A method for rapidly creating a waxy double recessive gene corn inbred line

Abstract

The application discloses a kind of sweet waxy double recessive gene corn inbred line's fast creation method, belong to the new variety breeding technical field of crop, the method includes selecting sweet, waxy corn and crossing, harvests F1 generation seed;F1 generation seed is selfed and obtains F2 generation seed, selects F2 generation waxy grain and is planted;Screening waxy grain contains homozygous waxy gene single plant as female parent, and hybridizes with corn induction line, obtains hybrid seed;In hybrid seed, screen double haploid seed and carry out doubling treatment, harvest double single line plant;Double single line plant is selfed, and obtains double single line seed, selects sweet seed in double single line seed, it is sweet waxy double recessive gene corn inbred line.This application provides method in the selection range of basic material used in the method is wide, inbred line creation efficiency is high, and character is stable, significantly shorten inbred line creation time, with significant popularization and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of new variety breeding of crops, in particular to a rapid creation method of a sweet waxy double recessive gene corn inbred line. BACKGROUND

[0002] Waxy corn is an important crop integrating food, fruits and vegetables, industrial raw materials, and silage feed, and has a very broad development prospect. With the improvement of living standards, consumers have higher requirements for fresh waxy corn, which should be both nutritious and delicious. Sweet waxy corn varieties contain both sweet corn and waxy corn kernels on the same corn cob, which are sweet and waxy, and have a unique taste, and are deeply loved by consumers.

[0003] Sweet waxy corn varieties are obtained by crossing a sweet waxy double recessive gene corn inbred line with a waxy corn inbred line to obtain F1 hybrid seeds. The F1 plants have both sweet corn kernels and waxy corn kernels on the fruiting ear, with a sweet kernel:waxy kernel ratio of 1:3. The sweet waxy double recessive gene corn inbred line is the basis for breeding sweet waxy corn varieties.

[0004] In addition to the conventional breeding process, the breeding of a sweet waxy double recessive gene corn inbred line also requires the addition of a sweet and waxy double recessive precise identification link, which significantly reduces the breeding cycle and efficiency. Therefore, improving the breeding efficiency, shortening the breeding cycle, and precisely identifying and rapidly creating a sweet waxy double recessive gene corn inbred line are of great significance for promoting the breeding of new sweet waxy corn varieties.

[0005] CN114747482A discloses a breeding method of a high-compatibility sweet waxy double recessive corn inbred line. The specific steps are as follows: first, corn materials with sweet and waxy genes are crossed to form an original population, and F2 generation seeds are obtained by selfing excellent single plants. Sweet kernel seeds are selected for planting, and molecular markers are used to select single plants with waxy genes. Single plants with waxy genes and excellent comprehensive traits are selected for selfing to obtain S1 generation seeds. S1 generation lines are obtained by planting S1 generation seeds, and excellent plants are selected for selfing. The remaining pollen is crossed with a waxy corn inbred line, and the lines are further screened based on the performance of the cross combination. This process is repeated in Sichuan and Yunnan to realize the simultaneous breeding of excellent inbred lines and sweet waxy corn combinations. Although this technology can breed a high-compatibility sweet waxy double recessive corn inbred line, the purification requires a long generation time, and the efficiency needs to be improved. SUMMARY

[0006] The present application provides a rapid creation method of a sweet waxy double recessive gene corn inbred line to solve the problems existing in the prior art. The method uses a wide range of base materials, has high inbred line creation efficiency, and stable traits, significantly shortens the inbred line creation time, and has significant application value.

[0007] To achieve the above object, the application provides the following scheme.

[0008] The application provides a method for rapidly creating a sweet and waxy double recessive gene corn inbred line, comprising the following steps:

[0009] (1) selecting sweet and waxy corns for hybridization, and harvesting F1 generation seeds;

[0010] (2) selfing the F1 generation seeds to obtain F2 generation seeds, and selecting waxy kernels in the F2 generation for planting;

[0011] (3) screening single plants containing homozygous waxy genes in the waxy kernels as female parents, and hybridizing the single plants with a corn inducing line to obtain hybrid seeds;

[0012] (4) screening and doubling haploid seeds in the hybrid seeds to obtain double inbred lines;

[0013] (5) selfing the double inbred lines to obtain double inbred line seeds, and selecting sweet seeds in the double inbred line seeds, i.e. the sweet and waxy double recessive gene corn inbred line.

[0014] Further, in step (3), the single plants containing homozygous waxy genes in the waxy kernels are screened by using molecular markers; and the reagent for detecting the molecular markers comprises primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or SEQ ID NO: 3-4.

