CN113957170B - Main effect QTL for regulating and controlling synergistic elongation of corn mesocotyl and coleoptile and molecular marker and application thereof - Google Patents
Main effect QTL for regulating and controlling synergistic elongation of corn mesocotyl and coleoptile 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 main effect QTL for determining the cooperative elongation of a maize mesocotyl and a coleoptile, which is one-factor multiple effect, by simultaneously regulating and controlling the length of the maize mesocotyl, the length of the coleoptile, the sum of the mesocotyl and the coleoptile, the ratio of the mesocotyl to the coleoptile and the like, and a molecular marker and application thereof. The molecular marker of the major QTL consists of three pairs of molecular marker primers, namely umc2047, umc2510 and bnlg 1597. The molecular marker disclosed by the invention is used for assisting in selecting the deep-sowing and drought-resistant corn material with long mesocotyl and coleoptile characteristics, the deep-sowing and drought-resistant performances of the corn material can be predicted by detecting the characteristic bands of the molecular marker, and the identification method is easy to operate, simple and feasible, high in selection efficiency and great in application potential in the field of deep-sowing and drought-resistant corn breeding.
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 the cooperative elongation of a corn mesocotyl and a coleoptile, namely a 'one-factor multiple-effect' main effect QTL for simultaneously regulating and controlling the length of the corn mesocotyl, the length of the coleoptile, the sum of the mesocotyl and the coleoptile and the ratio of the mesocotyl and the coleoptile, and a molecular marker and application thereof.
Background
As an ancient cultivation measure, the deep sowing of seeds plays a role in timely soil moisture deep sowing of grain crops such as corn (Zea mays L.) and wheat (Triticum aestivum L.) in stable dry areas, ensuring the safe emergence of seedlings and improving the drought resistance of the crops. In recent years, some excellent deep-sowing-resistant varieties are widely popularized and applied to drought-resistant and yield-increasing production of corn and wheat in arid regions, and play an important role in guaranteeing global grain production safety and promoting animal husbandry development. For example, the Indian maize variety "P1213733 (Komona)" which can be deeply sown by 30cm has been successfully bred and widely planted in the southwest and western dry farming areas of the United states, and can efficiently utilize deep soil moisture to ensure that maize seeds are swelled, germinated and planted, and finally exhibit good growth conditions, drought resistance and high yield (Zhao Xiaojiang and clock source, 2021; zhao et al, 2021; troyer1997). In areas with sudden lack of rainfall on loess plateau in China, after local farmers plant red mango wheat varieties through 10cm deep sowing, the varieties have excellent emergence rate in the deep sowing environment, and are still widely planted for many years (Takeda et al, 1995).
A large number of researches show that the cooperative elongation of the mesocotyl and the coleoptile is the main motive force for corn seed germination and ground breaking emergence under deep sowing stress, and the deep sowing resistance of corn is obviously determined (Zhao Xiaojiang and clock source, 2021; liu et al, 2017; zhao et al, 2021). The corn variety with longer mesocotyl and coleoptile can promote corn seeds to germinate and emerge quickly after deep sowing, has stronger breaking and emergence capability and higher seedling uniformity, and the corn root system is pricked deeper after deep sowing, so that the moisture and the nutrient of deep soil are absorbed more efficiently, and the comprehensive stress resistance of corn is improved finally. Interestingly, the elongation length (capacity) of maize mesocotyls and coleoptiles is a complex quantitative genetic trait controlled by multiple genes, and is easily affected by external environmental factors such as depth of sowing, temperature, moisture, nutrients and the like, so that research is difficult. In recent years, with rapid development of QTL (quantitative trait loci) analysis technology based on molecular marker linkage maps, the technology has been successfully applied to genetic mechanism studies of quantitative genetic traits such as maize (Zhang et al, 2012), wheat (sharp tip et al, 2021), barley (Hordeum vulgare l., takahashi et al, 2001), rice (Oryza sativa l., wang et al, 2021) crop hypocotyl and coleoptile elongation length. But the current crop has fewer QTL sites for regulating and controlling mesocotyls and coleoptiles with breeding application value. In addition, for the corn deep-sowing-resistant variety breeding, the corn deep-sowing-resistant drought-resistant variety breeding cost is high, the difficulty is high and the development is slow due to the fact that the deep-sowing-resistant material screening process is complex, time and labor are wasted, the efficiency is low, the assembly blindness of the deep-sowing-resistant variety is large, the production test period of the deep-sowing-resistant variety is long. Therefore, a QTL positioning group is constructed by using a corn inbred line with rich genetic background and large deep-sowing resistance difference (namely large difference between the mesocotyl and the coleoptile length), and a main effect QTL for stably regulating and controlling the mesoaxis length of the embryo and the coleoptile length in different deep-sowing environments is further excavated through QTL analysis, so that more valuable reference and technical support are provided for developing corn deep-sowing resistance and drought resistance molecular breeding.
Disclosure of Invention
The invention aims to provide a molecular marker for a main effect QTL for simultaneously regulating and controlling the length of a maize embryo axis, the length of a coleoptile, the sum of the mesoaxis and the coleoptile, the ratio of the mesoaxis to the coleoptile and the like in 3 deep sowing environments (3 cm, 15cm and 20 cm). The invention also provides a method for assisting in selecting the deep sowing and drought resisting corn material with long mesocotyl and coleoptile characteristics. The invention also provides application of the molecular marker of the main effect QTL for determining the cooperative extension of the mesocotyl and the coleoptile in deep sowing and drought resisting breeding of the corn by simultaneously regulating and controlling the mesocotyl length, the coleoptile length, the sum of the mesocotyl and the coleoptile and the like of the corn in the deep sowing environment.
