip of the corresponding SSR molecular marker. Can rapidly identify the corn genotypes with high photosynthetic performance and cold resistance, and eliminate other genotypes. The method is not only free from environmental influence when the corn genotype with excellent photosynthetic performance and cold resistance is selected, and has definite selection targets, but also can effectively reduce the cost of manpower and material resources, save time, and has great application potential and value in the cultivation of new variety of corn with excellent cold resistance.
Drawings
FIG. 1 shows the rate of change of 7 traits in 86 parts of maize genotype inbred line at 10deg.C (A) and 15deg.C (B) compared to normal temperature 25 deg.C treatment; wherein RC is the rate of change, SL is the seedling length, SW is the seedling weight, SB is the seedling biomass, SPAD is the chlorophyll SPAD value, pn is the net photosynthetic rate, MDA is malondialdehyde content, and MSI is the membrane stability index.
FIG. 2 is a personalized Pearson correlation analysis of 86 maize genotype inbred line 7 at 3 temperatures (10, 15, and 25 ℃) treatments; wherein SL is seedling length, SW is seedling weight, SB is seedling biomass, SPAD is chlorophyll SPAD value, pn is net photosynthetic rate, MDA is malondialdehyde content, MSI is membrane stability index, and curves between 2 traits represent that the 2 traits are obviously related at P <0.05 level.
FIG. 3 is an evaluation of inter-cluster cold resistance based on the individual mean cold resistance coefficients of 86 maize genotype inbred lines 7 at 10 and 15℃low temperature treatment; wherein I is a cold-sensitive genotype inbred line, II is a weak cold-resistant genotype inbred line, III is a cold-resistant genotype inbred line, IV is a moderate cold-resistant genotype inbred line, V is a strong cold-resistant genotype inbred line, and black triangles represent screening out 2 parent genotype inbred lines with larger cold resistance differences, which are subsequently used for constructing an F 2:3 group.
FIG. 4 is a bar graph (A) showing the photosynthetic characteristics of 5 seedlings of the parental genotypes N192 and Ji853 under 15 and 25 ℃ temperature treatment and a high-level box graph (B) showing the corresponding photosynthetic characteristics of the constructed F 2:3 positioning group family seedlings; wherein Pn is net photosynthetic rate, gs is stomatal conductance, ci is intercellular CO 2 concentration, tr is transpiration rate, rubisco is ribulose-1, 5-bisphosphate carboxylase activity, CK is normal temperature control treatment at 25 ℃, LT is low temperature stress treatment at 15 ℃, different lower case letters represent corresponding photosynthetic characteristics of different parental genotype inbred lines, and P <0.05 level difference is significant under all treatments.
FIG. 5 is a graph showing the skewness, kurtosis, genetic variation coefficient and generalized genetic power analysis of 5 photosynthetic characteristics of F 2:3 localized population family seedlings under 2 temperature (15 and 25 ℃) treatments; wherein Pn is net photosynthetic rate, gs is stomatal conductance, ci is intercellular CO 2 concentration, tr is transpiration rate, rubisco is ribulose-1, 5-bisphosphate carboxylase activity, CK is normal temperature control treatment at 25 ℃, LT is low temperature stress treatment at 15 ℃, CV g is genetic variation coefficient of corresponding photosynthetic characteristics of F 2:3 positioning group family seedlings under corresponding temperature treatment, kurtosis is Kurtosis, skewness is skewness, and H B is generalized genetic transmission of corresponding photosynthetic characteristics of F 2:3 positioning group family seedlings.
FIG. 6 shows principal component analysis and Pearson correlation coefficient analysis of 5 photosynthetic characteristics of the positioning population family seedlings of the parent genotypes N192, ji853 and F 2:3 under 2 temperature (15 and 25 ℃) treatment; wherein (A) is the characteristic value and the accumulated contribution rate of the principal component analysis of 5 photosynthetic characteristics of the positioning group family seedlings of the parent genotypes N192, ji853 and F 2:3 under 2 temperature (15 and 25 ℃), (B) is the load graph of the principal component analysis of 5 photosynthetic characteristics of the positioning group family seedlings of the genotypes N192, ji853 and F 2:3 under 25 ℃, and (C) is the load graph of the principal component analysis of 5 photosynthetic characteristics of the positioning group family seedlings of the genotypes N192, ji853 and F 2:3 under 15 ℃, (D) is a graph of 5 photosynthetic property Pearson correlation coefficients of genotype N192, ji853 and F 2:3 locating group family seedlings under the temperature treatment of 25 ℃, and (E) is a graph of 5 photosynthetic property Pearson correlation coefficients of genotype N192, ji853 and F 2:3 locating group family under the temperature treatment of 15 ℃, CK is the normal temperature control treatment of 25 ℃, LT is the low temperature stress treatment of 15 ℃, PCs is a main component, PC1 is a main component 1, PC2 is a main component 2, pn is a net photosynthetic rate, gs is stomatal conductivity, ci is the concentration of intercellular CO 2, tr is the transpiration rate, rubisco is the activity of ribulose-1, 5-bisphosphate carboxylase, and P <0.05 level between two characters is obviously related.
