AU2021106463A4 - Establishment and screening analysis method of .EMS mutant library of Panicum miliaceum L - Google Patents

Abstract

This invention discloses establishment and screening analysis method of EMS mutant library of Panicum miliaceum L, belonging to the field of agricultural breeding, a technical platform for EMS mutagenesis of Panicum miliaceum L was established by screening the best concentration of seeds treated by EMS. After planting 2333 M generation lines, 103 mutant lines were found, including leaf color, leaf type, plant height, fertility, ear type, ear color and grain color, with mutation frequency of 4.4%. The establishment of mutant library with abundant phenotype can provide abundant mutant resources and new germplasm for functional genomics research and breeding of Panicum miliaceum L.

Description

Establishment and screening analysis method of.EMS mutant library of Panicum miliaceum L

TECHNICAL FIELD The invention belongs to the field of agricultural breeding, and relates to the construction of Panicum miliaceum L mutant library, in particular to a method for establishing a mutant library by EMS induction aiming at Panicum miliaceum L

BACKGROUND Panicum miliaceum L belongs to the annual Gramineae genus Panicum, with chromosome number 2n=4x=36. It has the characteristics of short maturity, drought tolerance, barren tolerance, salt and alkali tolerance, etc. It is a characteristic coarse cereal crop and an ideal multiple cropping crop mainly cultivated by people in arid and semi-arid areas of northern China. After husking, Panicum miliaceum L is called yellow rice, which is rich in various protein, starch, fat, etc. needed by human body. It has high nutrition and health care value and can be processed into various foods for preventing and treating allergies, obesity and other related diseases; In addition, its stems and grains are high-quality forage for livestock, so Panicum miliaceum L plays an important role in meeting the diversified needs of mass food consumption and the development of animal husbandry. Mutation is an important driving force of biological evolution. Mutants can also provide materials for the study of gene function. With the continuous innovation and research of new technologies, many methods for analyzing and identifying gene functions have been developed, among which the most effective and direct method is to construct a saturated gene mutant library. Constructing a saturated mutant library is one of the direct and effective methods for the study of plant functional genomics and genetic breeding. Ethyl methanesulfonate (EMS), a chemical mutagen, is a stable and efficient chemical mutagen. EMS is widely used, and it can treat different tissues and organs such as seeds, seedlings, pollen or callus. EMS mutagenesis has a high mutation rate, does not need genetic transformation, and is easy to form point mutations, and most of them are dominant mutations, so it is easy to screen mutants and not easy to cause chromosome aberration. At present, EMS chemical mutagenesis has been successfully applied to Arabidopsis thaliana, rice, wheat, corn, sorghum and other plants, and a number of valuable innovative germplasm resources have been obtained. Significant progress has been made in improving plant growth period, disease resistance and stress resistance, seed setting rate and plant height. Screening and identification of these mutants provide rich material basis for future crop genomics research and crop genetic improvement, and have important scientific practical significance. After constructing EMS mutant library of Panicum miliaceum L, breeding materials for genetic improvement of this variety can be obtained through identification and screening of biological and agronomic characters, and at the same time, abundant research materials can be provided for forward genetics and functional genomics analysis of Panicum miliaceum L. Panicum miliaceum L is a good material for genome research because its water use efficiency and drought resistance are stronger than those of other C4 plants such as Panicum miliaceum L and sorghum. However, gene discovery and functional identification of Panicum miliaceum L started late in China, and there is no report on the construction, identification and functional genome of Panicum miliaceum L mutant library.