In order to solve the technical problems, the invention adopts the following technical scheme:
1. the molecular marker of the main effect QTL for the synergistic elongation of the maize mesocotyl and the coleoptile comprises three pairs of molecular marker primers of umc2047, umc2510 and bnlg1597, wherein the sequences of the primers are as follows:
as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of the SSR molecular marker primer umc2047 is as follows:
Forward:5’-GACAGACATTCCTCGCTACCTGAT-3’；
Reverse:5’-CTGCTAGCTACCAAACATTCCGAT-3’。
as shown in SEQ ID NO:3 and SEQ ID NO:4, the sequence of the SSR molecular marker primer umc2510 is as follows:
Forward:5’-TCTGCGAATAGAACTTAAACAGGTTG-3’；
Reverse:5’-AGAGGTCCGGGAAGAACAAGTAGT-3’。
as shown in SEQ ID NO:5 and SEQ ID NO:6, the sequence of the SSR molecular marker primer bnlg1597 is as follows:
Forward:5’-GATAATCTCGTCTCGCCAGG-3’；
Reverse:5’-CATAAAAGGATGCCGACGAC-3’。
the molecular marker of the main effect QTL for the cooperative extension of the maize mesocotyl and the coleoptile can simultaneously regulate and control the length of the maize mesocotyl, the length of the coleoptile, the sum of the mesocotyl and the coleoptile and the ratio of the mesocotyl and the coleoptile in a deep sowing environment.
2. The molecular marker of the main effect QTL with the synergistic elongation of the maize mesocotyl and the coleoptile is applied to deep-sowing and drought-resistant breeding of the maize, and the deep-sowing and drought-resistant maize material with the characteristics of the long mesocotyl and the coleoptile is selected in an auxiliary way to be applied to the deep-sowing and drought-resistant breeding of the maize.
3. A method for assisting in selecting deep-sowing drought-resistant corn materials with long mesocotyl and coleoptile characteristics, comprising the following steps: extracting genome DNA of corn materials to be detected; PCR amplification was performed with the molecular marker primers umc2047, umc2510 and bnlg 1597; when amplification products with lengths of 183bp, 206bp and 147bp are obtained, the corn to be detected is deep-sowing-resistant drought-resistant corn with long mesocotyl and coleoptile characteristics.
4. A method for evaluating deep sowing resistance of corn comprises the following specific steps: (1) material collection; (2) seed disinfection and soaking; (3) vermiculite matrix preparation; (4) 3cm and 20cm seed depth test; (5) analysis of results.
5. A method for obtaining a major QTL molecular marker for regulating and controlling the cooperative elongation of maize mesocotyl and coleoptile comprises the following specific detailed steps: (1) F1 hybrid and corresponding F2 and F2:3 population construction; (2) Parent inbred line and F2 group single seedling genome DNA extraction and concentration detection; (3) designing and synthesizing SSR molecular marker primers; (4) screening a parent-parent polymorphism SSR molecular marker primer; (5) F2, separating and mapping population whole genome scanning and genetic linkage map construction; (6) Measuring deep sowing resistance of F1 hybrid seeds, F2:3 populations and parent inbred lines under the deep sowing conditions of 3cm, 15cm and 20 cm; (7) F2:3 hybrid vigor and genetic force analysis of the deep sowing resistance character of the population; and (8) carrying out QTL positioning analysis on the deep-sowing-resistant character of the F2:3 population.
The invention has the beneficial effects that: the invention constructs 1 set of F containing 346 families by taking a maize inbred line W64A with strong deep-sowing resistance and other excellent agronomic characters as a female parent and K12 with sensitive deep sowing but good general coordination force as a male parent 2:3 The population is test materials, QTL detection analysis is carried out on the test materials by adopting a composite interval mapping method (composite interval mapping, CIM) under 3 environments such as a 3cm deep sowing environment (3 cm), a 15cm deep sowing environment (15 cm) and a 20cm deep sowing environment (20 cm), and finally a 'one-factor multiple-effect' main effect QTL (major QTL, mQTL) which is named as mL-MESCOL-Ch.1-1 exists between molecular markers of a Bin 1.09 region of a first chromosome of corn under 3 deep sowing environments such as 3cm, 15cm and 20cm and is regulated and controlled simultaneously under the conditions of the length of a maize Mesocotyl (MESL), the length of a coleoptile (MESL), the sum of the mesocotyl and the coleoptile (MESL+COLL) and the ratio of the mesocotyl and the coleoptile (MESL/COLL). The mQTL-MESCOL-Ch.1-1 had a cumulative phenotype contribution to the center axis length of the embryo of 21.24%, a cumulative phenotype contribution to the center axis length of the coleoptile of 36.65%, a cumulative phenotype contribution to the sum of center axis and coleoptile of 15.17%, and a cumulative phenotype contribution to the ratio of center axis to coleoptile of 20.39%. Analysis shows that the three pairs of SSR molecular markers are utilized to carry out PCR amplification on the corn material to be detected, and the cooperative elongation capability of the hypocotyl and the coleoptile in the corn to be detected can be predicted.
The SSR molecular markers disclosed by the invention are used for carrying out molecular marker assisted selection, and the characteristic amplification strips of the corresponding SSR molecular markers are detected, so that the mesoaxis length, the coleoptile length, the sum of the mesoaxis and the coleoptile, the ratio of the mesoaxis to the coleoptile and the like of corn can be predicted to determine the size of the cooperative elongation characteristic of the mesoaxis and the coleoptile. The method can rapidly identify the deep-sowing-resistant drought-resistant corn single plants with long mesocotyl and coleoptile characteristics, eliminate other single plants, has definite selection targets, is not influenced by environment, and effectively improves the breeding utilization value of candidate corn.