[0015] Further, the DNA of the waxy kernels in the F2 generation is used as a template, and primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or SEQ ID NO: 3-4 are used for PCR amplification, the amplification products are sequenced, and the material with 30bp deletion of the 7th exon and intron or 15bp deletion of the 10th exon of the wx gene is screened as the homozygous waxy gene material.

[0016] Further, in step (3), the corn inducing line is a corn inducing line with a purple marker.

[0017] Further, in step (1), the variety of the sweet corn includes a sweet corn inbred line BT708; and the variety of the waxy corn includes Haimai 515.

[0018] Further, in step (4), the doubling treatment comprises seed soaking treatment of the haploid seeds with 0.06% colchicine by mass percentage, and then flushing with water.

[0019] The application further provides a method for screening corns containing homozygous waxy genes, comprising the step of amplifying DNA of a corn to be tested by using primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or SEQ ID NO: 3-4.

[0020] The application also provides a product for screening corn containing homozygous waxy genes, comprising primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or SEQ ID NO: 3-4.

[0021] The application discloses the following technical effects:

[0022] (1) Wide selection range of base materials

[0023] According to breeding direction and target requirements, the waxy corn and sweet corn material with excellent quality, resistance, yield, and agronomic traits can be selected as the donor material, and the material can be an inbred line, a family variety, a population material, or a commercial hybrid, and there is no limitation on the grain color.

[0024] (2) Shortened time for creating inbred lines

[0025] The haploid technology is used to quickly purify the 100% genetically pure sweet waxy double recessive corn inbred line.

[0026] (3) High efficiency of creating inbred lines

[0027] The method of the application can obtain not only the sweet waxy double recessive corn inbred line, but also the homozygous waxy material.

[0028] (4) Stable traits of created inbred lines

[0029] The sweet waxy double recessive corn inbred line material created by the method of the application has stable traits, and is conducive to the breeding and popularization of subsequent commercial hybrid seeds. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0031] Figure 1 A schematic diagram of the method for quickly creating a sweet waxy double recessive gene corn inbred line;

[0032] Figure 2 A polyacrylamide gel electrophoresis diagram of DNA of waxy-7exon-1F / R amplified material;

[0033] Figure 3 A polyacrylamide gel electrophoresis diagram of DNA of waxy-10exon-1F / R amplified material;

[0034] Figure 4Part of the sequencing alignment results of the waxy-7 exon-1 F / R amplification product; Note: the dotted line in the figure is the 30bp deletion sequence of the 7th exon and the 7th intron of the wx gene;

[0035] Figure 5 Part of the sequencing alignment results of the waxy-10 exon-1 F / R amplification product; Note: the dotted line in the figure is the 15bp deletion sequence of the 10th exon of the wx gene;

[0036] Figure 6 The ear picture of the new sweet and waxy corn combination YN318;

[0037] Figure 7 The field planting picture of the new sweet and waxy corn combination YN318. DETAILED DESCRIPTION

[0038] The detailed description of various exemplary embodiments of the present application is now provided, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

[0039] It should be understood that the terms used in the present application are merely for describing the particular embodiments, and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range of values, and any other stated value or stated range of values within the stated range is also included within the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0041] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0042] As used herein, the terms "comprise", "comprising", "including", "include", "contain", "containing", "have", "having" or variants thereof are open-ended, and include the stated integer or group, but also any other integer or group.

[0043] The application provides a method for rapidly creating a sweet and waxy double recessive gene maize inbred line. The method comprises the following steps: first, hybridizing corn materials containing homozygous waxy genes and sweet genes to construct an original breeding population (F1), selfing excellent single plants of the F1 to obtain F2 seeds, selecting waxy grain seeds for planting, screening single plants with homozygous waxy genes by using molecular markers at the seedling stage, hybridizing the single plants with a haploid inducer line with purple markers as the female parent at the flowering stage, obtaining haploid seeds, and selecting a double haploid (DH line) with sweet waxy genes as the sweet and waxy double recessive inbred line material. The method for rapidly creating the sweet and waxy double recessive gene maize inbred line is shown in the schematic diagram of the application. Figure 1 The CTAB method used in the following examples comprises the following steps:

[0044] (1) cut 100 mg of young leaves, and grind them into powder with liquid nitrogen;

[0045] (2) move the powder material into a 1.5 ml centrifuge tube, add 0.5 ml of 2.0x CTAB extraction solution preheated at 80℃, shake well, and then place in a 65℃ constant temperature water bath for 30 min, and shake quickly every 8-10 min during the interval;

