CN110692512A - Method for rapidly predicting heterosis based on crop genome size - Google Patents
Method for rapidly predicting heterosis based on crop genome size Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
The invention discloses a method for rapidly predicting heterosis based on genome size, belonging to the technical field of crop breeding and comprising the following steps: s1, selecting a hybridization combination parent according to the relative difference percentage of genome sizes among crops and the Nei genetic distance; s2, emasculating and hybridizing the selected hybridization combination parent to obtain F1Generation; s3, for F1Carrying out heterosis analysis on generation target characters; s4. analysis F1Correlation of heterosis of generation target characters and Nei genetic distance between parents; s5. analysis F1Correlation of heterosis of generation target characters and absolute value of genome size difference between parents; s6, combining the analysis results, selecting and matching strong dominant hybridization combinations, and predicting F1Generation hybrid vigor. The breeding method provided by the invention is beneficial to improving the breeding efficiency and reducing the breeding cost, and has the advantage of wide applicability.
Description
Technical Field
The invention belongs to the technical field of crop breeding, and relates to a method for rapidly predicting heterosis based on crop genome size.
Background
Heterosis is a phenomenon that a hybrid produced by crossing two parents with different characters (or genetic compositions) exceeds the parents in the aspects of characters such as vitality, growth vigor, resistance, high yield and the like, and is a common biological rule. At present, crossbreeding by utilizing heterosis is one of the main ways for improving the yield, resistance and quality of crops. However, genetic traits of the F1 hybrid generation generated after hybridization of parents (male parent and female parent) are uncertain, not all filial generations have hybrid vigor, the hybrid vigor of the progeny generated by different parent combinations is also different, some hybrid vigors are obvious and some are not obvious, and even degeneration occurs.
In practical application of crop heterosis breeding, a great amount of combinations are prepared and then subjected to field phenotype screening, so that a combined variety with heterosis compared with a control is bred, and the defects of long period, large workload, strong blindness, low efficiency and the like exist, so that the breeding result is uncertain. The core of hybrid breeding is to utilize hybrid vigor, and the selection and matching of parents are key links for obtaining hybrid vigor. Conventionally, it has been a problem of great concern in hybrid breeding that how to obtain hybrid seeds having new or excellent traits meeting production requirements quickly and simply and how to predict the potential heterosis of combined parents in early generations of breeding or in the initial stages of innovation of hybrid parents.
The method for predicting the heterosis of the crops commonly adopted in the current production is essentially divided into two methods, wherein the first method is to predict the expression of hybrid progeny according to the genetic difference of hybrid parents and takes a genetic distance method as a representative method; the second method is to predict the performance of hybrid progeny according to the interaction effect of the hybrid parents, and the hybrid progeny is represented by a combining ability method. The first method only starts from the genetic difference between parent individuals and does not consider the interaction effect (additive effect and non-additive effect) between parent genes; the second method is generally characterized in that the measured combining ability is determined by the additive effect of genes, and can be expressed by the non-additive effect (allelic or non-allelic interaction) of parent genes only in a specific combination, and the field preparation combination is huge in workload, so that the comprehensive measurement of the interaction effect between parents is difficult under the current conditions. Therefore, neither of them can accurately explain the heterosis of crops completely and reasonably.
In addition, multiple disciplines such as phenomics, genomics, metabonomics, transcriptomics and the like are cross-linked by breeding means such as molecular markers, transgenosis, molecular design and the like, so that the screened regulatory gene of the target character and the constructed molecular regulatory network play a more key role in explaining the molecular mechanism of heterosis formation. However, most of the advantageous target traits are controlled by the quantitative traits of the micro-effective polygenes and have complex genetic background, and meanwhile, the genes are regulated and controlled by a huge amount of transcription factors, so that great challenges are still faced in the processes of mining excellent trait associated genes and researching the regulation and expression of the transcription factors. At present, only relevant transcription factor libraries are popularized and applied in rice and arabidopsis thaliana, and are still in an undeveloped or under development state in other crops, and meanwhile, the molecular biology breeding means and the multi-group chemistry combination need a special laboratory, relevant talents, a mature experimental technical system and a large amount of experimental consumables, so that the input cost is high, and the application of the molecular biology breeding means in production practice is greatly limited.