Drawings
FIG. 1 is a graph showing systematic clustering evaluation of deep sowing resistance according to the emergence rate of 100 corn inbred lines after germination for 10 days under the 3cm and 20cm deep sowing environments;
wherein I is a deep-sowing sensitive type, II is a weak deep-sowing resistant type, III is a medium deep-sowing resistant type, IV is a strong deep-sowing resistant type, and finally a strong deep-sowing resistant inbred line W64A (triangle mark) and a deep-sowing sensitive inbred line K12 (circular mark) for the research are selected;
FIG. 2 shows the growth conditions of mesocotyls and coleoptile after germination of the selected strong deep-sowing-resistant inbred line W64A and the deep-sowing-sensitive inbred line K12 in a deep sowing environment of 3cm and 20cm for 10 days;
wherein the arrow represents the coleoptile section;
FIG. 3 female inbred W64A, male inbred K12 and F assembled therefrom 1 Hybrid, constructed F 2 The air temperature and precipitation meteorological data of the test points of the group in the period of growing life of the test points of the Longxi of 2020 (4 months to 9 months of 2020);
FIG. 4 shows the growth performance of a plant in the flowering period and the ear performance after harvesting of a female parent inbred line W64A and a male parent inbred line K12 planted at a test point of the Longshi in 2020;
FIG. 5 maize F 2:3 Analyzing heterosis of the population in 3cm, 15cm and 20cm deep sowing environments, namely, the length of a mesocotyl, the length of a coleoptile, the sum of mesocotyls and the coleoptile and the ratio of mesocotyls to the coleoptile;
wherein MESL is the mesoaxis length, COLL is the coleoptile length, MESL+COLL is the sum of the mesoaxis and the coleoptile, MESL/COLL is the ratio of the mesoaxis to the coleoptile, HI is F 1 Heterosis index, RH is relative heterosis, MH is mesophilia, OH is superphilia, ARR is F 2:3 Reduced rate of heterosis;
FIG. 6 maize F 1 Hybrid seeds, F 2:3 Population, female parent inbred line W64A, male parent inbred line K12, medium embryo axis length, coleoptile length, sum of medium embryo axis and coleoptile in 3cm, 15cm and 20cm deep sowing environmentPearson-related analysis of the ratio of mesocotyl to coleoptile;
wherein, MESL is mesoaxis length, COLL is coleoptile length, mesl+coll is the sum of mesoaxis and coleoptile, MESL/COLL is the ratio of mesoaxis to coleoptile, r is Pearson correlation coefficient, and P <0.01 level is significantly correlated;
FIG. 7F constructed by hybridization of W64A with K12 2 Segregating mapping population genetic linkage maps;
FIG. 8 employs Composite Interval Mapping (CIM) F 2:3 The distribution of the 'one-factor multiple-effect' major QTL (mQTL-MESCOL-Ch.1-1) on corresponding chromosomes, wherein the distribution of the 'one-factor multiple-effect' major QTL (mQTL-MESCOL-Ch.1-1) is used for regulating and controlling the length of a mesoembryo axis, the length of a coleoptile, the sum of the mesocotyl and the coleoptile and the ratio of the mesocotyl and the coleoptile, and is positioned in the 3cm, 15cm and 20cm deep sowing environments;
wherein, MESL is the center axis length (rectangle mark), COLL is the coleoptile length (ellipse mark), MESL+COLL is the sum of center axis and coleoptile (crescent mark), MESL/COLL is the center axis to coleoptile ratio (diamond mark), and Node is coleoptile Node.
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 evaluating deep sowing resistance of corn, which comprises the following specific steps:
1. and (3) material collection: 100 corn inbred line seeds with larger genetic background difference of seed reproduction and harvest under the same-year same-place same-field management condition of the subject group are selected as test materials.
2. Seed disinfection and soaking: 180 seeds with uniform and consistent seed grains are selected from the 100 parts of corn inbred line seeds to weigh, and then the weight of the seeds is as follows: the seeds were sterilized by shaking continuously for 10min with a 0.5% volume NaClO solution at a ratio of 1g:10mL, followed by 250mL ddH 2 Washing the seeds for 3 times by shaking continuously with O water, and finally placing the sterilized seeds in an indoor environment at 20+/-1 ℃ according to the weight of the seeds: ddH 2 Soaking seeds in the proportion of 1g to 8mL of O water for 24 hours in a dark place to obtain soaked seeds for later use.
3. Preparing a vermiculite matrix: ddH 2 O water: the sterilized vermiculite is prepared into a vermiculite matrix by uniformly stirring according to the proportion of 5g to 1mL for later use.
4.3 cm and 20cm seed depth test: selecting a seed deep-sowing test device (authorized bulletin number: CN 209768182U) with an inner diameter of 17cm and a height of 50cm as test equipment, layering and loading vermiculite matrixes into the seed deep-sowing test device, uniformly sowing 30 grains of the soaked seeds on the surface of the vermiculite matrixes, covering the surface of the seeds with the vermiculite matrixes of 3cm and 20cm respectively, and then filling the vermiculite matrixes just with the seed deep-sowing test device, so as to obtain 2 kinds of deep-sowing test treatment, namely 3cm deep-sowing (normal deep-sowing; CK) and 20cm deep-sowing (deep-sowing stress; 20 cm) treatment, wherein each treatment is biologically repeated for 3 times. The seed deep-sowing test device under the corresponding treatment is then displaced into an intelligent artificial climate chamber (12 h illumination, 600 mu mol/(s m) 2 ) Constant temperature of 22+ -1deg.C, 65% relative humidity), and supplementing ddH to each seed deep-seeding test device every 2d 2 O water 20mL. After 100 corn inbred line seeds germinate for 10 days under 2 sowing depth treatments, the emergence rate (RAT; RAT=10 d emergence seed number/total sowing seed number×100%) of the corresponding corn inbred line under each sowing depth treatment is counted, and vermiculite matrix at the root of the seedling is quickly washed out with clear water for photographing.
5. Analysis of results: the emergence rate of each maize inbred line under 2 deep sowing treatments was systematically cluster-analyzed using IBM SPSS 19.0 (SPSS inc., chicago, IL, USA) software, and the deep sowing resistance of 100 maize inbred lines was comprehensively evaluated.
Example 2
The invention provides a method for obtaining a major QTL molecular marker for regulating and controlling the cooperative elongation of corn mesocotyl and coleoptile, which comprises the following specific steps:
1.F 1 hybrid and corresponding F 2 And F 2:3 Group construction: the strong deep-sowing-resistant corn inbred line W64A is used as a female parent, the deep-sowing-sensitive corn inbred line K12 is used as a male parent for field planting, and the F is obtained by strictly bagging, pollinating and hybridizing in the flowering phase 1 Hybrid (abbreviated as H-w×k). Continuing to plant F in the field in the next year 1 Hybrid seeds, in-flowering-period strict bagging pollination selfing and harvesting 1F containing 346 seeds 2 The mapping population (abbreviated as F2-W x K) was isolated. F containing 346 grains continued for 4 months in 2020 2 The mapping segregating population seeds and W64A and K12 inbred line seeds were sown at the Sichuan village corn test points (34.97 DEG N;104.40 DEG E; altitude 2074 m) in Gansu Long-Sangxi according to a planting density of 4500 plants/mu. The film is uniformly covered (film length is 120cm, film thickness is 0.02 mm) before sowing, and other management measures are the same as those of the local general field management. Three leaves of seedling are cut off in one heart period 2 Leaves of single plants, W64A and K12 seedlings were quick frozen with liquid nitrogen at-80℃for later use. Red hanging tag F 2 The order of the single plants. Before-flowers each F 2 The single plant is strictly bagged and pollinated to ensure that the single plant is strictly selfed, and each F is counted after flowers 2 Agronomic traits of individual plants include Plant Height (PH), ear Height (EH), stem thickness (stem diameter). Harvesting at 9 months to obtain F 2:3 Colony families (abbreviated as P-W.times.K) clusters were allowed to air dry and examined naturally and hundred grain weights (KW) were determined.