SUMMARY To solve the problems existing in the prior art, the purpose of the present invention is to provide a method for establishing and screening and analyzing the EMS mutant library of Panicum miliaceum L, to establish a mutant library of Panicum miliaceum L with rich phenotype, which is helpful to speed up the process of discovering genes with many excellent traits of Panicum miliaceum L, and to provide abundant mutant materials for functional genomics research and breeding of Panicum miliaceum L. This invention provides stablishment and screening analysis method of .EMS mutant library of Panicum miliaceum L is characterized in comprising the following steps: (1) Seed preparation: selecting the seeds of red Panicum miliaceum L with full and uniform grains, adding distilled water twice the volume of the seeds of red Panicum miliaceum L, shaking evenly, soaking in a refrigerator at 4C for 6-8h, taking out, and standing at room temperature for 10min. (2) EMS induction: under the condition of fume hood, 1% of EMS solution was added into seeds, and mixed and oscillated at 300rpm/min for 1Oh at room temperature. Take out the EMS solution treatment container, add 0.2M NaSO with the same volume as EMS solution for neutralization, mix it upside down for 7-10 times, and then stand for min. Filter out the neutralized EMS aqueous solution with a filter screen, pour the waste liquid into the lower water of the fume hood, wash the seeds with distilled water for 5 times, and filter the water with a filter screen every time until the effluent solution is not foamy. Dump the treated seeds in the filter screen, take them out of the fume hood and put them in the washing pool, rinse them with running water for 5 min, and hang them in the air to control the moisture. (3) Mutation seed planting: the seeds induced by EMS in step (2) are called M generation seeds, and the M-generation seeds are planted with a row spacing of 30cm and a plant spacing of 6 cm. The damage and mutation of M plants were observed and recorded during the whole growth period, and single seeds were harvested in 10 months, which were called M generation. (4) Investigation on related traits of mutant plants: Planting M generations of mutant library obtained in step (3), with 15-20 seedlings per plant line, carefully observing the growth variation of M generations in the field, and observing the morphological characteristics of plants in different periods after seed germination: counting the ratio of albino seedlings to etiolated seedlings in seedling stage, observing the disease of plants at jointing stage, counting the ratio of dwarfed seedlings at booting and heading stage. The mutants with special morphology were observed in each period, the mutation characteristics and types were recorded in detail, the mutation frequency was counted, and the strains with obvious phenotypic changes were harvested per plant. (5) Formula for calculating M generation mutation frequency: M generation mutation frequency = M generation mutant strain /M generation population strain coefficientX 100%.

Preferably, in step (4), the related traits are the decrease or absence of chlorophyll content in leaves, the increase or decrease of plant height, the change of ear type, ear color and grain color, the early or late maturity and the change of fertility. The invention has the beneficial effects that: According to the method provided by the invention, the best concentration of EMS treated Yi xuan red Panicum miliaceum L seeds is systematically studied and screened by taking Yi xuan red Panicum miliaceum L as a material, the technical platform for EMS mutagenesis of Panicum miliaceum L is established for the first time, and library of Panicum miliaceum L mutants with rich phenotypes is obtained. The observation statistics of 2333 M generations during the whole growth period showed that there were abundant mutation types of materials, and 103 mutant lines with morphological characters were found, including leaf color, leaf type, plant height, ear type, ear color, grain color and fertility. The method provided by the invention can obtain a Panicum miliaceum L mutant library with rich phenotype in a short time, which not only helps to accelerate the process of discovering many excellent trait genes in Panicum miliaceum L, but also provides rich mutant materials for functional genomics research and breeding of Panicum miliaceum L. Dwarfing and obtaining spike mutants lay a material foundation for solving the problems of lodging and falling grains in production as soon as possible.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a morphological characteristic diagram of etiolated seedlings and albino seedlings of Yi xuan red Panicum miliaceum L selected from Iraq according to the present invention. Fig. 2 is a graph showing the incidence of Yi xuan red Panicum miliaceum L according to the present invention. Fig. 3 is a diagram of an early maturing mutant of Yi xuan red Panicum miliaceum L according to the present invention. Fig. 4 is a diagram of dwarf mutant of Yi xuan red Panicum miliaceum L in accordance with that present invention.

Fig. 5 is a diagram of spike mutant of Yi xuan red Panicum miliaceum L according to the present invention.