In the prior art, patent application with publication number CN105900698A discloses a method for predicting heterosis by grafting, which comprises the following specific steps: selecting a test site; designing the number of cells and the number of repetitions; grafting two or more than two plants; after grafting, managing according to the requirements of grafting crops; measuring the target yield and related physiological indexes of the grafting types during harvesting; and predicting the grafting combination by adopting a hybrid vigor prediction method. According to the technical scheme, the parent-parent value class, the affinity value class and the interaction value class are grafted on two varieties, the target yield and related physiological indexes of different grafting types are calculated, the heterosis prediction of different varieties is realized, the cross breeding efficiency is improved, but the method needs to carry out various types of grafting and culture management on parents, and is not suitable for crops which cannot be bred through grafting.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for rapidly predicting heterosis based on the size of a crop genome.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly predicting heterosis based on crop genome size comprises the following steps:
s1, preparing a plurality of parent crops with target traits and stable heredity, and determining and calculating the genome size of the parent crops and the Nei genetic distance between the parent crops; judging whether the parent crops meet the following conditions: a. the relative difference percentage of the genome sizes of the two parent crops is 2-20%; b. the Nei genetic distance between two parent crops is more than or equal to 0.35. Selecting the hybrid combination which meets the conditions a and b simultaneously; and if the condition a or b is not met, replacing the parent crop and repeating the steps. Wherein, the relative difference percentage of the genome sizes of the two parent crops in the condition a is 2-20%, which means that the relative difference percentage of the genome sizes of the two parent crops is 2-20% according to the value obtained by multiplying (1-the ratio of the smaller genome size to the larger genome size) by 100%. The parent crop is the same subspecies crop with the same ploidy. In the hybrid combination, the crop with larger genome is the female parent, and the crop with smaller genome is the male parent.
S2, emasculating and hybridizing the hybridization combination parents selected in the step S1 to obtain F1Generation, pair F1Planting the generation and hybrid parent inbred line and the local main-push hybrid variety, and eliminating the hybridAnd (5) strain. The local dominant hybrid is the best variety suitable for local production.
S3, after the crops without the mixed strains planted in the step S2 are mature, respectively aligning the crops with the F1Performing target character investigation on the generation and hybrid parent inbred lines and the local main-push hybrid varieties, and calculating F1Respective target character mean values of generation and hybrid parent inbred lines and local main-boost hybrid varieties, and F is analyzed1Heterosis of generation target characters. Heterosis includes mesophilic advantage, super-philic advantage and over-standard advantage.
S4, combining Nei genetic distance between hybridization parents with F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the heterosis of the generation target character and the Nei genetic distance between the hybrid parents are in a linear significant or extremely significant relationship or not is determined, wherein the P value of the significant relationship is less than or equal to 0.05, and the P value of the extremely significant relationship is less than or equal to 0.01.
S5, comparing the absolute value of the genome size difference between the hybridization parents with F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the heterosis of the generation target character and the absolute value of the genome size difference between the hybridization parents are in a linear significant or extremely significant relationship. The absolute value of the difference in genome size between the two parents to be hybridized herein means the difference in measured genome size between the two parents to be hybridized.
S6, combining the judgment results of the steps S4 and S5, F1Predicting F when the heterosis of the generation target character and the Nei genetic distance between the hybrid parents and the absolute value of the genome size difference are in linear significant or extremely significant relation1Generation hybrid vigor.
In the breeding method, the crops as parents are judged and selected, and then the combination is configured. The genome size is the most basic and important biodiversity parameter of plants, and due to the influences of factors such as the growth environment and cultivation habit of the plants, the chromatin three-dimensional space structure, the number of non-coding DNA repetitive sequences and the like among the same subspecies crops with the same ploidy are greatly different, so that the genome size shows irregular difference. ByThe genome size of the two parent crops is different, the chromosome structures of cells such as sperm cells, egg cells and the like are necessarily completely different, and the characteristics of the chromosome structures of the cells are the basis of the gene expression and inhibition of the crops. Therefore, when parents with different genome sizes are hybridized, the genome between the parents generates interaction, and the three-dimensional structure of the chromosome is changed in the process of forming a gametophyte into a zygote, which is beneficial to the activation and expression of the zygote genome, so that the originally inhibited gene is expressed after the zygote genome is activated, and the genetic basis of heterosis is generated, thereby ensuring that the hybrid F has the advantages of high efficiency, high yield and high quality1Representing the heterosis with higher characters. Meanwhile, significant or extremely significant correlation exists between the Nei genetic distance and the hybrid vigor of the hybrid parents, and the influence of the genetic difference and the interaction effect of the parents on the hybridization can be comprehensively considered by combining the genome size difference and the Nei genetic distance between two parent crops, so that the parent combination meeting the conditions a and b on the basis that the parents have target traits is realized, and the variety with the hybrid vigor can be obtained by hybridization more easily. In addition, the target character is the parent material with the character which is consistent with the breeding purpose, the parent material has the advantages of consistent growth period, strong resistance, high yield and good quality of two parent materials in the hybridization combination. The invention judges and screens the hybrid parents in the early stage of breeding, and can obtain the parents with higher heterosis varieties after the hybridization to prepare the hybrid combination, thereby being beneficial to shortening the breeding period, improving the breeding efficiency and reducing the breeding cost.