2. Parental inbred line and F 2 Group individual seedling genome DNA extraction and concentration detection: extracting the female parent inbred line W64A, the male parent inbred line K12 and each F in the step 1 by adopting a CTAB (hexadecyl trimethyl ammonium bromide) method 2 Genomic DNA of individual seedlings was detected by 1% agarose gel electrophoresis for DNA quality, and NanoDrop was used TM The DNA concentration is detected by an One/OneC ultra-micro ultraviolet spectrophotometer, and the corresponding DNA concentration is diluted to 50 ng/mu L for standby.
SSR molecular marker primer design and synthesis: from the maize genome database MaizeGDB website (http:// www.maizegdb.org /), 1000 pairs of SSR molecular markers uniformly distributed on 10 chromosomes of maize were selected, synthesized by Sangon in Shanghai, the SSR primers were synthesized in a purification manner of HAP, and the concentration of each Primer consisting of Primer 1 and Primer2 was diluted to 1mmol/L for use.
4. Screening parent-parent polymorphism SSR molecular marker primers: and (3) taking the genomic DNA of W64A and K12 extracted in the step (2) as a template, designing a synthesized SSR molecular marker as a primer, and carrying out PCR amplification by a Biometra-T1 PCR instrument. The PCR amplification used a reaction system of 20. Mu.L: 1.4. Mu.L of DNA (50 ng/. Mu.L), 0.6. Mu.L of Primer 1 (1 mmol/L), 0.6. Mu.L of Primer2 (1 mmol/L), 2X Power Taq PCR Master Mix 10.0.10.0. Mu.L, UPH 2 O7.4. Mu.L. The PCR reaction procedure was: pre-denaturation at 95℃5.0min,1 cycle; denaturation at 94℃for 0.5min, annealing at 50.0-60.3℃for 0.5min and extension at 72℃for 0.5min for 36 cycles; finally, the extension is carried out for 10min at 72 ℃, and the preservation is finished for 60min at 4 ℃. Performing gel running and silver dyeing on the PCR amplification product by 8% non-denaturing polyacrylamide gel electrophoresis on the amplification products of the W64A and K12 genome DNA, and screening SSR markers of polymorphism between the parent W64A and K12 to prepare F 2 And (5) carrying out whole genome scanning and genetic linkage map construction on the segregation mapping population.
5.F 2 And (3) performing whole genome scanning and genetic linkage map construction on the segregation mapping population: f obtained by extraction in the step 2 2 Isolating genome DNA of mapping population, parent W64A and K12 seedling, carrying out whole genome scanning by using polymorphism SSR marker between two parents according to the method of the step 4, and analyzing F 2 Genotype banding of the population at each SSR site: the band derived from the female parent W64A is denoted as "A", the band of the male parent K12 is denoted as "B", the heterozygous is denoted as "H", and the delegation is denoted as "-". The distribution of each SSR-tagged band in the population was subjected to a card square test, whose significance was checked and obtained to match the female parent: heterozygous: SS with 1:2:1 segregation ratio for male parentR marks information and constructs F using JoinMap4.0 software (http:// www.kyazma.nl/index. Php/mc. JoinMap/sc. Evaluation /) 2 The genetic linkage map of the segregating population is calculated by using a Kosambi function. F of construction 2 Genetic linkage map of segregating populations for subsequent F 2:3 QTL localization analysis of the population families.
F under 6.3 cm, 15cm and 20cm deep sowing 1 Hybrid seeds, F 2:3 And (3) determining deep sowing resistance of the population and parent inbred line: each F harvested in the above step 1 2:3 Group family, F 1 The hybrid and the seeds of the parent W64A and K12 were each 270-seed weighed, and then according to the seed weight: the seeds were sterilized by shaking continuously for 10min with a 0.5% volume NaClO solution at a ratio of 1g:10mL, followed by 375mL ddH 2 Washing the seeds for 3 times by shaking continuously with O water, and finally placing the sterilized seeds in an indoor environment at 20+/-1 ℃ according to the weight of the seeds: ddH 2 Soaking seeds in the dark for 24 hours according to the proportion of 1g to 8mL of O water to obtain soaked seeds; pressing ddH before sowing 2 O water: uniformly stirring sterilized vermiculite at a ratio of 5g to 1mL to prepare a vermiculite matrix; selecting a seed deep-sowing test device (authorized bulletin number: CN 209768182U) with an inner diameter of 17cm and a height of 50cm as test equipment, layering and loading vermiculite matrixes into the seed deep-sowing test device, uniformly sowing 30 grains of the seed after seed soaking on the surface of the vermiculite matrixes, and covering the surface of the seed with the vermiculite matrixes of 3cm, 15cm and 20cm respectively, wherein the vermiculite matrixes are just filled with the seed deep-sowing test device, so that 3 seed deep-sowing test treatments, namely 3cm deep-sowing (normal deep-sowing; CK), 15cm deep-sowing (deep-sowing stress of 1;15 cm) and 20cm deep-sowing (deep-sowing stress of 2;20 cm) are obtained, and each treatment is biologically repeated for 3 times. The seed deep-sowing test device under the corresponding treatment is then displaced into an intelligent artificial climate chamber (12 h illumination, 600 mu mol/(s m) 2 ) Constant temperature of 22+ -1deg.C, 65% relative humidity), and supplementing ddH to each seed deep-seeding test device every 2d 2 O water 20mL. To treat each F under 3 kinds of deep sowing 2:3 Group family, F 1 After the seeds of the hybrid seeds and W64A, K are germinated for 10 days, the vermiculite matrix at the root of the seedling is quickly washed off by clean water, and the seedling 1 with the integral consistent growth vigor is selectedStrain 0 was determined in which the embryo axis length (MESL; MESL is the distance from the seed to the coleoptile node), the coleoptile length (coleoptile length, COLL; COLL is the distance between the coleoptile node and the top of the coleoptile), and the sum of the embryo axis and the coleoptile (mesocotyl and coleoptile total lengty, mesl+coll), and the ratio of mesoaxis to coleoptile (ratio of mesocotyl to coleoptile, MESL/COLL) were calculated.