FIG. 7 is a genetic linkage map of an F 2 segregating population containing 282 individuals constructed using polymorphic SSR molecular markers; wherein ch.1, ch.2, ch.3, ch.4, ch.5, ch.6, ch.7, ch.8, ch.9, ch.10 are chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, respectively.
FIG. 8 is a statistical analysis of the locating results of 5 photosynthetic characteristic QTL of F 2:3 locating population families under 2 temperature (15 and 25 ℃) treatment using a composite interval mapping method (CIM); where Pn is the net photosynthetic rate, gs is the stomatal conductance, ci is the intercellular CO 2 concentration, tr is the transpiration rate, rubisco is the ribulose-1, 5-bisphosphate carboxylase activity, PVE is the phenotype contribution rate of the QTL, ch.1, ch.2, ch.3, ch.4, ch.5, ch.6, ch.7, ch.8, ch.9, ch.10 are chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, respectively.
FIG. 9 is a schematic diagram (B) of the distribution of the multi-effect main QTL on the linkage group (A) of corn F 2:3 positioning group family seedlings under low temperature stress and simultaneously regulating and controlling the activity of ribulose-1, 5-bisphosphate carboxylase, stomatal conductance and transpiration rate and the application of the molecular marker of the multi-effect main QTL in the breeding of the high-photosynthetic-efficiency cold-resistant corn by using corn light or auxiliary selection of the genotype of the cold-resistant corn; wherein Pn is net photosynthetic rate, gs is stomatal conductance, ci is intercellular CO 2 concentration, tr is transpiration rate, rubisco is ribulose-1, 5-bisphosphate carboxylase activity, CK is normal temperature control treatment at 25 ℃, LT is low temperature stress treatment at 15 ℃, G1, G2, G3 and G4 are corn genotype 1, genotype 2, genotype 3 and genotype 4 respectively, and MAS is molecular marker assisted selective breeding.
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 corn seedling cold-resistant genotype evaluation method, which comprises the following specific steps:
1. Collecting corn inbred line seeds of different genotypes: 86 parts of corn genotype inbred line seeds planted in the same test point in the same year and tightly bagging and self-pollinating are collected.
2. Seed pretreatment: and (2) using 70% ethanol (v/v) as a disinfectant for disinfecting the seeds for 10min according to the proportion of the dry seed mass to the disinfectant volume of 1g to 25mL for each genotype self-bred line seed with plump and uniform seeds in the step (1), flushing the seeds for 5 times by using ddH 2 O water, and soaking the seeds of the corresponding genotype self-bred line with ddH 2 O water as a seed soaking liquid according to the proportion of the dry seed mass to the seed soaking liquid volume of 1g to 25mL at normal temperature and in a dark place for 24h for later use.
3. Corn seedling different temperature gradient test: each 30 seeds of the maize genotype inbred line well soaked in the step 2 are uniformly sown in a plastic flowerpot (with the inner diameter of 20cm and the height of 14 cm) filled with sterilized vermiculite, the plastic flowerpot is placed in a climatic incubator (the illumination time is 12 hours per day, the illumination temperature is 25+/-0.5 ℃, the illumination intensity is 300 mu mol/s.m 2 and the relative humidity is 70%) and cultivated for 15d at constant temperature, then maize seedlings are respectively placed under 3 temperature gradient treatments of 10+/-0.5 ℃, 15+/-0.5 ℃ and 25+/-0.5 ℃ and the like for continuous cultivation for 7d, each treatment is biologically repeated for 3 times, and 30mL of Hoagland nutrient solution is poured every 2d during the seedling cultivation.
4. Cold resistance property determination: selecting 6 seedlings with the same overall growth vigor from each corn genotype seedling under 3 temperature gradient treatments, and measuring growth parameters such as seedling length (SL, SEEDLING LENGTH), seedling weight (SEEDLING WEIGHT, SW), seedling biomass (seedling biomass, SB) and the like; measuring chlorophyll SPAD value of 3 rd leaf of seedling by SPAD-502 chlorophyll meter of japan meridad; the net photosynthetic rate of leaf 3 of seedlings (net photosynthetic rate, pn) was determined using an LI-6400/XT portable photosynthetic apparatus, at which time the photosynthetically active radiation (photosynthetic active radiation, PAR) was set to 1000 μ M m -2s-1 and the CO 2 concentration was replenished by a CO 2 steel cylinder; referring to the method of Ahmad et al (2022), malondialdehyde (MDA) content and membrane stability index (membrane stability index, MSI) of the 3 rd leaf of seedlings were determined.
5. Data statistical analysis and cold resistance evaluation: all test data determined in step 4 above were collated using Excel 2016. The rate of change (RATE CHANGE, RC) and cold resistance coefficient (cold-RESISTANCE COEFFICIENT, CRC) for each trait under low temperature stress treatment at 10 or 15℃were calculated with reference to the method of Zhao et al (2022), namely: RC= (T LT-TCK)/TCK x 100% (1) and CRC=T LT/TCK (2). In the formula, T LT is a corresponding property measurement value under 10 or 15 ℃ low temperature stress treatment, T CK is a corresponding property measurement value under 25 ℃ normal temperature treatment, 86 parts of maize genotype inbred seedlings 7 at 3 temperature treatments are subjected to ANOVA variance analysis by using IBM SPSS16.0 software and to Pearson correlation analysis by GENESCLOUD on-line software, and average cold resistance coefficients of the 7 parts of maize genotype inbred seedlings at 10 and 15 ℃ low temperature stress treatment are subjected to clustering evaluation on cold resistance between the groups of IBM SPSS16.0 software.
