阅读说明：本技术 一种玉米单倍体诱导系的选育方法 (Breeding method of corn haploid induction line ) 是由 赵连坤 王建民 付建征 韩福祥 付俊清 李娜 张泽伟 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种玉米单倍体诱导系的选育方法,包括以下步骤：步骤一：以青贮玉米杂交种和多抗普通玉米杂交种分别与JNY23组配F1;步骤二：A×JNY23和B×JNY23各进行1-2代自交,同时使用单倍体诱导率测验系KVC19测定A×JNY23和B×JNY23分离后代的诱导率；步骤五：将(A×JNY23)F3BC1或(A×JNY23)F4BC1,和((B×JNY23)F3BC1或((B×JNY23)F4BC1中分离得到的诱导系进行杂交,分离稳定出新型诱导系。通过自交多代稳定株做诱导系,产生的诱导系具有根系发达、耐旱抗倒、抗高温耐高湿、花粉量大、散粉时间长的优点,在开展单倍体工程化育种时可有效扩大规模应用,降低风险。(The invention discloses a method for breeding a corn haploid induction line, which comprises the following steps: the method comprises the following steps: f1 is combined by silage corn hybrid and multi-resistance common corn hybrid with JNY23 respectively, and the second step is: carrying out 1-2 generations of selfing on A × JNY23 and B × JNY23 respectively, and simultaneously determining the induction rates of the A × JNY23 and B × JNY23 separated generations by using a haploid induction rate test system KVC 19; step five: the induction lines separated from (A x JNY 23) F3BC1 or (A x JNY 23) F4BC1 and ((B x JNY 23) F3BC1 or ((B x JNY 23) F4BC 1) are hybridized to separate and stabilize a new induction line, and the stable plants are used as the induction lines through selfing for multiple generations.)

1. A method for breeding a corn haploid induction line is characterized by comprising the following steps: the method comprises the following steps: f1 is combined by respectively mixing silage corn hybrid and multi-resistance common corn hybrid with JNY23, and the composition mode of F1 is A multiplied by JNY23 and B multiplied by JNY23;

step two: carrying out 1-2 generations of selfing on Ax JNY23 and Bx JNY23 respectively, simultaneously measuring the induction rates of the A x JNY23 and B x JNY23 separated generations by using a haploid induction rate test system KVC19, and respectively selecting the single plant with the highest induction rate in 2 populations;

step three: selecting a single plant with the highest induction rate from the separated offspring of Ax JNY23 as a female parent, and carrying out backcross by using JNY23 as a male parent to obtain (Ax JNY 23) F3BC1 or (Ax JNY 23) F4BCl, (B x JNY 23) F3BC1 or (B x JNY 23) F4BC 1) by the same method, wherein the backcross is generally carried out in F3 or F4 generations and is specifically determined according to the measurement result of the induction rate of the single plant;

step four: selfing (a × JNY 23) F3BC1 or (a × JNY 23) F4BC1 and ((B × JNY 23) F3BC1 or ((B × JNY 23) F4BC 1) for 1 generation, while measuring the induction rate of the segregating progeny using haploid induction rate test line CVK19, selecting the individual with the highest induction rate;

step five: hybridizing an induction line separated from (A x JNY 23) F3BC1 or (A x JNY 23) F4BC1 and ((B x JNY 23) F3BC1 or ((B x JNY 23) F4BC 1), continuously measuring the induction rate, carrying out 3-4 generation self-separation while measuring the induction rate and other comprehensive properties, selecting a line with the induction rate of more than 10 percent, detecting the pollen scattering time, pollen quantity, lodging resistance and disease resistance, and separating and stabilizing to obtain a novel induction line.

2. The method for breeding the corn haploid inducer line as claimed in claim 1, wherein in the first step, the JNY23 is the inducer line which is hoped to be bred by the agriculture industry independently, and is a stable inbred line, and the induction rate is about 10%.