7.F 2:3 Analyzing heterosis and genetic power of the deep sowing resistance character of the population: f under the single deep-cast environment measured in the above step 6 using the GLM (general linear model for univariate) model in IBM-SPSS 19.0 (SPSS Inc., chicago, IL, USA) software (https:// www.ibm.com/products/SPSS-statics) 2:3 Colony, F 1 The 4 deep-cast-resistant traits of 2 parental inbred lines such as the hybrid and W64A and K12 were analyzed by variance, and kurtosis (kurtosis) and skewness (skewness) were calculated. F is calculated according to the following formula 2 : 3 Generalized genetic transmission (h) of 4 deep-sowing-resistant characters of population 2 ) And generalized genetic forces of interaction
Namely: />
And->
Wherein: />
For genotype variance, ++>
For genotype-environment interaction variance, +.>
For the ambient variance>
N (n=3) is the number of depth,r (r=10) is a repetition number. Hybrid vigor of 4 deep-sowing-resistant traits is calculated according to the following formula: f (F) 1 Heterosis index (F) 1 heterosis index,HI；HI＝F 1 /MP x 100%), relative heterosis (relative heterosis, RH; RH= (F) 1 -MP)/F 1 X 100%), medium dominant (mid-parent split, MH; mh= (F) 1 -MP)/mp×100%, super-parent dominance (OH; oh= (F) 1 -P H )/P H ×100％)，F 2:3 Dominance decrease rate (F) 2:3 advantage reduction rate,ARR；ARR＝(F 2:3 -F 1 )/F 1 X 100%). Wherein MP is the average value of parents, P H Is a high-value parent.
8.F 2:3 QTL positioning analysis of the deep-sowing-resistant character of the population: f under 3 kinds of deep sowing environments in combination with step 6 2:3 4 deep-sowing-resistant traits of 346 families of the population and F constructed in step 5 2 Population genetic linkage map information, F in a single deep-sowing environment was mapped by a composite interval mapping method (compositive interval mapping, CIM) in Windows QTL Cartographer version 2.5.5 software (http:// statgen. Ncsu. Edu/qtgcart/WQTLcarbot. Htm) 2:3 QTL positioning was performed for 4 deep-sowing-resistant traits of the population. For CIM, genotype scans were performed for 4 deep-broadcast-tolerant traits every 0.5cM using the Zmapqtl program module Model 6, window size 10.0cM, and LOD threshold (LOD) was determined by 1000 samples>3.0). Estimating QTL genetic effects from the absolute value of the ratio of dominant effect (D) to additive effect (a): the |d/a|=0.00-0.20 is additive (a), the |d/a|=0.21-0.80 is Partially Dominant (PD), the |d/a|=0.81-1.20 is dominant (D), and the |d/a|is dominant (D)>1.20 is super dominant (OD). And generating SSR molecular marker sequence tables (shown as sequence tables) at two ends of the main effect QTL by using patent version 3.5 software.
Example 3
The invention provides an evaluation result of a corn deep sowing resistance evaluation method, which comprises the following specific results:
1.3cm and 20cm deep sowing environment, 100 parts of maize inbred line are subjected to seedling rate analysis: the emergence rate of 100 parts of maize inbred line decreased significantly as the depth of sowing increased from 3cm to 20cm (Table 1). The method shows that the sowing depth obviously influences the top soil emergence condition of the corn seeds, and the emergence rate of the corn seeds can be used as a reliable index for evaluating the deep sowing resistance of the corn. Overall, the coefficient of variation between 100 maize inbred seedlings was 19.08% at 3cm depth of sowing, while the coefficient of variation between 100 maize inbred seedlings was 118.08% at 20cm depth of sowing (table 1). Suggesting that the deep-sowing resistance is different among different corn inbred lines. Therefore, the emergence rates of different corn inbred lines are obviously different in different sowing depth environments.
TABLE 1 statistical analysis of the emergence rate after 10d germination of 100 maize inbred lines under 3cm and 20cm depth-of-field treatments
2. Systematic clustering evaluation of emergence rates of 100 corn inbred lines in combination with 3cm and 20cm deep sowing environments: further, the deep sowing resistance of 100 parts of corn inbred line is objectively reflected. Thus, the study conducted systematic cluster evaluation analysis (FIG. 1) of the emergence rate of 100 maize inbred lines in combination with 3cm and 20cm deep-sowing environments. The results show that these 100 parts of maize inbred line are divided into 4 types, of which type I is a deep-sowing sensitive type, comprising 50 parts of maize inbred line, accounting for 50.0% of the test maize inbred line; type II is a weak, deep-sowing-tolerant type comprising 30 parts of maize inbred line, accounting for 30.0% of the test maize inbred line; type III is a medium deep-sowing-resistant type comprising 5 parts of maize inbred, 5.0% of the test maize inbred, and type IV is a strong deep-sowing-resistant type comprising 15 parts of maize inbred, 15.0% of the test maize inbred. And 1 part of strong deep-sowing-resistant inbred line W64A and 1 part of deep-sowing-sensitive inbred line K12 are obtained by screening. In comparison to the 3cm deep-sowing control, the strong deep-sowing resistant inbred line W64A and the deep-sowing sensitive inbred line K12 were significantly elongated at 20cm deep, with both the hypocotyl and the coleoptile being significantly longer than K12 (fig. 2). Therefore, the 2 corn inbred lines with large deep-sowing resistance difference can be used as F for subsequent deep-sowing resistance trait QTL detection 1 Hybrid seeds, F 2 And F 2:3 Reliable parent material for population construction.