Example 2
The invention provides a method for simultaneously regulating and controlling the activity of ribulose-1, 5-biphosphate carboxylase, pore conductance and transpiration rate of maize seedlings under low-temperature stress, which comprises the following specific steps:
F 1 hybrid, F 2 segregating population and F 2:3 locating population construction: when the field reaches the maize flowering phase, the moderate cold-resistant genotype inbred line N192 is taken as a female parent, the cold-sensitive genotype inbred line Ji853 is taken as a male parent, and the F 1 hybrid is obtained by strictly bagging and hybridizing. F 1 hybrid seeds are planted in the field of the next year, the flowering phase is strictly bagged, pollinated and selfed, and 1F 2 separated population containing 282 seeds is obtained. N192, ji853 and 282 parts of F 2 segregating group single plants are planted in the field of the next year, when the seedlings grow to five leaves and one heart period, each single plant of the F 2 group is numbered in sequence, a third leaf is cut off, and the third leaf is placed into a self-sealing bag and stored at the temperature of minus 70 ℃. And (3) when the flowering period is reached, carrying out the strict bagging self-pollination on each single plant of the F 2 segregation population for 3 times, deriving to obtain a corresponding F 2:3 positioning population family, harvesting each F 2:3 positioning population family after the physiological maturity of corn, and carrying out seed examination after natural air drying.
2. Parental genotype inbred line and F 2 isolated population genome DNA extraction and quality inspection: and (3) extracting genome DNA of the single plant five-leaf one-heart seedling samples of the female parent N192, the male parent Ji853 and each F 2 in the step 1 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.
SSR molecular marker primer is obtained: the maize genome database MaizeGDB (http:// www.maizegdb.org /) website downloads SSR molecular marker primer 1200 pairs uniformly distributed on 10 chromosomes of maize, which are synthesized by the division of biological engineering (Shanghai). The synthesis of the SSR molecular marker primers selects an HAP purification mode, and the concentration of each pair of SSR molecular marker primers (each pair of SSR molecular marker primers consists of a Primer 1 and a Primer 2) is diluted to 1mmol/L for standby.
4. Screening a parent genotype inbred line polymorphism SSR molecular marker primer: 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 20 mu L of a reaction system of a Biometra-T1 PCR instrument produced in Germany is used for PCR amplification. The reaction of 20. Mu.L of the reaction system is specifically ：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 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. The PCR amplified products of the N192 and Ji853 parent genotypes genome DNA are subjected to gel running and silver staining by 8% non-denaturing polyacrylamide gel electrophoresis, and SSR markers with clear stripes and obvious polymorphism between the parent genotypes N192 and Ji853 are screened out for standby.
5. F 2 segregating population whole genome scanning and genetic linkage map drawing based on SSR molecular markers: and (3) selecting 282 parts of F 2 segregating group single plants extracted in the step (2) and genome DNA of parent genotypes N192 and Ji853 seedlings, carrying out whole genome scanning on 282 parts of F 2 segregating group single plants 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 single plant on each pair of SSR molecular markers. Carrying out a square test on the distribution condition of genotypes of each pair of SSR molecular markers in F 2 segregating population individuals, and further obtaining maternal genotypes: heterozygous genotype: the segregation ratio of the male parent genotypes accords with 1:2:1 SSR molecular marker information. Genetic maps of the F 2 isolate were constructed using the jonmap4.0 genetic map mapping software, and map distances (cM) were calculated by the Kosambi function.