DESCRIPTION OF THE INVENTION In the following, the technical scheme in the embodiments of the present invention will be described clearly and completely in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative labor belong to the scope of protection of the present invention. Embodiment 1 1. Materials to be tested: Yi xuan red Panicum miliaceum L 2. Experimental design Six concentration gradients were set up in the experiment, and each concentration was set up with three repetitions, and each repetition treated 300 seeds of Yi xuan red Panicum miliaceum L and one clear water control. 3. Test method (1) Select the seeds with full and uniform grains, adding distilled water with twice the volume of the seeds, shaking evenly, and soaking in a refrigerator at 4Cfor 6h. (2) Prepare EMS solutions with concentrations of 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2% respectively. (3) Treat300 seeds of Yi xuan red Panicum miliaceum L with prepared EMS solution respectively, mixing and oscillating at 300rpm/min for 10 on an oscillator at room temperature. (4) Add 0.2M NaSO with the same volume as EMS solution for neutralization, mixing upside down for 7-10 times, and stand for 15min. (5) Filter out the neutralized EMS aqueous solution with a filter screen, pour the waste liquid into the lower water of the fume hood, wash the seeds with distilled water for 5 times, and filter the water with a filter screen every time until the effluent solution is free of foam. Dump the treated seeds in a filter screen (seed net bag), take them out from a fume hood and put them in a washing pool, rinse them with running water for 5 min, and hang them in the air to control the moisture. (6) The seeds induced by EMS in step (5) are called M-generation seeds, and the M-generation seeds are planted with a row spacing of 30cm and a plant spacing of 6cm. The damage of M plants, such as albino seedlings and etiolated seedlings, emergence and mutation, is observed and recorded during the whole growth period. A total of 2,333 seeds per plant were harvested in 10 months, and these seeds were called M generation. 4. Statistics of experimental results After emergence, count the seedling number of seeds treated with different EMS concentrations, and calculate the seedling rate. The results are shown in Table 1 below. Table 1 Effect of different EMS concentration treatments on seedling rate of Yi xuan red Panicum miliaceum L seeds

EMS~neeutraion EMS Concerntration (%) M The number of seeds treated (ri) (Grain) The number of seeds treated (strain) Seedling rate (%)

0 (CK) 300 294 98 0.2 300 292 97.3 0.4 300 255 85 0.6 300 219 73 0. 8 300 191 637 1.0 300 139 46.3 1.2 300 88 29.3

5. Determination of the optimum concentration of 4.EMS It is generally considered that the semi-lethal dose (that is, the dose at which plants survive 50% after EMS mutagenesis) is the best mutagen amount and the energy threshold that should be absorbed to cause hereditary variation. It can be seen from Table 1 that the lethal rate of Izakami is the closest to 50% after 1.0% EMS treatment, so 1.0% EMS concentration is determined as the optimum treatment concentration. 6. M2 generation seed planting

Planting M2 generation seeds of the mutant library obtained in step (6), with 15-20 seedlings per plant line, and carefully observing the growth variation of M2 generation in the field with wild Yi xuan red Panicum miliaceum L as control material, and observing the morphological characteristics of plants in different periods after the seeds germinate: At seedling stage, the ratio of albino seedlings to etiolated seedlings is 2%, and the morphological characteristics of etiolated seedlings and albino seedlings are shown in Fig.1. Observe the disease of plants at jointing stage. The specific morphology is shown in Fig. 2. The results show that plants are susceptible to diseases and broken tips at jointing stage. The proportion of dwarfing seedlings and the time of maturity were counted at booting and heading stage. Among them, a total of 15 strains in M2 generation population were found to have mutations related to plant height, with a mutation frequency of 0.6%. In addition, three early-maturing and late-maturing mutant strains were observed respectively, with a mutation rate of 0.03%. Mutants with special morphology were observed at seedling stage, jointing stage, booting and heading stage and mature stage. The early-maturing mutant is shown in Fig.3, and one strain is super-early-maturing mutant, which starts heading only about 24 days after sowing, about 30 days earlier than the heading time of the control materials in the same plot. Dwarfing mutants are shown in fig. 4, with short stem mutation: due to the abundant rain in that year, the average plant height of Yixuandahongmi reached 167cm, and it was observed that there were dwarfing mutants in 8 strains, the highest one was 141cm, and the shortest one was 89cm, which was only 1/2 of the wild type. The spike type mutant was shown in fig. 5, and the spike type of wild type Yixuandahongmi was lateral spike type, and the whole spike drooped sideways after heading. The average ear length is 33.2cm and the average ear weight is 6.5g. Four types of mutants were observed in 15 mutant lines related to panicle type found in M2 generation from heading date: (1) Panicle-dense mutant: a total of 5 mutant lines, which showed that although the whole panicle spread in the same direction as the wild type, the branches on the panicle were shorter and the spikelets were arranged compactly. The average ear length of the mutant with dense ear is 30.4cm, and the average single ear weight is 10.5g. (2) Ear-erect mutant: There are 3 mutant lines, showing that the whole ear is thin and long and erect from heading to maturity. The average ear length of the erect panicle mutant is 30.4cm, and the average single panicle weight is 1.9g. (3) Panicle-wide mutant: There are two mutant lines in total. Compared with the lateral spike type of wild-type Yixuandahongmi, the panicle-wide mutant shows that the branches on the panicle are very short from heading, which makes the spikelets of each branch arrange very compactly and upward like sorghum panicles. The average ear length and single ear weight of the mutant with thick ear are 17.7cm and 5.1g, respectively. (4) Spike radiation mutant: There are 5 mutant lines, which scatter irregularly from heading to maturity The mutation frequency was counted, and the plants with obvious phenotypic changes were harvested. The formula of M2 mutation frequency is: M2 mutation frequency=M2 mutant strain /M2 population strain coefficient X 100%. The observation statistics of 2333 M2 generations during the whole growth period showed that there were abundant mutation types in materials, and 103 mutant lines with morphological characters were found, with a mutation frequency of 4.4%, including leaf color, leaf shape, plant height, fertility, ear type, ear color and grain color. The specific statistics are shown in Table 2. Table 2 Statistics of phenotypic mutation typesof M2 mutant population mutation Mutant Trait mutation type Mutant Trait type coefficient coefficient leaf colour Albino 4 Spike type diaer 2 chlorisis 16 spike density 5 albino leaf core 1 radaion 5 chlorisis leaf 1 Spike erect 3 core Implicit stripe 6 Maturity Premature 3 leaf Striped leaf 9 Late mature 3 leafshape Spotted leaf 8 Seed color purple 3 Leaves 2 white 4 stagnate green leaf tip broke 1 yellow 5 Rotten leaf 2 Spike color red 2 height of Tall stalk 7 Low fertility 3 stalk ________