In summary, 2 typical sowing depths such as normal sowing depth of 3cm of conventional corn and sowing depth of 20cm of deep sowing resistant corn are selected for test treatment, the emergence rate of corn seed vitality, emergence speed and seed soil-lifting capacity are objectively reflected as deep sowing resistant evaluation indexes, and F for constructing a corn deep sowing resistant character QTL positioning research is scientifically and accurately screened by intuitively and rapidly counting the emergence rate of 100 corn inbred lines under the 2 sowing depth conditions and further performing systematic clustering evaluation 1 Hybrid seeds, F 2 And F 2:3 Parental inbred lines W64A and K12 of the population, F constructed using the same 1 Hybrid seeds, F 2 And F 2:3 The colony can lay a foundation for QTL positioning analysis of the corn deep-sowing-resistant character under different deep-sowing environments.
Example 4
The invention provides a result of a main effect QTL molecular marker for regulating and controlling the cooperative extension of corn mesocotyl and coleoptile, which comprises the following specific results:
1. parent in field, F 1 Hybrid seeds and F 2 Meteorological data for test points during the growth period of the population planting: parental inbred lines W64A and K12, F assembled by using the same 1 Hybrid and F constructed by selfing 2 The colony is planted in the corn test points in Gansu Long-Xuancun in the middle of 4 months to 9 months in the last ten days of 2020. The average temperature of the test point in the growth period of the corn is 16.3 ℃, the highest temperature is 24.4 ℃, the lowest temperature is 1.0 ℃, and the total precipitation is 414.1mm (figure 3), thereby meeting the temperature and moisture requirements required by the normal growth and development of the corn.
2.F 1 Hybrid seeds, F 2 Representation of agronomic and grain traits in the population and parent fields: the field measurement of the plant height, the spike height, the stem thickness, the hundred grain weight and other characters of 2 parental inbred lines shows that the 2 inbred lines have large differences among the characters and rich genetic background, and F is assembled by the 2 inbred lines 1 The hybrid can show strong hybrid vigor, F 2 The variation types between the populations were numerous (Table 2 and FIG. 4). Thus, F constructed with these 2 inbred lines with large genetic differences 2:3 The group can be rich in variation types among different characters, especially in deep-sowing-resistant character aspectOverdetermined F 2:3 The variety types of the deep-sowing-resistant traits of the population in different deep-sowing environments can truly locate the QTL locus of the deep-sowing-resistant traits of the corn.
Table 2 maize parent and F of its combination 1 Hybrid seeds, F 2 Agronomic and seed performance of populations
Note that: different lowercase letters represent significant differences between parental inbred lines at P <0.05 levels.
F under 3.3 cm, 15cm and 20cm deep sowing environment 2:3 Identification of deep sowing resistance of the population: the 2 parental inbred lines showed significant differences in 4 deep-cast resistance traits such as mesoaxis length, coleoptile length, mesoaxis-to-coleoptile sum, mesoaxis-to-coleoptile ratio, etc. in the 3, 15 and 20cm deep-cast environments (table 3), indicating the 2 inbred lines were different in deep-cast resistance. When the depth of the sowing is increased to 15cm and 20cm compared with the normal depth of the sowing of 3cm, the 2 parts of parent inbred lines W64A, K12 and F 1 Hybrid seeds and F 2:3 The average mesoaxis lengths of the populations were elongated by 169.1 and 232.5%, 41.4 and 124.1%, 233.8 and 293.8%, 192.8 and 250.0%, respectively, the average coleoptile lengths were elongated by 30.9 and 55.1%, 110.9 and 120.4%, 73.4 and 93.7%, 71.3 and 113.0%, respectively, the sum of the average mesoaxis and coleoptile was elongated by 106.8 and 152.4%, 80.4 and 122.1%, 161.7 and 204.0%, 151.5 and 225.3%, respectively, and the average mesoaxis to coleoptile ratios were increased by 108.2 and 114.8%, respectively, -32.9 and 1.3%, 92.6 and 103.3%, 11.3 and 32.3%, respectively (table 3). The deep sowing can obviously influence the development of the maize mesocotyl and coleoptile, and further promote the deep sowing of seedlings of the maize, so that the characters can be used as evaluation indexes of the deep sowing resistance of the maize. In addition, F in the 3 kinds of deep-sowing environments 2:3 The kurtosis and skewness of the 4 deep-sowing-resistant characters of the population are basically between-1.0 and 1.0(Table 3), is typical of quantitative genetic characteristics, and is suitable for QTL analysis.
Table 3 maize parent and F of its combination 1 Hybrid seeds, F 2:3 Determination of 4 deep-sowing-resistant traits of population
Note that: MESL: center axis length, COLL: coleoptile length, mesl+coll: the sum of the mesocotyl and coleoptile, MESL/COLL: the ratio of mesocotyl to coleoptile, CV: coefficient of variation.
4. Heterosis analysis of 4 deep-sowing-resistant traits of corn: further, under the conditions of 3cm, 15cm and 20cm depth, the method comprises the steps of selfing 2 parts of parents into a line F 1 Hybrid seeds and F 2:3 Heterosis analysis of 4 deep-sowing-resistant traits of the population shows that F of the 4 traits 1 The heterosis index is 106.0 percent (coleoptile length) to 161.4 percent (mesocotyl length), the relative heterosis is 5.7 percent to 36.2 percent (mesocotyl length), the mesophilic dominance is 6.0 percent (coleoptile length) to 61.4 percent (mesocotyl length), the sum of the mesocotyl length, the mesocotyl and the coleoptile is 15.1 percent and 16.3 percent respectively, the ratio of the coleoptile length, the mesocotyl and the coleoptile is minus super-philic dominance is minus 5.5 percent and minus 4.3 percent respectively, and F is the sum of the mesocotyl length, the mesocotyl and the coleoptile is plus super-philic dominance of minus 5.5 percent and minus 4.3 percent respectively 2:3 The dominance reduction rates are reduced to different degrees, the length of the mesocotyl, the length of the coleoptile, the sum of the mesocotyl and the coleoptile and the ratio F of the mesocotyl and the coleoptile 2:3 The dominant reduction was 10.4%, 4.6%, 11.5% and 24.5%, respectively (FIG. 5). The heterosis size of these 4 deep-sowing-resistant traits of maize is different, wherein F is the sum of mesocotyl length, mesocotyl and coleoptile 1 The heterosis index, relative heterosis, super-and mesophilic dominance are all stronger, and the central embryo axis length and the hybrid vigor performance of the sum of the central embryo axis and coleoptile should be considered in breeding.