6. Determination and statistical analysis of photosynthetic characteristics of corn F 2:3 positioning group seedlings under different temperature treatments: and (2) weighing 20 seeds of each F 2:3 positioning group family and the parental genotypes N192 and Ji853 obtained in the step (1) in sequence, disinfecting the seeds for 10min according to the proportion of dry seed mass to disinfectant volume of 1g to 25mL, flushing the seeds for 5 times by using ddH 2 O water, and soaking the seeds for 24h at normal temperature and in a dark place according to the proportion of dry seed mass to seed soaking volume of 1g to 25mL by using ddH 2 O water as seed soaking liquid to obtain the soaked seeds. 20 seeds of each genotype parent and F 2:3 family seed which are well soaked are uniformly sown in a plastic flowerpot (with the inner diameter of 20cm and the height of 14 cm) filled with sterilized vermiculite, the plastic flowerpot is placed in a climatic incubator (the illumination time is 12 hours per day, the temperature is 25+/-0.5 ℃, the illumination intensity is 300 mu mol/s.m 2 and the relative humidity is 70%) and cultivated for 15 days at constant temperature, and then the corn seedlings corresponding to the three leaf period are respectively placed under low temperature stress (low temperature stress and LT) at 15+/-0.5 ℃ and normal temperature Control (CK) at 25+/-0.5 ℃ for 2 treatments to continue to cultivate for 7 days, each treatment is repeated biologically, and 30mL of Hoagland nutrient solution is poured every 2 days during the cultivation of the seedlings. All F 2:3 -localized colony family and parental genotype trefoil-heart seedlings after completion of the incubation treatments at different temperatures, the net photosynthetic rate (net photosynthetic rate, pn), stomatal conductance (stomatal conductance, gs), transpiration rate (transpiration rate, tr), intercellular CO 2 concentration (intercellular CO 2 concentration, ci) of the 3 rd leaf of 5 seedlings was determined using a us LI-6400/XT portable photosynthetic apparatus, each temperature treatment, and trefoil-heart seedling ribulose-1, 5-bisphosphate carboxylase (ribulose 1,5-biphosphate carboxylase, rubisco) activity was calculated by reference to the method of Zhao et al (2022), namely: rubisco=pn/Ci (3). Where Pn is the net photosynthetic rate and Ci is the intercellular CO 2 concentration. Photosynthetically active radiation (photosynthetic active radiation, PAR) was set at 1000 μ M m -2s-1 and CO 2 concentration was supplemented by CO 2 cylinders at the time of measurement of the photosynthetically properties. 5 photosynthetic characteristics of each F 2:3 locating population family, female parent N192, male parent Ji853 genotype seedling were measured at each temperature treatment, sorted with Excel 2016 software, and the mean of 5 photosynthetic characteristics of F 2:3 locating population at 2 temperature treatments was calculated with statistical analysis software IBM-SPSS16.0 (SPSS Inc., chicago, IL, USA) (mean,) Standard deviation (standard deviation, SD), kurtosis (kurtosis) and skewness (skewness), genotype variance (genotypic variance, V G), environment variance (environmental variance, V E), genotype-to-environment interaction variance (variance of genotype x environment interaction, V GEI), error of machinery (error of variance, vepsilon), reference to the method of zhao et al (2023) and calculate the generalized genetic transmission (broad-sense heritability, H B) and genetic coefficient of variation (genetic variation coefficient, CV g) of 5 photosynthetic characteristics of F 2:3 -localized family seedlings, namely: h B＝VG/(VG+VGEI/n+V epsilon/nr). Times.100% (4) and/>Wherein H B is generalized genetic force, V G is genotype variance, V GEI is genotype and environment interaction variance, n (n=2) is temperature treatment, r (r=5) is the number of seedlings of each F 2:3 positioning population family measured under each temperature treatment, CV g is genetic variation coefficient, SD is standard deviation,/>Is the mean value. High-level box-shaped graphs and Pearson correlation coefficient graphs of 5 photosynthetic characteristics of F 2:3 positioning population families under 2 temperature treatments are drawn by adopting GENESCLOUD on-line software. Principal component analysis (PRINCIPAL COMPONENT ANALYSIS, PCA) was performed using IBM-SPSS16.0 (SPSS inc., chicago, IL, USA) software on the 5 photosynthetic characteristics of F 2:3 -located group families, parental genotypes N192 and Ji853 at 2 temperature treatments.
7. Corn F 2:3 positioning group seedling photosynthetic property QTL positioning under different temperature treatments: combining the F 2:3 positioning group 282 family seedlings 5 photosynthetic characteristics (net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, transpiration rate, ribulose-1, 5-bisphosphate carboxylase activity) at2 temperatures (15 and 25 ℃) and the F 2 segregation group linkage map information constructed in the step 5, carrying out QTL positioning analysis on the 5 photosynthetic characteristics of the F 2:3 positioning group seedlings at a single temperature by adopting a composite interval mapping method (compositive INTERVAL MAPPING, CIM) in Windows QTL Cartographer version 2.5 software. For CIM, genotype scans were performed for 5 photosynthetic characteristics every 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 genetic effects of the 5 photosynthetic property QTLs from the absolute value of the ratio of dominant effect (dominance effect, d) to additive effect (ADDITIVE EFFECT, a), namely: the sum of D/a (0.00-0.20) is additive effect (ADDITIVE EFFECT, A), D/a (0.21-0.80) is partial dominant effect (partial-dominance effect, PD), D/a (0.81-1.20) is dominant effect (dominance effect, D), and D/a (1.20) is overdominant effect (over-dominance effect, OD).
8. Simultaneously regulating and controlling multiple photosynthetic characteristics of corn seedlings under low-temperature stress, namely 'one-factor multiple-effect' major QTL identification: under single (15 or 25 ℃) temperature treatment, the F 2:3 positioning group family seedlings in the step 7 are identified as a main effect QTL when the phenotype contribution rate (phenotypic variation explained, PVE) of the QTL for regulating and controlling the single photosynthetic property is more than or equal to 10%. When the same main effect QTL interval regulates and controls 2 or more than 2 photosynthetic characteristics at the same time, the main effect QTL is called a 'one-factor multiple-effect' main effect QTL.