3. The method for breeding the corn haploid inducer line as claimed in claim 1, wherein in the first step, the silage corn and the multi-resistance common corn hybrid have the advantages of developed root system, drought resistance, lodging resistance, high temperature and humidity resistance, large pollen amount and long pollen scattering time.

4. The method for breeding the corn haploid inducer line as claimed in claim 1, wherein in step four, CVK19 Ji nong's breeding industry self-selects the hybrid test species, the endosperm aleurone layer and the embryo tip purple marker of the hybrid seed after pollination by the inducer line are particularly obvious.

Technical Field

The invention relates to the technical field of breeding, in particular to a breeding method of a corn haploid induction line.

Background

The corn haploid breeding is a breeding method which utilizes a corn haploid induction line to pollinate a base material, induces and generates haploid grains, selects the haploid grains according to the marking properties, restores normal chromosome number of the haploid chromosomes by chromosome doubling means such as artificial chemical agent doubling and the like, and becomes a selfing line (DH line). Haploid breeding technology is applied in practice by more and more breeders due to the advantages of rapidness and high efficiency.

The induction rates of different induction systems are different, the induction rate is higher than 16%, and the induction rate is lower by about 1%. Most of the induction systems have weak root systems, thin and soft stems, poor plant types and easy lodging when the planting density is higher, and meanwhile, the male spikes have few branches, the pollen quantity is small, and the pollen activity is poor in a high-temperature environment. Most of the haploids are artificially pollinated when in use, and the scale is difficult to be effectively enlarged when haploid engineering breeding is carried out. In order to solve the problems, a new idea is needed to be utilized to breed a novel induction line for solving the problem of haploid breeding scale. As the induction lines are basically derived from Stock6, when most breeders improve the induction lines, more induction lines from different sources are improved by hybridization, the special comprehensive characters of common corn are not increased, and the use risk is still high.

Disclosure of Invention

The invention aims to provide a method for breeding a corn haploid inducer line, which solves the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for breeding a corn haploid induction line comprises the following steps: the method comprises the following steps: f1 is combined by respectively mixing silage corn hybrid and multi-resistance common corn hybrid with JNY23, and the composition mode of F1 is A multiplied by JNY23 and B multiplied by JNY23;

step two: carrying out 1-2 generations of selfing on Ax JNY23 and Bx JNY23 respectively, simultaneously measuring the induction rates of the A x JNY23 and B x JNY23 separated generations by using a haploid induction rate test system KVC19, and respectively selecting the single plant with the highest induction rate in 2 populations;

step three: selecting a single plant with the highest induction rate from the separated offspring of Ax JNY23 as a female parent, and carrying out backcross by using JNY23 as a male parent to obtain (Ax JNY 23) F3BC1 or (Ax JNY 23) F4BCl, (B x JNY 23) F3BC1 or (B x JNY 23) F4BC 1) by the same method, wherein the backcross is generally carried out in F3 or F4 generations and is specifically determined according to the measurement result of the induction rate of the single plant;

step four: selfing (a × JNY 23) F3BC1 or (a × JNY 23) F4BC1 and ((B × JNY 23) F3BC1 or ((B × JNY 23) F4BC 1) for 1 generation, while measuring the induction rate of the segregating progeny using haploid induction rate test line CVK19, selecting the individual with the highest induction rate;

step five: hybridizing an induction line separated from (A x JNY 23) F3BC1 or (A x JNY 23) F4BC1 and ((B x JNY 23) F3BC1 or ((B x JNY 23) F4BC 1), continuously measuring the induction rate, carrying out 3-4 generation self-separation while measuring the induction rate and other comprehensive properties, selecting a line with the induction rate of more than 10 percent, detecting the pollen scattering time, pollen quantity, lodging resistance and disease resistance, and separating and stabilizing to obtain a novel induction line.