Further, in the step S1,
the reference crop is a congeneric or congeneric plant of known genomic size.
The flow cytometry technique can rapidly and quantitatively determine and analyze parameters such as cell size, cell granularity, cell surface area, nucleoplasm proportion, DNA content, cell cycle, RNA content, protein content, cell activity, intracellular cytokine, enzyme activity and the like of single cells or other biological particles marked by fluorescence. When the genome size (namely DNA content) of crops is measured, quantitative fresh leaves of plants are weighed, mechanically crushed in a cell lysis buffer solution and then filtered, a specific fluorescent dye mixed with RNA enzyme is added into the filtrate to dye the cell nucleus of a sample to be measured, and the DNA content of the cell nucleus is in direct proportion to the fluorescent molecular binding amount. Under certain pressure, the stained cell nucleuses pass through the instrument flow chamber one by one, then are irradiated by laser, and the excited fluorescence is converted into an electric signal, so that the relative value of the cell nucleuses DNA reflected by the fluorescence intensity (namely the fluorescence intensity value of the crop G1 stage) can be obtained.
Further, in step S1, the Nei genetic distance is calculated as a result of genotyping the crop using the SNP chip. And (3) carrying out genotyping on the genomic DNA of the parent crop by using a high-density SNP chip, and finally obtaining the Nei genetic distance of the SNP marker by using special computer analysis software on the data obtained by genotyping.
Further, in step S2, F13-10 rows of the generation parent selfing line, the hybrid parent selfing line and the local main-push hybrid variety are respectively planted, 5-6 plants are planted in each row, and the plant spacing is 18-25 cm. Each F1The generation, the cross parent inbred line and the local main-push hybrid variety are planted with the crops in the number, so that enough strains with consistent purity can be obtained after the hybrid strains are eliminated.
Further, in step S2, the hybrid line is eliminated by identifying the inbred line and F of the hybrid parent through morphology and molecular markers1The strain purity of the generation, and strains and mixed strains with inconsistent shapes are screened out. The purity of the strain can be identified through molecular markers, such as simple repeat sequence markers (SSR) or single nucleotide polymorphism markers (SNP markers), the SSR is also called microsatellite DNA, the microsatellite is in co-dominant inheritance and can identify heterozygotes and homozygotes, and the single nucleotide polymorphism markers (SSRP markers) can distinguish the difference of individual genetic materials and can realize the identification of the purity of the strain.
Further, in the above step S3, the objective trait is investigated as F1Generation, cross parent inbred line andrandomly selecting 5-10 plants from the local main-push hybrid variety material for investigation. The objective characters after eliminating the hybrid lines are investigated, and the average value of the investigation results is used as the objective character value of each material, wherein the objective characters can be plant height, thousand seed weight and the like.
Furthermore, the parent crops are stable sterile lines of quality sterility or nuclear sterility of the same subspecies with the same ploidy, stable restorer lines corresponding to sterility types and conventional fertile materials in crop materials bred by utilizing heterosis. The crop material comprises crops with wider heterosis utilization in the production of rice, rape, wheat, corn, sorghum, soybean and the like.
The invention has the beneficial effects that:
1) the method is easy to master and strong in operability, does not need to prepare a large number of hybrid combinations in the field to determine the combining ability, saves a large number of breeding processes of multi-generation hybridization and selfing, can quickly predict the parent hybrid combination with heterosis, and is beneficial to shortening the breeding period, improving the breeding efficiency and reducing the breeding cost.
2) The method is suitable for crop materials through cross breeding, namely, the crops through cross breeding can be bred by the method, and the method has extremely strong wide applicability.
3) The invention adopts the flow cytometry technology for the determination of the genome size, does not need to obtain the genome size by a high-throughput genome sequencing mode, can complete the determination in a common laboratory, and is convenient and efficient.
4) The method of the invention considers the genetic difference of the hybrid parents and the interaction effect between the parents, more comprehensively predicts the crop heterosis and is beneficial to improving the accuracy of the selection of the hybrid combination.