5. Analysis of variance and genetic force detection of 4 deep-sowing-resistant traits of corn: further, F is carried out under 3 kinds of deep sowing environments 2:3 The 4 deep-sowing-resistant character combined analysis of variance of the population shows that F is constructed 2:3 The 4 deep-sowing-resistant characters of the population reach P among genotypes, environments and genotype and environment interactions<The 0.01 level difference was significant (table 4). The 4 deep-sowing-resistant traits of the corn are obviously influenced by deep sowing environment and interaction effect of the deep sowing environment and the deep sowing environment besides being obviously controlled by self inheritance. The generalized genetic transmission of the 4 characters is higher and is between 80.88 and 91.55 percent, and the generalized genetic transmission of the genotype and environment interaction is lower and is between 8.32 and 18.09 percent (Table 4). Thus QTL localization for these 4 deep-sowing-resistant traits of maize is feasible.
TABLE 4F under 3cm, 15cm and 20cm depth of sowing 2:3 Analysis of variance and genetic force detection of 4 deep-sowing-resistant characters of population
Note that: * Indicating significant differences in P <0.01 levels.
6. Pearson correlation analysis of 4 deep-sowing-resistant traits of corn: further, the parental inbred lines W64A, K12 and F in 3 deep sowing environments 1 Hybrid seeds, F 2:3 Pearson correlation analysis of the 4 deep-cast resistance traits of the population shows that the mesoaxis length and the coleoptile length, the sum of the mesoaxis and the coleoptile and the ratio of the mesoaxis and the coleoptile are all obviously and positively correlated, the correlation coefficient (r) is respectively 0.746, 0.981 and 0.825, the correlation coefficient is obviously and positively correlated, the correlation coefficient is 0.857, the correlation coefficient is obviously and positively correlated, and the correlation coefficient is 0.733 (figure 6). The mesocotyl and coleoptile of the corn are developed cooperatively, and corn seedlings are finally sent out of the ground surface through the cooperative promotion development under the deep sowing environment, so that the purpose of sprouting and emergence of the seedlings is achieved.
7. Parental polymorphism SSR marker screening and F 2 Constructing a genetic linkage map of the isolated population: 1000 pairs of SSR primers are designed and synthesized in a corn MaizeGDB database, and are utilized to carry out polymorphic SSR primer screening between parent inbred lines W64A and K12, and finally SSR primer 254 pairs with obvious polymorphism are screened out, wherein the screening rate is that25.4%. Further incorporating these polymorphic SSR primer pairs with F of 346 individuals 2 And (5) carrying out whole genome scanning and genetic linkage map construction on the population. The square test of the card shows that 13 pairs of SSR primers are partially separated, the frequency is 5.12 percent, the residual 241 pairs of SSR primers accord with the separation ratio of 1:2:1, and a genetic linkage map containing 235 pairs of SSR markers and covering 10 chromosomes of corn is constructed (figure 7). The genetic linkage map was constructed to have a full length of 1667.0cM and an average spacing between markers of 7.09cM (FIG. 7). Compared with the reference map IBM2 2008Neighbors (http:// www.maizegdb.org), most of the marker Bin positions and sequences are highly consistent with the reference map. Description of constructed F 2 The genetic linkage map of the segregating population is reliable and can be used for subsequent QTL positioning research.
8.1 positioning of major QTL (mQTL-MESCOL-Ch.1-1) regulating the synergistic elongation of maize mesocotyl and coleoptile: f by QTL positioning analysis 2:3 The colony finally locates 1 main effect QTL of 'one factor multiple effect' for regulating and controlling the length of embryo axis, the length of coleoptile, the sum of mesocotyl and coleoptile and the ratio of mesocotyl and coleoptile in corn under 3 kinds of deep sowing environments of 3cm, 15cm, 20cm and the like, and the main effect QTL is named mQTL-MESCOL-Ch.1-1. The major mQTL-MESCOL-Ch.1-1 is located at Bin 1.09 of the first chromosome and is between SSR molecular markers umc2047-umc2510-umc 2012. The main effect mQTL-MESCOL-Ch.1-1 shows additive effects on the genetic effects of the center axis length, the coleoptile length, the sum of the center axis and the coleoptile, and the ratio of the center axis to the coleoptile, and alleles for increasing the center axis length, the coleoptile length, the sum of the center axis and the coleoptile, and the ratio of the center axis and the coleoptile are all from a strong deep-cast female parent inbred line W64A. The cumulative phenotype contribution rate of the major mQTL-MESCOL-Ch.1-1 to the center axis length is 21.24%, the cumulative phenotype contribution rate to the coleoptile length is 36.65%, the cumulative phenotype contribution rate of the sum of the center axis and the coleoptile is 15.17%, the cumulative phenotype contribution rate of the ratio of the center axis to the coleoptile is 20.39%, and the phenotype contribution rate of the major mQTL-MESCOL-Ch.1-1 in the 15cm and 20cm deep sowing environment is larger (Table 5 and FIG. 8). Therefore, the main effect mQTL-MESCOL-Ch.1-1 can be used for deep-sowing and drought-resistant corn materials with long mesocotyl and coleoptile characteristicsAnd the genetic effect of the main effect mQTL-MESCOL-Ch.1-1 is an additive effect, so that the accumulated effect of the mesocotyl and the coleoptile is fully considered when the deep-sowing and drought-resistant corn varieties with the characteristics of the long mesocotyl and the coleoptile are assembled, particularly the influence of the genetic effect of the female parent is fully considered, and further, a new excellent corn variety with strong deep-sowing resistance and drought resistance is obtained.