[0046] (3) add 0.5 ml of phenol chloroform, mix well by inverting up and down for 5 min, and centrifuge at 20℃ and 12000 r / min for 10 min;

[0047] (4) take the supernatant to another centrifuge tube, add an equal volume of nucleic acid extraction solution (chloroform: isopropyl alcohol is 24:1), and shake on a shaker for 10 min (keep the centrifuge tube horizontally placed);

[0048] (5) place in a 4℃ centrifuge, centrifuge at 12000 r / min for 10 min;

[0049] (6) take the supernatant to another centrifuge tube, add an equal volume of isopropyl alcohol, mix well until the milk-white flocculent precipitate appears;

[0050] (7) centrifuge to discard the supernatant, transfer to a 1.5 ml centrifuge tube, and wash the precipitate once with 70% (v / v) ethanol;

[0051] (8) centrifuge at 12000 r / min for 1 min, discard the supernatant, and keep the DNA in the tube and dry;

[0052] (9) add 0.1-0.3 ml of ddH2O to dissolve fully, and store at -20℃ for standby use.

[0053] The corn materials used in the following examples are all stored in Chongqing Academy of Agricultural Sciences.

[0054] The specific research is as follows:

[0055] A method for rapid creation of a waxy double recessive gene maize inbred line

[0056] comprising the following steps:

[0057] (1) Hybridize waxy maize Heymai 515 as the female parent and sweet maize inbred line BT708 as the male parent, adjust the planting date according to the growth period of the female and male parents to make the male parent anthesis meet the female parent silking and hybridize to obtain F1 generation seeds;

[0058] (2) Plant the F1 generation seeds in the field, bag pollination and strictly self-pollinate to obtain F2 generation seeds (waxy seeds, sweet seeds, and powdery seeds);

[0059] (3) Select the waxy seeds in the F2 generation and plant them in the field, take the tender leaves at the 6-7 leaf stage, extract DNA using the CTAB method, and further screen single plants containing homozygous waxy genes using molecular marker technology;

[0060] (4) Hybridize the single plants containing homozygous waxy genes screened in step (3) as the female parent with the purple marker-induced line 2061 (Qi Z.Y., Yang H., Qiu Z.G., Zhang Y.Q., Yuan L., Cai Z.R., Wang N., Jin C., and Li S.J., 2012, Study on induction effect of haploid in different maize genotypes, Xinan Nongye Xue bao (Southwest China Journal of Agricultural Sciences), 2012, 25(4): 1152-1158) as the male parent to obtain hybrid seeds and harvest and dry them;

[0061] (5) Screen the purple-topped white embryo pseudo-haploid seeds obtained in step (4) according to the purple marker;

[0062] (6) Soak the seeds obtained in step (5) with 0.06% (w / w) colchicine for 12 h, then rinse with running water for 2 h for doubling treatment;

[0063] (7) Planting the seeds doubled in step (6), selecting the plants with light color, weak growth, narrow leaves and straight leaves (the doubled haploid plants) for strict single bag self-pollination to obtain the doubled haploid (DH line) seeds (with sweet and waxy);

[0064] (8) The sweet seeds obtained in step (7) are the sweet and waxy double recessive gene corn inbred line (CS2675).

[0065] In the step (3), the single plant containing the homozygous waxy gene is further screened by the molecular marker technology, and the steps are as follows:

[0066] wx-D7 (30 bp deletion in the 7th exon) and wx-D10 (15 bp deletion in the 10th exon) are two main genotypes used in waxy corn breeding, and other genotypes account for a small proportion in breeding. Therefore, the primers are designed to amplify these two exons, and the primer sequences are as follows:

[0067] Table 1 primer sequence information

[0068]

[0069] Genotype identification results:

[0070] The 170 materials are amplified by using the waxy-7exon-1F / R and waxy-10exon-1F / R primers, the PCR products are subjected to polyacrylamide gel electrophoresis, and the PCR amplification products of B73 and Mo17 DNA are used as controls, and the electrophoresis results are shown in Figure 2 and Figure 3 By comparing with the PCR amplification products of B73 and Mo17 DNA, the small PCR products are selected for sequencing in Shanghai Biosciences, and the sequencing comparison results of the PCR products are shown in Figure 4 and Figure 5 Figure 4 It is part of the sequencing comparison results of the waxy-7exon-1F / R amplification products; note: the dotted line part in the box in the figure is the deletion sequence (30 bp) of the 7th exon and the 7th intron of the wx gene, which is different from wx-D7 (the deletion sequence is only in the exon), and the lack of sequence is located at the 3' end of the 7th exon and the 5' end of the 7th intron, which is a new waxy corn germplasm. Figure 5 It is part of the sequencing comparison results of the waxy-10exon-1F / R amplification products; note: the dotted line part in the figure is the deletion of 15 bp of the 10th exon of the wx gene.