Drawings
FIG. 1 is a flow chart of the present invention for predicting heterosis.
FIG. 2 is a flowchart illustrating the method for predicting rape heterosis according to an embodiment of the present invention.
FIG. 3 shows the measurement of ZS11 and cabbage type oil as reference crops in the first embodiment of the present inventionVegetable 4300, B0933 and hybrid F thereof1Flow cytometric histograms of surrogate genome size.
FIG. 4 is a cross F of a cross of brassica napus 4300 and B0933 identified in the first embodiment of the present invention1The genuineness and purity of the individual plants are shown in the figure.
FIG. 5 is a flowchart of rice heterosis prediction according to the second embodiment of the present invention.
FIG. 6 shows the results of the determination of reference crops 93-11, indica 2254, 2261 and hybrids F thereof in the second embodiment of the present invention1Flow cytometric histograms of surrogate genome size.
FIG. 7 shows a cross F of positive and negative crosses for identifying indica rice 2254 and 2262 in the second embodiment of the present invention1The genuineness and purity of the individual plants are shown in the figure.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
As shown in fig. 1, the present invention provides a method for rapidly predicting heterosis based on genome size, comprising the steps of:
s1, preparing a plurality of parent crops which have target traits and are stable in heredity, and determining and calculating the genome size of the parent crops and the Nei genetic distance between the parent crops. The genome size can be determined by using the flow cytometry, taking the same species or the same genus plant with known genome size as a reference crop, and multiplying the ratio of the fluorescence intensity of the parent crop to the fluorescence crop of the reference crop by the genome size of the reference crop to obtain the genome size of the parent crop. The Nei genetic distance can be obtained by adopting an SNP chip to carry out genotyping result calculation, carrying out genotyping on parental genome DNA by utilizing a high-density SNP chip, and combining with special computer analysis software to obtain the Nei genetic distance marked by the SNP.
Judging whether the parent combination meets the following conditions: a. the relative difference percentage of the genome sizes of the two parent crops is 2-20%; b. the Nei genetic distance between two parent crops is more than or equal to 0.35. Wherein, the relative difference percentage of the genome size in the condition a is 2-20% (1-the ratio of the smaller genome size to the larger genome size) x 100%, and then is 2-20%. Two parent crops simultaneously meet the conditions a and b, and are selected as a hybrid combination; and if the condition a or b is not met, replacing the parent crop and repeating the steps. The parent crop is the same subspecies crop with the same ploidy, and the selected hybrid combination has larger genome as female parent and smaller genome as male parent.
S2, emasculating and hybridizing the hybridization combination parents selected in the step S1 to obtain F1Generation, pair F1And (3) planting 3-10 rows of each material, 5-6 plants in each row, and the plant spacing is 18-25 cm, so that hybrid lines with inconsistent shapes or inconsistent purity are eliminated. Selecting strains with regular shape and consistent purity of molecular marker identification strains, and determining average genome size and F between crossing parent inbred lines by flow cytometry1Mean genome size between generations, analysis of the hybrid parents and F1And (5) generating genome size change.
S3, after the crops without the mixed strains planted in the step S2 are mature, respectively aligning the F1Carrying out target character investigation on the generation and hybrid parent inbred lines and local main-push hybrid varieties, such as plant height, thousand seed weight and the like, randomly selecting 5-10 plants from each material for investigation, and then calculating F1Respective target character mean values of generation and hybrid parent inbred lines and local main-boost hybrid varieties, and F is analyzed1Heterosis of generation target characters. Here, the hybrid superiority includes mesophilic superiority, super-philic superiority and super-dominant superiority:
S4, combining Nei genetic distance between hybridization parents with F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the heterosis of the generation target character and the Nei genetic distance between the hybridization parents are in a linear significant or extremely significant relation or not is evaluated by adopting a P value in statistics, the significant degree of the calculation result of the correlation degree is evaluated, the P value is not more than 0.05, and the P value is not more than 0.01.
S5, comparing the absolute value of the genome size difference between the hybridized parents (namely, the genome size difference between the parents) with the F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the heterosis of the generation target character and the absolute value of the genome size difference between the hybridization parents are in a linear significant or extremely significant relationship.
S6, combining the judgment results of the steps S4 and S5, F1When the heterosis of the generation target character and the Nei genetic distance between the hybrid parents and the absolute value of the genome size difference are in linear significant or extremely significant relation, the method provided by the invention can accurately predict F1The hybrid vigor with convenient target characters can be generated, and the hybrid combination with the hybrid vigor can be quickly predicted by adopting the method of the invention.