TABLE 5F under 3cm, 15cm and 20cm depth of sowing 2:3 Positioning information of major QTL (mQMESCOL-Ch.1-1) for synergistic elongation of hypocotyl and coleoptile in population
Note that: MESL: center axis length, COLL: coleoptile length, mesl+coll: the sum of the mesocotyl and coleoptile, MESL/COLL: ratio of mesocotyl to coleoptile, a: additive effects.
Example 5
The invention provides an application method of a molecular marker for regulating and controlling a main effect QTL of corn mesocotyl and coleoptile synergistic elongation in corn molecular marker assisted selection breeding, which comprises the following specific steps:
1. the application method of the molecular marker for regulating and controlling the main effect QTL of the synergistic elongation of the hypocotyl and the coleoptile in the deep-sowing-resistant drought-resistant molecular marker assisted selective breeding of the corn comprises the following steps: the main effect QTL (mQTL-MESCOL-Ch.1-1) for regulating the cooperative extension of the maize mesocotyl and the coleoptile is specifically 1 'one-factor multiple-effect' main effect QTL for simultaneously controlling the length of the maize mesocotyl, the length of the coleoptile, the sum of the mesocotyl and the coleoptile, the ratio of the mesocotyl and the coleoptile and the like to determine the cooperative extension of the maize mesocotyl and the coleoptile. The SSR molecular marker of the main effect QTL consists of three pairs of SSR molecular markers of umc2047, umc2510 and bnlg1597, wherein the sequence of an SSR molecular marker primer umc2047 is as follows:
Forward:5’-GACAGACATTCCTCGCTACCTGAT-3’；
Reverse:5’-CTGCTAGCTACCAAACATTCCGAT-3’；
the sequence of the SSR molecular marker primer umc2510 is as follows:
Forward:5’-TCTGCGAATAGAACTTAAACAGGTTG-3’；
Reverse:5’-AGAGGTCCGGGAAGAACAAGTAGT-3’；
the sequence of the SSR molecular marker primer bnlg1597 is as follows:
Forward:5’-GATAATCTCGTCTCGCCAGG-3’；
Reverse:5’-CATAAAAGGATGCCGACGAC-3’。
as can be seen from the above embodiments, the method for selecting the deep-sowing and drought-resistant corn material with the characteristics of long mesocotyl and coleoptile by using the SSR molecular markers for simultaneously controlling the mesocotyl length, the coleoptile length, the sum of the mesocotyl and the coleoptile, the ratio of the mesocotyl to the coleoptile and the like of the corn to determine the main QTL for the cooperative elongation of the mesocotyl and the coleoptile comprises the following steps: extracting genome DNA of corn to be detected, and carrying out PCR amplification by using SSR molecular marker primers umc2047, umc2510 and bnlg 1597; when amplification products with lengths of 183bp, 206bp and 147bp are obtained, the corn to be detected is a candidate deep-sowing and drought-resistant corn single plant with long mesocotyl and coleoptile characteristics, other single plants are eliminated, hybridization combination is purposefully assembled, and a new excellent variety of the deep-sowing and drought-resistant corn is bred, so that the method has great application potential when applied to drought-resistant safety production of corn in drought areas.
<110> Gansu agricultural university
<120> a major QTL for regulating and controlling synergistic elongation of maize mesocotyl and coleoptile, and molecular marker and application thereof
<160>6
<210>1
<211>24
<212>DNA
<213> corn (Zea mays L.)
<400>1
gacagacatt cctcgctacc tgat 24
<210>2
<211>24
<212>DNA
<213> corn (Zea mays L.)
<400>2
ctgctagcta ccaaacattc cgat 24
<210>3
<211>26
<212>DNA
<213> corn (Zea mays L.)
<400>3
tctgcgaata gaacttaaac aggttg 26
<210>4
<211>24
<212>DNA
<213> corn (Zea mays L.)
<400>4
afaggtccgg gaagaacaag tagt 24
<210>5
<211>20
<212>DNA
<213> corn (Zea mays L.)
<400>5
<210>6
<211>20
<212>DNA
<213> corn (Zea mays L.)
<400>6
Claims (2)
1. The application of the molecular marker of the main effect QTL with the synergistic elongation of the maize mesocotyl and the coleoptile in the deep-sowing and drought-resistant breeding of the maize is characterized in that the molecular marker is used for assisting in selecting the deep-sowing and drought-resistant maize material with the characteristics of the long mesocotyl and the coleoptile to be applied to the deep-sowing and drought-resistant breeding of the maize; the molecular marker consists of three pairs of molecular markers of umc2047, umc2510 and bnlg1597, and the primer sequence of the molecular marker umc2047 is shown as SEQ ID NO:1 and SEQ ID NO:2, the primer sequence of the molecular marker umc2510 is shown in a sequence table SEQ ID NO:3 and SEQ ID NO:4, the primer sequence of the molecular marker bnlg1597 is shown as a sequence table SEQ ID NO:5 and SEQ ID NO: shown at 6.
2. A method for assisting in selecting deep-sowing drought-resistant corn material with long mesocotyl and coleoptile characteristics, which is characterized by comprising the following steps: extracting genome DNA of corn materials to be detected; PCR amplification was performed using primer sequences of molecular markers umc2047, umc2510 and bnlg 1597; when amplification products with lengths of 183bp, 206bp and 147bp are obtained, the corn to be detected is deep-sowing-resistant drought-resistant corn with long mesocotyl and coleoptile characteristics; the primer sequence of the molecular marker umc2047 is shown in a sequence table SEQ ID NO:1 and SEQ ID NO:2, the primer sequence of the molecular marker umc2510 is shown in a sequence table SEQ ID NO:3 and SEQ ID NO:4, the primer sequence of the molecular marker bnlg1597 is shown as a sequence table SEQ ID NO:5 and SEQ ID NO: shown at 6.
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| CN112997879A (en) * | 2021-03-22 | 2021-06-22 | 甘肃农业大学 | Method for evaluating maximum elongation characteristics of hypocotyl and coleoptile of corn |
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| CN111247901A (en) * | 2020-02-14 | 2020-06-09 | 甘肃农业大学 | Method for evaluating deep sowing resistance of corn |
| CN112997879A (en) * | 2021-03-22 | 2021-06-22 | 甘肃农业大学 | Method for evaluating maximum elongation characteristics of hypocotyl and coleoptile of corn |
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