9. Simultaneously regulating and controlling a plurality of photosynthetic characteristics of corn seedlings under low-temperature stress, and searching molecular marker information of a multi-effect main effect QTL: searching SSR molecular marker information on two sides of a main effect QTL of multiple cause multiple effects of a plurality of photosynthetic characteristics of corn seedlings under the low-temperature stress identified in the step 8 at a corn genome database (http:// www.maizegdb.org /) website, wherein the SSR molecular marker information comprises chromosome, cross-stack group (mail B73 Genome sequencing Project contig 2005), bin position, chromosome arm and repeated sequence information of the SSR molecular markers; the physical location of the corresponding SSR molecular markers was found in the maize B73 RefGen_v3 (https:// www.maizegdb.org/genome /) physical reference genome.
Example 3
The invention provides a result of evaluating cold-resistant genotypes of corn seedlings, which comprises the following specific results:
1. Joint variance analysis between traits of 86 maize genotype inbred seedlings under different temperature gradient treatments: 10. the joint analysis of variance (Table 1) was performed on 3 growth parameters (seedling length, seedling weight, seedling biomass), 2 photosynthetic characteristics (chlorophyll SPAD value, net photosynthetic rate), 2 membrane physiological characteristics (malondialdehyde content, membrane stability index) of 86 maize genotype inbred seedlings at 3 temperature treatments of 15 and 25 ℃. The results indicate that the 7 traits all differ significantly at P <0.01 level between genotypes, between temperatures, between genotype and temperature interactions. The growth phenotype, photosynthetic property and membrane physiological change of corn seedlings under different temperature treatments are jointly regulated and controlled by inherent genetic property, environmental temperature and interaction effect of corn genotypes.
Table 13 Joint ANOVA analysis of variance between traits of 86 maize genotype inbred seedlings at temperature (10, 15, and 25 ℃) gradients
Note that: f is the F-value for ANOVA analysis of variance, which represents that the variance was significant at P <0.01 levels.
2. Rate of change of traits measured in 86 maize genotype inbred seedlings treated at low temperature 10 and 15 ℃): as shown in FIGS. 1A and 1B, 86 parts of maize genotype inbred seedlings were reduced in seedling length, seedling weight, seedling biomass, chlorophyll SPAD value, net photosynthetic rate and film stability index, and increased in malondialdehyde content compared to the normal temperature 25℃treatment, and their Rate of Change (RC) at the low temperature 10 and 15℃treatment was expressed as malondialdehyde content > seedling length > film stability index > seedling weight > chlorophyll SPAD value > seedling biomass > net photosynthetic rate.
3. Pearson correlation analysis between traits of 86 maize genotype inbred seedlings under different temperature gradient treatments: pearson correlation analysis was performed between 7 traits of 86 maize genotype inbred seedlings at 3 temperatures (10, 15 and 25 ℃) (FIG. 2). The results show that 17 pairs of traits are obviously positively or negatively correlated (P < 0.05), which indicates that 3 growth parameters (seedling length, seedling weight and seedling biomass) of the selfing seedlings of different corn genotypes, 2 photosynthetic characteristics (chlorophyll SPAD value and net photosynthetic rate) and 2 membrane physiological characteristics (malondialdehyde content and membrane stability index) are mutually synergistic or antagonistic to each other under different temperature treatment to jointly form the cold resistance difference of the selfing seedlings of different corn genotypes.
4. Comprehensive evaluation of cold resistance of 86 maize genotype inbred lines based on inter-group cluster analysis: according to the individual character expression of 86 corn genotype inbred seedlings 7 under the temperature treatment of 10, 15 and 25 ℃, the average cold resistance coefficient of 7 characters of 86 corn genotype inbred seedlings under the low temperature stress treatment of 10 and 15 ℃ is calculated, and the cold resistance of 86 corn genotype inbred seedlings is comprehensively evaluated by using the average cold resistance coefficient of 7 characters under the low temperature stress treatment of 10 and 15 ℃ through inter-group cluster analysis (figure 3). The results show that 86 corn genotype inbred lines can be divided into 5 types when the Euclidean distance is 5, namely the type I is a cold sensitive genotype inbred line, and the corn genotype inbred line comprises 12 corn genotype inbred lines accounting for 13.9% of the tested materials; class II is a weak cold-resistant genotype inbred line, comprising 32 maize genotype inbred lines, accounting for 37.2% of the tested material; class III is a cold-resistant genotype inbred line, comprising 21 maize genotype inbred lines, accounting for 24.1% of the tested material; class IV is a moderate cold resistant genotype inbred line, comprising 19 maize genotype inbred lines, accounting for 22.1% of the tested material; class V is a strong cold resistant genotype inbred line that contains 2 maize genotype inbred lines, accounting for 2.3% of the tested material. On the basis, the subject group further selects a moderate cold-resistant genotype inbred line N192 and a cold-sensitive genotype inbred line Ji853, and the photosynthesis performance (net photosynthetic rate and chlorophyll SPAD value) of the 2 genotype inbred lines under different temperature treatments is obvious. Therefore, the subject group selects the moderate cold-resistant genotype inbred line N192 as a female parent and the cold-sensitive genotype inbred line Ji853 as a male parent to be used for subsequently positioning the main effect QTL of the photosynthetic characteristics of the corn under the low-temperature stress by constructing F 1 hybrid, F 2 segregating group and F 2:3 positioning group.