Example one
As shown in fig. 2, taking brassica napus as an example, the present embodiment provides a method for rapidly predicting heterosis based on genome size, comprising the following steps:
s1, taking 12 genetically stable tetraploid brassica napus as a parent crop, determining the genome size of the 12 materials through a flow cytometer, and calculating the relative difference percentage of the genome size between every two materials in the 12 materials to be within the range of 0.7-18.9%. And obtaining the Nei genetic distance between every two materials in the range of 0.33-0.74 through SNP chip typing. 48 groups of hybridization combinations which simultaneously meet the conditions that the relative difference percentage of the genome sizes of two parent crops is 2-20% and the Nei genetic distance of the two parent crops is more than or equal to 0.35 are selected, and 23 groups of hybridization combinations with consistent growth period, strong resistance, high yield and good quality (with target characters) are selected by combining the character conditions (such as growth period, resistance, yield, quality and the like) of 12 parts of materials, and the 23 groups are selected as the hybridization combinations to be prepared.
When the genome size of 12 parts of materials is measured by a flow cytometer, an improved LB01 flow cell lysate is adopted, and the formula of the cell lysate is as follows: 15mmol/L tris (hydroxymethyl) aminomethane, 2mmol/L disodium ethylenediaminetetraacetate, 0.5mmol/L spermine tetrahydrate, 80mmol/L potassium chloride, 20mmol/L sodium chloride, 10mmol/L magnesium sulfate, 0.1% (v/v) polyethylene glycol octylphenyl ether, 15mmol/L beta-mercaptoethanol, 0.05% tween 20 and 0.1% (m/v) polyvinylpyrrolidone, pH 7.5. The method comprises the steps of carrying out cell lysis on rape leaves, mechanically crushing the rape leaves in a cell lysate, filtering, adding a mixed solution of Propidium Iodide (PI) and RNase 0.5mg/mL respectively into the filtrate, keeping out of the sun for 15min, detecting by using a C6Plus flow cytometer produced by American BD company, and calculating the average genome size of each parent material by using double 11(ZS11) in tetraploid brassica napus with known genome size as a reference crop.
S2, performing emasculation hybridization on the parent materials of the 23 groups of hybridization combinations selected in the step S1 to obtain F1And (4) generation. During hybridization, bagging parent material main inflorescence for selfing, emasculating its lateral branch, and positive and negative cross hybridizing to obtain 46 portions of hybrid F1Generation, collection of hybrid F1Generation and parental inbred line seeds. During planting, extracting DNA of leaves of parent materials, and screening the DNA by SSR molecular marker amplification difference strip primers.
For the collected F1And planting the generation and parent inbred lines and the local main-push hybrid variety with the cotton seed oil 18, wherein 5 rows are planted, 5 plants are planted in each row, the plant spacing is 25cm, the rice is used as the previous rice, and the soil fertility is uniform. Investigating parental inbred lines, hybrid F1The uniformity of the seedling generation period and the moss period is random from strains with consistent uniformitySelecting 10 strain number tags, collecting the leaves of each strain to extract DNA, and performing SSR molecular marker purity identification by using early-stage screened primers, as shown in FIG. 4, the forward-reverse cross hybridization F of brassica napus 4300 and B0933 is shown1And (5) generation identification results. Eliminating mixed strain to obtain hybrid F with consistent form and purity1And (4) generation strain. Measurement of F1The genome size of the generative lines, as shown in FIG. 3, FIG. 3 lists the parental Brassica napus 4300, B0933 and F thereof in 23 sets of crossing combinations1When the genome size of the reference crop ZS11 is measured, a flow cytometry is used for measuring the fluorescence intensity of each material in the G1 phase by a flow cytometer. It can be seen from the analysis and calculation of the results in FIG. 3 that when the size of the maternal genome in the hybridization combination is larger than that of the paternal genome, F is obtained by hybridization1The subgenomic group is larger in size.