[0071] ​According to the electrophoresis results and sequencing results, in the 170 materials, the 7th exon and intron deletion of 30 bp is W7, W8, W11-W41, W47-W60, W95-W110, W129-W136, W148-W170, and the 10th exon deletion of 15 bp is W1-W6; these materials are all homozygous waxy maize germplasm.

[0072] The step (3) of the embodiment selects the F2 generation waxy grain in the field, and the advantage of the waxy grain compared with the sweet grain of the prior art is that the waxy grain has a higher seedling rate, a stronger seed topsoil capacity, and a stronger seedling viability, which can significantly improve the success rate of field selection of excellent single plants and shorten the creation period.

[0073] Effect verification

[0074] The created sweet waxy double recessive gene maize inbred line CS2675 is crossed with the waxy maize inbred line Y407 to form a sweet plus waxy maize new combination YN318. Figure 6 The fruit ear picture of the sweet plus waxy maize new combination YN318.

[0075] The sweet plus waxy maize new combination YN318 participates in the waxy maize new combination observation test of the Chongqing Academy of Agricultural Sciences in 2020, the waxy maize new variety comparison test of the Chongqing Academy of Agricultural Sciences in 2021, the multi-point test of the Chongqing Academy of Agricultural Sciences in 2022, and the multi-point test of the Chongqing Academy of Agricultural Sciences in 2023.

[0076] According to the multi-point test results in 2022-2023, the average fresh ear yield of YN318 is 12860 kg / hm 2 , the plant height is 250 cm, the ear position height is 112 cm, the ear shape is conical, the ear length is 18.2 cm, the ear thickness is 4.9 cm, the bare tip length is 0.2 cm, the ear row number is 12.8 cm, the row grain number is 39.6 grains, the grain depth is 1.1 cm, the hundred-grain weight is 293 g, the grain is white, and the ear axis is white. The grain is sweet plus waxy type, and the ratio of waxy grain to sweet grain on the ear is 3:1. Amylopectin / rough starch is 100%. After steaming and cooking, the taste is waxy with a little sweetness, the flavor is unique, and it is better than the control Yunu 7. Figure 7 The field planting picture of the sweet plus waxy maize new combination YN318.

[0077] The above-described embodiments only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for rapidly creating a sweet and sticky double recessive gene corn inbred line, characterized in that: The following steps are involved: (1) Select sweet corn and glutinous corn for hybridization and harvest F1 generation seeds; (2) F2 generation seeds were obtained by self-pollination of F1 generation seeds, and the waxy grains of F2 generation were selected for planting; (3) selecting a single plant containing a homozygous waxy gene in waxy grains as the female parent and hybridizing it with the corn inducer line to obtain hybrid seeds; (4) selecting pseudohaploid seeds from hybrid seeds for doubling treatment and harvesting double monoploid plants; (5) self-pollinating the double monoline plants to obtain double monoline seeds, and selecting the sweet seeds among the double monoline seeds, which are the sweet and sticky double recessive gene corn inbred lines; In step (3), molecular markers are used to screen waxy grains for individual plants containing homozygous waxy genes; the reagents for detecting the molecular markers include primers having nucleotide sequences as shown in SEQ ID NOs: 1-2 and / or SEQ ID NOs: 3-4; Using DNA from waxy grains in the F2 generation as a template, PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NOs: 1-2 and / or SEQ ID NOs: 3-4. The amplified products were sequenced, and materials with a 30 bp deletion in the 7th exon and intron of the wx gene or a 15 bp deletion in the 10th exon were screened as homozygous waxy gene materials.

2. The rapid creation method according to claim 1, characterized in that: In step (3), the maize induction line is a maize induction line with a purple mark.

3. The rapid creation method according to claim 1, characterized in that: In step (1), the variety of the sweet corn includes the sweet corn inbred line BT708; the variety of the glutinous corn includes Haimai 515.

4. The rapid creation method according to claim 1, characterized in that: In step (4), the doubling treatment includes: soaking the pseudohaploid seeds with 0.06% by mass of colchicine, and then washing them with running water.

5. A method for screening corn containing a homozygous waxy gene, characterized in that: The method comprises the steps of amplifying the corn DNA to be tested by using primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and / or SEQ ID NO: 3-4.

6. A product for screening corn containing a homozygous waxy gene, characterized in that: The primers comprise nucleotide sequences as shown in SEQ ID NOs: 1-2 and / or SEQ ID NOs: 3-4.

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