In conclusion, corn seedlings can be damaged by low-temperature cold injury when the environmental temperature is lower than 15 ℃ during the period of seed germination to seedling morphogenesis. Therefore, the research explains the response mechanism of 86 corn genotype seedlings to low-temperature cold injury from the angles of corn seedling growth phenotype, photosynthetic property, membrane physiological metabolism and the like by setting 3 temperature (10, 15 and 25 ℃) gradients, and evaluates the cold resistance performance of 86 corn genotype inbred line seedlings by adopting an inter-group cluster analysis method system according to the cold resistance coefficient of the corresponding characters. The method can objectively and accurately reflect the integral expression of cold resistance of the seedlings of the selfing lines with different corn genotypes, and the screened N192 and Ji853 corn genotypes with larger cold resistance difference provide reliable research materials for positioning main effect QTL of photosynthetic characteristics of corn under subsequent low-temperature stress.
Example 4
The invention provides a result of simultaneous regulation and control of ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate of maize seedlings under low-temperature stress, which is a result of multi-effect main effect QTL molecular marking, and specifically comprises the following steps:
1. Maize parent genotypes and F 2:3 positioning group family seedlings photosynthetic property expression under different temperature treatments: in the case of the parental genotypes N192 and Ji853 (FIG. 4A), the net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, transpiration rate, ribulose-1, 5-bisphosphate carboxylase activity exhibited significant differences between the parental genotypes N192 and Ji853 seedlings (P < 0.05) at the same temperature (15 or 25 ℃). Furthermore, the net photosynthetic rate, stomatal conductance, transpiration rate and ribulose-1, 5-bisphosphate carboxylase activity of the parent genotypes N192 and Ji853 seedlings were all significantly reduced (P < 0.05) after low temperature stress treatment at 15 ℃, which was reduced by 29.91 and 60.28%, 21.88 and 61.91%, 19.28 and 5.43%, 38.76 and 65.36%, respectively; while the intercellular CO 2 concentrations of both the parental genotypes N192 and Ji853 seedlings were significantly increased (P < 0.05), which were increased by 15.03 and 22.93%, respectively. The method shows that the parental genotype inbred lines have abundant photosynthetic property genetic variation, and compared with the parental genotype inbred line Ji853, the maternal genotype inbred line N192 can maintain higher photosynthetic property under low-temperature stress at 15 ℃. Therefore, the genetic population constructed by using the 2 parental genotype inbred lines can carry out quantitative trait genetic analysis on the photosynthetic characteristics of the corn under different temperature treatments. Similarly, 282 parts of F 2:3 -localized group family seedlings had a net photosynthetic rate, stomatal conductance, transpiration rate and ribulose-1, 5-bisphosphate carboxylase activity reduced by 21.80%, 11.18%, 134.10%, 38.74% and their seedlings had an increase in intracellular CO 2 concentration of 5.53% at 15℃low temperature stress compared to 25℃normal temperature control treatment, respectively (FIG. 4B). The 5 photosynthetic characteristics of the corn can be used as important indication characters of the cold resistance of the corn seedlings, and the detection of the main effect QTL locus with high contribution rate can lay a foundation for corn cold resistance molecular Marker Assisted Selection (MAS) breeding.
2. Normal distribution detection of photosynthetic characteristics of maize F 2:3 positioning group family seedlings under different temperature treatments, genetic variation coefficients and generalized genetic force analysis: except that the stomatal conductance (skewness =1.067) and the deflection of ribulose-1, 5-bisphosphate carboxylase activity (skewness =1.072) of 282 parts of F 2:3 -localized group family seedlings at 25 ℃ are slightly more than 1, the net photosynthetic rate, the concentration of intercellular CO 2, the kurtosis and the deflection of the transpiration rate of 282 parts of F 2:3 -localized group family seedlings at 2 temperatures (15 and 25 ℃) are all between-1.0 and 1.0 (figure 5), and the characteristic normal distribution characteristic is shown as quantitative genetic characteristics. In addition, 282 parts of F 2:3 at normal temperature treatment at 25 ℃ locate the genetic variation coefficient (CV g) of the 5 photosynthetic characteristics of the group family seedlings between 42.02 and 60.31%, CV g of the group family seedlings between 48.76 and 84.78% (figure 5) at 15 ℃ low temperature treatment, and the generalized genetic variation (H B) of the 5 photosynthetic characteristics are all larger and between 69.18 and 84.85% (figure 5). Therefore QTL localization of these 5 photosynthetic characteristics is feasible.