S3, after the crops screened in the step S2 grow to the maturity period, randomly selecting 10 individual plants from each material, carrying out statistical investigation on agronomic characters such as the root diameter, the plant height, the number of effective branches in one time, the length of fruiting section of the main sequence, the number of effective siliques of the whole plants and the like, and calculating an average value. Meanwhile, randomly selecting 30 fruits from each part of material, counting the average number of the fruits, and weighing thousand seeds by using a one-ten-thousandth balance after the seeds are harvested, dried and moisturized. The quality characters of the seeds, such as erucic acid content, glucosinolate content, oil content, protein content and the like, are measured by adopting an RT3700 near infrared instrument produced by FOSS company in America. Each part of the materials comprises 23 groups of screened parent inbred lines after combination and 46 parts of positive and negative hybrid F1Generation and local promotion of the material of the hybrid variety rong oil 18. Analysis and calculation of F1The hybrid vigor of generation character, the calculation result shows that the hybrid F1The high plant height in the generation, the effective branch number at one time, the fruit grain number per horn, the thousand grain weight, the single plant yield and other properties show higher positive over-standard advantages. Wherein, F is obtained by combining 23 parents with larger maternal genome size1The generation has the advantages over the standard superior to the parent combination with smaller maternal genome size, and proves that the F with larger genome size is obtained by hybridization by taking the crop with larger genome size as the female parent1The generation hybrid has better heterosis.
S4, comparing the Nei genetic distance between 23 groups of parent crops with F1The heterosis of the generation target characters is subjected to correlation analysis, and the result shows that F1The relative or super-relative advantages of the characters such as the fruit grain number of each generation, the single plant yield, the root and stem thickness and the like and the Nei genetic distance between the hybridization parents are in a significant or extremely significant relationship.
S5, comparing the absolute value of the genome size difference between 23 groups of parent crops with F1The heterosis of the generation target characters is subjected to correlation analysis, and the result shows that F1The absolute value of the difference between the parent size and the genome size of the hybrid parents and the relative or super-relative advantages of the generation, the branch height, the effective branch at one time, the thousand kernel weight, the fruit kernel number per horn, the thioglycoside and other characters are in a significant or extremely significant relationship.
And S6, combining the judgment results of the steps S4 and S5, wherein the heterosis of the characters such as the fruit grain number per horn, the single plant yield, the thousand kernel weight and the like for the purpose of crossbreeding is in linear significant or extremely significant relation with the absolute values of the genetic distance between the parent crops and the genome size difference. By adopting the method, the rape strong-superiority hybrid combination can be better prepared by taking the genome size difference between parents and the Nei genetic distance as reference bases. Meanwhile, the invention aims to better predict rape hybrid F1The above character heterosis of the generation provides a method, which is beneficial to reducing the breeding cost and accelerating the breeding progress.
Example two
As shown in fig. 5, the present embodiment provides a method for rapidly predicting heterosis based on genome size, taking rice as an example, comprising the following steps:
s1, taking 16 genetically stable diploid indica rice varieties as parent crops. The 16 material genome sizes were determined by flow cytometry. Calculating to obtain the relative difference percentage of the genome size between every two materials in 16 parts of materials within the range of 0.3-15.2%, and obtaining the Nei genetic distance between every two materials within the range of 0.24-0.67 by typing of a 50K SNP chip of rice. The method comprises the following steps of firstly, selecting 34 groups of hybridization combinations which simultaneously meet the conditions that the percentage of relative difference of genome sizes of two parent crops is 2-20% and the Nei genetic distance of the two parent crops is more than or equal to 0.35, then, screening out 34 groups of hybridization combinations which have consistent growth period, strong resistance, high yield and good quality (with target characters) and meet the requirements of the percentage of relative difference of genome sizes and the Nei genetic distance by combining the character conditions (growth period, resistance, yield, quality and the like) of 16 parts of materials, and selecting the 34 groups as the hybridization combinations to be prepared.
When the size of 16 material genomes is determined by a flow cytometer, an improved Galbraith flow cell lysate is adopted, and the lysate formula is as follows: 30mmol/L sodium citrate, 20mmol/L propanesulfonic morphine, 45mmol/L magnesium chloride, 15mmol/L sodium chloride, 0.1% (v/v) polyethylene glycol octylphenyl ether, 0.05% tween 20 and 0.1% (m/v) polyvinylpyrrolidone, pH 7.0. And (2) carrying out cell lysis on rice leaves, mechanically crushing the rice leaves in a cell lysate, filtering, adding a mixed solution of Propidium Iodide (PI) and RNase, wherein the mixed solution is 0.05mg/mL respectively, keeping out of the sun for 15min, detecting by using a flow cytometer produced by Hisenmeikang Japan, and calculating the average genome size of each parent material by using a diploid indica rice variety 93-11 with a known genome size as a reference crop.
S2, performing emasculation hybridization on the parent materials of the 34 groups of hybridization combinations selected in the step S1 to obtain F1And (4) generation. During hybridization, the partial tillering emasculation hybridization and the rest tillering bagging selfing of the parent material are carried out to obtain 68 parts of hybrid F after positive and negative hybridization1Generation, collection of hybrid F1Generation and parental inbred line seeds. During planting, extracting DNA of leaves of parent materials, and screening the DNA by SSR molecular marker amplification difference strip primers.