As a further scheme of the invention, in the step one, the JNY23 is an induction line of Ji farming breeding independently, is a stable inbred line, and has the induction rate of about 10 percent;

as a further scheme of the invention, in the step one, the silage corn and multi-resistance common corn hybrid has the advantages of developed root system, drought resistance, lodging resistance, high temperature and humidity resistance, large pollen amount and long pollen scattering time.

As a further aspect of the invention, in step four, the CVK19 Ji race industry self-selected hybrid test seeds were marked with endosperm aleurone layer and embryo tip purple color of hybrid seeds after pollination with the induction line.

Compared with the prior art, the invention has the beneficial effects that: the invention selects an induction line JNY23, silage corns with large pollen amount and humidity resistance popularized in production and common corn hybrids with multiple disease resistance, selects CVK19 as a haploid induction rate test line, utilizes the silage corns and the multiple resistance corn hybrids to transform the induction line, uses hybridization, selfing and backcross means in the transformation process, combines single plant induction rate measurement and seed and plant color marking to select in the process, carries out hybridization on the transformed induction line A and the induction line B of two different groups, and uses self-bred multi-generation stable plants as the induction line, so that the generated induction line has the advantages of developed root system, drought resistance, lodging resistance, high temperature and humidity resistance, large pollen amount and long pollen dispersion time, and can effectively expand the scale application and reduce the risk when carrying out haploid engineering breeding.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. A method for breeding a corn haploid induction line comprises the following steps: the selected silage maize Yayu silage 26 and multi-resistance common maize Liyu 88 are respectively combined with JNY23 to form F1 and F1, the Yayu silage 26 is multiplied by JNY23, the Liyu 88 is multiplied by JNY23, and during harvesting, the panning is carried out, wherein the embryo tips are non-purple grains (haploid grains).

Performing white crossing on the Yayu silage 26 multiplied by JNY23 and the Liyu 88 multiplied by JNY23 to obtain F2 grains, and selecting the grains with purple endosperm aleurone layers and embryo tips and obvious colors. In order to ensure that the offspring can be separated into single plants with higher induction rate, more than 50 grains are selected from each F2 generation of grains. The larger the population, the better the goal, but the corresponding increase in workload.

The selected Yanyu silage 26 multiplied by JNY23 and Liyu 88 multiplied by JNY 23F 2 seeds are planted singly, and meanwhile, the number of plants is about 10 times of the number of the F2 seeds when a test strain CVK19 is planted. If 50 grains are planted in each F2 generation of seeds and 100 grains are planted in two F2 generations of seeds, 1000 plants are planted in CVK 19.

Before castration, the No. 26X JNY23 No. F2 of Yayu silage and the No. 88X JNY23 No. F2 of Liyu silage are eliminated, and only the No. 26X JNY23 No. F2-1, (26X JNY 23) F2-2, … …, (26X JNY 23) F2-N, (88X JNY 23) F2-1, (88X JNY 23) F2-2, … …, (88X JNY 23) F2-N are reserved.

When selfing is carried out on each individual plant of (Yayu silage 26X JNY 23) F2, a test line CVK19 is used as a female parent, each individual plant of (Yayu silage 26X JNY 23) F2 is used as a male parent, hybridization is carried out, pollination is carried out for 10 ears, the number of haploid seeds obtained by induction of each individual plant and the total number of seeds are counted during harvesting, the induction rate of each individual plant is calculated, 5F 2 individual plants with the highest induction rate are selected, 10 seeds with purple embryos, grain powder layers and embryo tips are selected from the 5 harvested individual ears, and 50 (Yayu silage 26X JNY 23) F3 seeds are obtained.

Meanwhile, the test line CVK19 is taken as a female parent, each single plant of (Liyu 88X JNY 23) F2 is taken as a male parent for hybridization, pollination is carried out for 10 ears, the number of haploid seeds and the total seeds obtained by induction of each single plant are counted during harvesting, the induction rate of each single plant is calculated, 5F 2 single plants with the highest induction rate are selected, 10 grains with obvious colors and endosperm aleurone layers and embryo tips are selected from 5 rows of the harvested single ears, and 50 grains of (Liyu 88X JNY 23) F3 seeds are obtained in total.