3. Relationship between photosynthetic characteristics of maize parent genotypes N192, ji853 and F 2:3 positioning population family seedlings under different temperature treatments: principal Component (PCA) and Pearson correlation analysis were performed on 5 photosynthetic characteristics of 2 parental genotype inbred lines N192, ji853 and F 2:3 locating group family seedlings under normal temperature at 25℃and low temperature stress treatment, respectively. From the initial characteristic values of principal component analysis and the characteristic values after factor extraction, it is known that the characteristic values of the first 2 principal components in 5 principal components under stress treatment at normal temperature of 25 ℃ and low temperature of 15 ℃ are all greater than 1, and the cumulative contribution rates reach 80.835% and 81.448%, respectively (fig. 6A). It is explained that the first 2 main components under different temperature treatments can represent most of information of photosynthetic characteristics of corn seedlings. And at these 2 temperature treatments, the primary pore conductance, transpiration rate, net photosynthetic rate and ribulose-1, 5-bisphosphate carboxylase activity of the first principal component (PC 1) are determined, while the primary intercellular CO 2 concentration of the second principal component (PC 2) is determined (FIGS. 6B and 6C). Further Pearson correlation analysis showed that there was an extremely complex linear correlation between 5 photosynthetic characteristics of maize parental genotype inbred lines N192, ji853 and F 2:3 localized population seedlings at 2 temperature treatments of 15 and 25 ℃ and 8 significant (P < 0.05) correlations between traits at all 2 temperature treatments (figures 6D and 6E). The phenomenon that linkage inheritance can exist among photosynthetic characteristics of corn is described, and detection of the 'one-cause multiple-effect' QTL has important significance for revealing genetic loci of the photosynthetic characteristics of the corn.
4. Screening a polymorphism SSR molecular marker primer between parental genotype inbred lines and constructing an F 2 separation group linkage map: the subject group designs and synthesizes 1200 pairs of SSR molecular marker primers from a corn MaizeGDB database, and uses the primers to screen polymorphic SSR molecular marker primers between a parental genotype inbred line N192 and Ji 853. And (3) screening out SSR molecular marker primer 260 pairs with clear bands and obvious polymorphism. Further utilizing the polymorphic SSR molecular marker primer pairs to carry out SSR molecular marker whole genome scanning on F 2 segregation population containing 282 single plants, and carrying out square detection on the card to propose a 25 pairs of partial segregation SSR molecular markers, so that 1 set of F 2 segregation population linkage map with the total length of 1668.0cM (shown in figure 7) is finally constructed and used for positioning the photosynthetic characteristic QTL of the maize F 2:3 positioning population family under the subsequent different temperature treatments.
5. Corn F 2:3 positioning group family seedling photosynthetic property QTL positioning under different temperature treatments: by adopting a composite interval mapping method (CIM), 6 regulating net photosynthetic rate QTLs, 3 regulating pore conductance QTLs, 4 regulating intercellular CO 2 concentration QTLs, 4 regulating transpiration rate QTLs and 4 regulating ribulose-1, 5-bisphosphate carboxylase activity QTLs (figure 8A) are detected among 5 photosynthetic characteristics of F 2:3 positioning groups under normal temperature control at 25 ℃ and low temperature stress treatment at 15 ℃, and are respectively positioned on 1, 2, 3, 4, 5, 6, 7, 8 and 10 chromosomes (figure 8C) of corn, and the phenotype contribution rate of single QTLs under 2 temperature treatments is between 4.35 and 15.17% (figure 8B).
6. Corn F 2:3 positioning group family seedlings under low temperature stress simultaneously regulate and control ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate as one-factor multiple-effect main QTL identification and SSR molecular marker information: further analysis found that the bnlg1443-umc1020 region located in the Bin 6.05 region of the corn chromosome 6 ctg404-ctg373 cascade could stably detect 1 gene-multi-effect QTL that was responsible for the ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate of the maize seedlings at 2 temperatures (15 and 25 ℃) while stably regulating and controlling the F 2:3 -located population (tables 2 and 3; the cumulative phenotypic contribution of the gene-multi-effect QTL to ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate of the maize seedlings at 2 temperature environments was 24.16%, 9.43% and 15.54%, respectively (tables 2 and 3; fig. 9A). In addition, the genetic effects of the "one-cause multiple-effect" major QTL on maize seedling ribulose-1, 5-bisphosphate carboxylase activity and transpiration rate were both shown to be dominant, the genetic effects on stomatal conductance were shown to be partially dominant, and alleles that increased ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate were all from the maternal genotype inbred line N192 (table 2).
TABLE 2 corn F 2:3 positioning population family seedlings under Low temperature stress while regulating ribulose-1, 5-bisphosphate carboxylase Activity, stomatal conductivity and transpiration Rate "one-factor multiple-effect" major QTL information
Note that: rubisco is ribulose-1, 5-bisphosphate carboxylase, tr is transpiration rate, gs is stomatal conductance, LT is low temperature stress treatment at 15 ℃, CK is normal temperature control treatment at 25 ℃, bin is the position of chromosome Bin, LOD is the log value of likelihood function ratio, and PVE is the phenotype contribution rate of QTL. Positive values of additive effect (a) indicate that the allele from male parent Ji853 is potentiated, whereas negative values of additive effect indicate that the allele from female parent N192 is potentiated.
TABLE 3 SSR molecular marker information of maize F 2:3 positioning population family seedlings under low temperature stress while regulating and controlling ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate "one-factor multiple-effect QTL
In conclusion, the research detects that 1 'one-factor multiple-effect' major QTL with high contribution rate is detected by simultaneously and stably regulating and controlling F 2:3 positioning group seedling ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate under 2 temperature (15 and 25 ℃) treatments, and the 'one-factor multiple-effect' major QTL is used for excavating major genes of regulating and controlling photosynthetic performance of corn seedlings under different temperature treatments, especially under low temperature stress, and provides reliable SSR marker information for cultivating a new excellent variety MAS of corn with cold resistance and high light efficiency, thereby having huge application potential.