For the collected F1And (3) planting the generation and parent inbred lines and the local main-push hybrid indica rice variety Luoyou838, wherein 5 rows are planted, 6 plants are planted in each row, the plant spacing is 18cm, and the soil fertility is uniform. Investigating parental inbred lines, hybrid F1The uniformity of the seedling generation period and the heading period is determined by randomly selecting 12 strain number tags from strains with consistent uniformity, collecting leaves of each strain to extract DNA, and performing SSR molecular marker purity identification by using primers screened at the early stage, as shown in FIG. 7, FIG. 7 is a positive and negative cross hybridization F of indica rice 2254 and 22621Generation identification result, eliminating mixed strain and obtaining hybrid vigorHybridization F1And (4) generation strain. Measurement of F1The genome size of the generation line is shown in FIG. 6, and FIG. 6 lists 34 sets of hybrid combination parent indica rice 2254, 2261 and their reciprocal hybrids F1When the genome size of the generation and reference crops 93-11 is measured, a flow cytometry is used for measuring the fluorescence intensity of each material in the G1 phase by a flow cytometer. From the analysis and calculation of the results in FIG. 6, it can be seen that when the size of the maternal genome in the hybridization combination is larger than that of the paternal genome, F is obtained by hybridization1The subgenomic group is larger in size.
S3, after the crop screened in the step S2 grows to a mature period, randomly selecting 10 single plants from each material, carrying out statistical investigation on the agronomic characters such as plant height, sword leaf length, sword leaf width, primary branch number, secondary branch number, effective spike number and the like, and calculating the average value. Meanwhile, 30 rice ears are randomly selected for each part of material, the average ear length, the ear number and the ear weight are counted, after the seeds are harvested and dried, the thousand seed weight is weighed by a one-ten-thousandth balance, and the quality characters such as chalky grain number, chalky degree, special aspect ratio and the like of the seeds are measured. Each part of the materials comprises a parent inbred line and a positive and negative hybrid F which are screened from 34 groups of combinations1Generation and local promotion of hybrid indica rice variety Luoyou838. Analysis and calculation of hybridization F1The hybrid vigor of generation target characters shows that the hybrid F1The characteristics of high plant height, effective spike number, grain number, chalky grain rate, single plant yield and the like in the generation have obvious positive over-standard advantages. Wherein 34 parents with larger maternal genome size are hybridized in a combined way F1The overproof advantages of the generations are all superior to those of the parent combination with smaller maternal genome size, and the results prove that the hybrids with larger genome size obtained by hybridization have better hybrid advantages by taking the crops with larger genome size as the maternal plant.
S4, combining the Nei genetic distance between 34 groups of combined parent crops with 68 parts of F1Correlation analysis is carried out on the heterosis of the generation target character, and the result shows that the Nei genetic distance and the F between the hybridized parent crops1The parent or super-parent advantages of the traits of the ear number, the ear weight, the single plant yield and the like are in a significant or extremely significant relationship.
S5, combining 34 groups of parent crop genome sizesAbsolute value of difference to 68 parts of F1The heterosis of the generation target characters is subjected to correlation analysis, and the result shows that the absolute value of the genome size difference between the hybridized parent crops and F1The parent or super-parent advantages of the characteristics such as plant height, branch number at one time, effective spike number, spike weight, chalky grain rate, special aspect ratio and the like are in a significant or extremely significant relationship.
S6, combining the judgment results of the steps S4 and S5, the heterosis of the characters such as spike grain number, spike weight, single plant yield, chalky grain rate and the like as the goal of cross breeding respectively has linear obvious or extremely obvious relationship with the absolute values of the genetic distance between parent crops and the genome size difference, the method can quickly and accurately predict the heterosis of the characters of rice, and obtains the hybrid F with higher heterosis1Parent hybrid combination of generations.