50 selected (Yayu silage 26X JNY 23) F3 seeds, (Liyu 88X JNY 23) F3 seeds, about 1000 test lines CVK19, and about 20 plants JNY 23.

(Yuyu Qing 26X JNY 23) F3 and (Liyu 88X JNY 23) F3 before castration, the green plant of the stalk is eliminated, only the purple plant of the stalk is reserved, and the single plant is numbered as (Yayu Qing 26X JNY 23) F3-1, (Yayu Qing 26X JNY 23) F3-2, … …, (Yayu Qing 26X JNY 23) F3-N, (Liyu 88X JNY 23) F3-1, (Liyu 88X JNY 23) F3-2 and … (Liyu 88X JNY 23) F3-N.

Backcross is carried out by taking each single plant of (Yayu silage 26X JNY 23) F3 as a female parent and JNY23 as a male parent to obtain seeds of (Yayu silage 26X JNY 23) F3-1/JNY23, (Yayu silage 26X JNY 23) E3-2/JNY23 …, (Yayu silage 26X JNY 23) F3-N/JNY 23. Meanwhile, a test line CVK19 is used as a female parent, each single plant of (Yayu silage 26 x JNY 23) F3 is used as a male parent for hybridization, pollination is carried out for 10 ears, the number of haploid seeds and the total number of seeds obtained by induction of each single plant are counted during harvesting, the induction rate of each single plant is calculated, 5F 3 single plants with the highest induction rate are selected, 10 seeds with obvious purple mesh colors in endosperm aleurone layers and liver tips are selected from the 5 harvested single ears, and 50 seeds of (Yayu silage 26 x JNY 23) F3/JNY23 are obtained.

Meanwhile, backcross was carried out using individuals of (Liyu 88X JNY 23) F3 as female parents and the induction line JNY23 as male parents to obtain (Liyu 88X JNY 23) F3-1/JNY23, (Liyu 88X JNY 23) F3-2/JNY23, … …, (Liyu 88X JNY 23) F3-N/JNY23 seeds. The test line CVK19 was used as the female parent, each individual plant of (Liyu 88X JNY 23) F3 was used as the male parent to perform crossing, pollination was performed for 10 ears, the number of haploid seeds and total seeds obtained during harvesting were counted, the inductivity of each individual plant was calculated, 5F 3 individual plants with the highest inductivity were selected, 10 seeds with purple endosperm aleurone layer and embryo tip and obvious color were selected from each of the 5 harvested individual ears, and 50 seeds of (Liyu 88X JNY 23) F3/JNY23 were obtained in total.

50 selected (Yayu silage 26 multiplied by JNY 23) F3/JNY23 seeds, (Liyu 88 multiplied by JNY 23) F3/JNY23 seeds are planted, the Yayu silage 26 multiplied by JNY 23) F3/JNY23 is used as a female parent, the Liyu 88 multiplied by JNY 23) F3/JNY23 is used as a male parent for cross pollination, meanwhile, the stalks are eliminated to be green plants, and only the purple stalks are reserved.

The filial generation of the hybrid of (Yayu silage 26X JNY 23) F3/JNY23 and (Liyu 88X JNY 23) F3/JNY23 is sowed in single seed, selfed, the stem is eliminated as green plant, only the purple plant is kept, and the excellent single plant number is YLY-1, YLY-2.

When each individual plant of YLY-1, YLY-2,.. YLY-N is selfed, a test line CVK19 is used as a female parent, each individual plant of YLY-1, YLY-2,.. YLY-N is used as a male parent for hybridization, pollination is carried out for 10 ears, the number of haploid seeds and the total seeds obtained by induction of each individual plant are counted during harvesting, the induction rate of each individual plant is calculated, 5 individual plants with the highest corresponding induction rate are selected, 20 seeds with obvious purple mesh colors of endosperm aleurone layers and embryo tips are respectively selected from the 5 harvested individual ears, and 100 selfed seeds are obtained.