Example 5
The invention provides an application of a molecular marker for simultaneously regulating and controlling the activity of ribulose-1, 5-bisphosphate carboxylase, air pore conductivity and transpiration rate of corn seedlings 'multi-effect main effect QTL' in corn light efficient utilization cold-resistant corn breeding under low-temperature stress, which comprises the following specific steps:
1. The application of the molecular marker for simultaneously regulating and controlling the activity of ribulose-1, 5-biphosphate carboxylase, air pore conductivity and transpiration rate of the main effect QTL of maize seedlings in the breeding of the cold-resistant maize by efficiently utilizing the maize light under low-temperature stress: the multi-effect main effect QTL for one factor is used for simultaneously regulating and controlling the activity of ribulose-1, 5-bisphosphate carboxylase, the air pore conductivity and the transpiration rate of the corn seedlings under the low-temperature stress, and has 1 multi-effect main effect QTL for 1 factor which is used for simultaneously and stably regulating and controlling the activity of ribulose-1, 5-bisphosphate carboxylase, the air pore conductivity and the transpiration rate of the corn seedlings under the low-temperature stress and the normal-temperature contrast treatment at the temperature of 15 ℃. The SSR molecular marker of the 'one-factor' main effect QTL consists of two pairs of SSR molecular markers (shown in figure 9B) of bnlg1443 and umc1020, wherein the sequence of the SSR molecular marker primer bnlg1443 is as follows:
Forward:5’-TACCGGAATCCTCTTTGGTG-3’；
Reverse:5’-TTTGACAACCTCTTCCAGGG-3’；
The sequence of the SSR molecular marker primer umc1020 is as follows:
Forward:5’-CCTGGAGAGCCACTACAAGGAA-3’；
Reverse:5’-TCAGCCTGAGCTCACATCATCT-3’；
From the above examples, the method for selecting the maize genotype with high photosynthetic performance and cold resistance by using the molecular marker of the multi-effect main QTL for simultaneously controlling the activity of ribulose-1, 5-bisphosphate carboxylase, stomatal conductance and transpiration rate of maize seedlings under the low temperature stress comprises the following steps: extracting genome DNA of a corn genotype to be detected; PCR amplification is carried out by using SSR molecular marker primers bnlg1443 and umc 1020; when amplification products with lengths of 183bp and 216bp are obtained, corn to be detected is a corn genotype (figure 9B) with high photosynthetic performance and cold resistance, other genotype materials are eliminated, hybridization combination is purposefully assembled, a new variety or new line with high light utilization and good cold resistance is bred, and the method is further applied to northern spring sowing corn areas or high-altitude corn areas to realize safe production of corn, so that huge economic, ecological and social benefits are generated.
Claims (4)
1. The maize multiple-effect QTL molecular marker is characterized in that the molecular marker is identified by primers bnlg1443 and umc 1020; bnlg1443 is shown in a sequence table SEQ ID NO:1 and SEQ ID NO:2, umc1020 is shown as a sequence table SEQ ID NO:3 and SEQ ID NO: 4.
2. The maize multiple-effect QTL molecular marker of claim 1, wherein said molecular marker simultaneously modulates maize seedling ribulose-1, 5-bisphosphate carboxylase activity, stomatal conductance and transpiration rate under low temperature stress.
3. The application of the maize multiple-effect QTL molecular marker in the selection of cold-resistant maize genotypes, which is characterized in that primers bnlg1443 and umc1020 are adopted for PCR amplification; when amplification products with lengths of 183bp and 216bp are respectively obtained, the corn to be detected is a corn genotype with high photosynthetic performance and cold resistance.
4. A screening method for obtaining a multi-effect main QTL molecular marker for simultaneously regulating and controlling the activity, stomatal conductance and transpiration rate of ribulose-1, 5-bisphosphate carboxylase of corn seedlings under low-temperature stress is characterized by comprising the following steps: (1) F 1 hybrid, F 2 segregation population and F 2:3 positioning population construction; (2) Extracting genomic DNA of a parental genotype inbred line and F 2 isolated population and detecting quality; (3) obtaining SSR molecular marker primers; (4) Screening a parent genotype inbred line polymorphism SSR molecular marker primer; (5) F 2 segregating population whole genome scanning and genetic linkage map drawing based on SSR molecular markers; (6) Determining photosynthetic characteristics and statistically analyzing the F 2:3 positioning group seedlings of the corn under different temperature treatments; (7) Corn F 2:3 positioning group seedling photosynthetic characteristic QTL positioning under different temperature treatments; (8) Simultaneously regulating and controlling the identification of a plurality of photosynthetic characteristics 'one-factor multiple-effect' major QTL of corn seedlings under low-temperature stress; (9) And (3) simultaneously regulating and controlling the molecular marker information retrieval of a plurality of photosynthetic characteristics 'one-factor multiple-effect' major QTL of the maize seedlings under low-temperature stress.
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