Comparative example
The difference between the comparative example and the second example is mainly as follows: the screening conditions for preparing the hybridization combination of the comparative example only comprise the Nei genetic distance between parents and the parent target characters, namely two parent crops which meet the condition that the Nei genetic distance between the parents is more than or equal to 0.35 and have the target characters are selected as the hybridization combination, and the genome size difference (namely the relative difference percentage of the genome sizes) between the parent crops is not considered. Based on the above conditions, 74 hybrid combinations were selected from 16 genetically stable diploid indica varieties identical to those in the examples, and the parental materials of the 74 hybrid combinations were subjected to emasculation hybridization to obtain F1Generation, collection F1Generation and parental inbred line seeds. Through pair F1The generation and the parent inbred line and the local main-push hybrid variety radiance 838 are planted and verified, and F with good hybrid vigor can be obtained1The parental combination of the generation has 36 groups, wherein 34 groups are the same as the parental combination configured in the second embodiment, namely, the combination satisfying the condition of genome size difference accounts for 94.4% of the parental combinations of 36 groups. Therefore, the method of the invention obviously improves the accuracy of preparing the hybrid combination with heterosis, is beneficial to reducing manpower and material resources consumed in the breeding process and reducing the breeding cost.
In other embodiments, other types of crops, such as crops with more extensive heterosis utilization of wheat, corn, sorghum, and soybean, can be used, and the breeding method is similar to the above embodiments, and is not listed here.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for rapidly predicting heterosis based on crop genome size is characterized in that: the method comprises the following steps:
s1, preparing a plurality of parent crops with target traits and stable heredity, and determining and calculating the genome size of the parent crops and the Nei genetic distance between the parent crops; judging whether the parent crops meet the following conditions: a. the relative difference percentage of the genome sizes of the two parent crops is 2-20%; b. the Nei genetic distance between two parent crops is more than or equal to 0.35; selecting the hybrid combination which meets the conditions a and b simultaneously; if the condition a or b is not met, replacing the parent crop and repeating the steps; the parent crops are the same subspecies crops with the same ploidy;
s2, emasculating and hybridizing the hybridization combination parents selected in the step S1 to obtain F1Generation, pair F1Planting the generation and hybrid parent inbred lines and local main-push hybrid varieties, and eliminating hybrid strains;
s3, after the crops without the mixed strains planted in the step S2 are mature, respectively aligning the crops with the F1Performing target character investigation on the generation and hybrid parent inbred lines and the local main-push hybrid varieties, and calculating F1Respective target character mean values of generation and hybrid parent inbred lines and local main-boost hybrid varieties, and F is analyzed1Heterosis of generation target character；
S4, combining Nei genetic distance between hybridization parents with F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the heterosis of the generation target character and the Nei genetic distance between the hybridization parents are in a linear significant or extremely significant relationship, wherein the P value of the significant relationship is less than or equal to 0.05, and the P value of the extremely significant relationship is less than or equal to 0.01;
s5, comparing the absolute value of the genome size difference between the hybridization parents with F1Carrying out correlation analysis on heterosis of generation target characters, and judging F1Whether the absolute value of the size difference of the genome between the heterosis of the generation target character and the hybrid parent is in a linear significant or extremely significant relation or not;
s6, combining the judgment results of the steps S4 and S5, F1Predicting F when the heterosis of the generation target character and the Nei genetic distance between the hybridization parents and the absolute value of the genome size difference are in linear significant or extremely significant relation1Generation hybrid vigor.
2. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in the hybrid parents, the larger genome is used as a female parent, and the smaller genome is used as a male parent.
3. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 2, wherein: in the step S1, the genome size of the parent crop is determined by adopting the flow cytometry,
the reference crop is a congeneric or congeneric plant of known genomic size.
4. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in step S1, the Nei genetic distance is calculated as a result of genotyping the crop using the SNP chip.
5. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in the step S2, F13-10 rows of the generation parent selfing line, the hybrid parent selfing line and the local main-push hybrid variety are respectively planted, 5-6 plants are planted in each row, and the plant spacing is 18-25 cm.
6. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in the step S2, hybrid lines are eliminated, namely hybrid parent inbred lines and hybrid parent inbred lines are identified through morphology and molecular markers1The strain purity of the generation, and strains and mixed strains with inconsistent shapes are screened out.
7. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in step S3, the objective trait is investigated as F1And randomly selecting 5-10 plants from the generation parent inbred line, the hybrid parent inbred line and the local main-push hybrid variety material for investigation.
8. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: in step S3, the heterosis includes mesophilic advantage, super-philic advantage and super-dominant advantage.
9. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 1, wherein: the parent crops are stable sterile lines of the same subspecies with the same ploidy or stable genic sterile, stable restorer lines corresponding to sterile types and conventional fertile materials in crop materials bred by utilizing heterosis.
10. The method for rapidly predicting heterosis based on crop genome size as claimed in claim 9, wherein: the crop material includes rice, rape, wheat, corn, sorghum and soybean.
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