Planting 100 selected YLY-1, YLY-2, YLY-N seeds, sowing the seeds in a single seed, and planting CVK19 test lines, wherein the number of the seeds is about 2000.

Before the YIL-1, YLY-2, YIL-N is castrated, green plants of stalks can be eliminated, only purple plants of stalks are reserved, and the number of each plant is numbered as YLY-1F2-1, YLY-1F2-2, … …, YLY-1F 2-1-N, YLY-2F2-1, YLY-2F2-2, … …, YLY-2F2-N, YLY-NF2-1, YLY-NF2-2, YLY-N-F2-N.

When each individual plant is selfed, a test line CVK19 is used as a female parent, each individual plant is used as a male parent for hybridization, pollination is carried out for 10 ears, the haploid grain number and the total grain number obtained by induction of each individual plant are counted during harvesting, the induction rate of each individual plant is calculated, 5 individual plants with the highest corresponding induction rate are selected, 20 grains with purple endosperm aleurone layers and obvious embryo tips are selected from the 5 harvested individual ears, and 100 grains of the YLYF3 selfed seeds are obtained.

100 selected YLYF3 seeds are planted, and CVK19 test lines are planted, and the number of the plants is about 2000.

Before the male extraction of YLEF 3, green plants of stalks can be eliminated, only purple plants of stalks are reserved, and the single plants are numbered as YLYF3-1, YLEF 3-2, … … and YLF 3-N.

When each individual plant of YLEF 3 is selfed, a test line CVK19 is used as a female parent, each individual plant of YLEF 3 is used as a male parent for hybridization, pollination is carried out on 10 ears, the number of haploid seeds and the total number of seeds obtained by inducing each individual plant are counted during harvesting, the induction rate of each individual plant is calculated, 5 seeds with the highest induction rate are selected, 20 seeds with purple endosperm aleurone layers and obvious embryo tips are selected from the 5 harvested individual ears, and 100 seeds of YLEF 4 are obtained.

100 selected YLYF4 seeds are planted, and CVK19 test lines are planted, and the number of the plants is about 2000.

When each individual plant of YLEF 4 is selfed, a test line CVK19 is used as a female parent, each individual plant of YLEF 4 is used as a male parent for hybridization, pollination is carried out on 10 ears, the number of haploid seeds and the total number of seeds obtained by inducing each individual plant are counted during harvesting, the induction rate of each individual plant is calculated, 5 seeds with the highest induction rate are selected, 20 seeds with purple endosperm aleurone layers and obvious embryo tips are selected from the 5 harvested individual ears, and 100 seeds of YLEF 5 are obtained.

100 selected YLYF5 seeds are planted, and CVK19 test lines are planted, and the number of the plants is about 2000.

When each individual plant of YLEF 5 is selfed, a test line CVK19 is used as a female parent, each individual plant of YLEF 5 is used as a male parent for hybridization, pollination is carried out on 10 ears, the number of haploid seeds and the total number of seeds obtained by inducing each individual plant are counted during harvesting, the induction rate of each individual plant is calculated, 5 seeds with the highest induction rate are selected, 20 seeds with purple endosperm aleurone layers and obvious embryo tips are selected from the 5 harvested individual ears, and 100 seeds of YLEF 6 are obtained.

The obtained YLEF 6 seeds are selected out with the induction rate of more than 10 percent, selfed and stored, and a new induction line is obtained.

In the breeding process, the maize Yayu silage 26 and the Liyu 88 are hybridized and backcross is carried out by high inductivity JNY23, so the form of the induced line YLY is high, and the induced line YLY is used as an induced line mainly utilizing pollen, has strong advantages and large pollen quantity.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.