Low stalk 8 Susceptible Susceptible 1 todisease todisease

The above embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention. On the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.

Claims (2)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Observation method of .EMS mutant library of Panicum miliaceum L is characterized in comprising the following steps: (6) Seed observation: observing the seeds of red Panicum miliaceum L with full and uniform grains, adding distilled water twice the volume of the seeds of red Panicum miliaceum L, shaking evenly, soaking in a refrigerator at 4C for 6-8h, taking out, and standing at room temperature for 10min. (7) EMS observation: under the condition of fume hood, 1% of EMS solution was added into seeds, and mixed and oscillated at 300rpm/min for 1Oh at room temperature. Take out the EMS solution treatment container, add 0.2M NaSO with the same volume as EMS solution for neutralization, mix it upside down for 7-10 times, and then stand for min. Filter out the neutralized EMS aqueous solution with a filter screen, pour the waste liquid into the lower water of the fume hood, wash the seeds with distilled water for 5 times, and filter the water with a filter screen every time until the effluent solution is not foamy. Dump the treated seeds in the filter screen, take them out of the fume hood and put them in the washing pool, rinse them with running water for 5 min, and hang them in the air to control the moisture. (8) Mutation seed observation: the seeds observed in the EMS in step (2) are called M-generation seeds, and the M-generation seeds are observed with a row spacing of cm and a plant spacing of 6 cm. The damage and mutation of M plants were observed and recorded during the whole growth period, and single seeds were observed in 10 months, which were called M generation. (9) Investigation on related traits of observed plants: Observing M generations of mutant library obtained in step (3), with 15-20 seedlings per plant line, carefully observing the growth variation of M generations in the field, and observing the morphological characteristics of plants in different periods after seed germination: counting the ratio of albino seedlings to etiolated seedlings in seedling stage, observing the disease of plants at jointing stage, counting the ratio of dwarfed seedlings at booting and heading stage. The mutants with special morphology were observed in each period, the mutation characteristics and types were recorded in detail, the mutation frequency was counted, and the strains with obvious phenotypic changes were harvested per plant.

   (10) Formula for calculating M generation mutation frequency: M generation mutation frequency = M generation mutant strain /M generation population strain coefficient X 100%.
2. 2. The method for establishing, screening and analyzing the EMS mutant library of Panicum miliaceum L according to claim 1, which is characterized in that in step (4), the related traits are the decrease or absence of chlorophyll content in leaves, the increase or decrease of plant height, the change of ear type, ear color and grain color, the advance or delay of maturity and the change of fertility.