CN104025998B - Utilize the method for hybridized induction monoploid selecting and breeding corn inbred line - Google Patents

Abstract

The invention discloses a method for breeding maize inbred lines by using hybridization induced haploid, and belongs to the field of crop breeding methods. The method of the present invention is to use Jigaoyu line No. 31 to pollinate the basic population or hybrid species that meet the breeding objectives, select the grains with purple on the top of the aleurone layer and no purple on the germ tip; then naturally or artificially double the obtained materials, Carry out selfing again; Then select the panicle row whose progeny is uniform and unseparated, which is a new inbred line. The haploid induction rate of the method of the present invention is high, which can reach more than 13%; secondly, the selection marker is obvious, the screening is easy and the accuracy is high; in addition, the inducible line used in the present invention is moderate in plant height and long in flowering time. Moreover, the amount of pollen is large, natural pollination can be carried out to induce haploids, and artificial pollination is avoided, which not only saves labor, but also is suitable for large-scale colony operations; the method of the invention also has the characteristics of high breeding efficiency, simple procedure, and fast breeding speed.

Description

A Method for Breeding Maize Inbred Lines Using Hybridization Induced Haploid

technical field

The invention belongs to a method for crop breeding, in particular to a method for breeding maize inbred lines by using hybridization to induce haploid.

Background technique

Breeding is a cyclical and long-term work. It generally takes 10-15 years to breed a variety. Not only is the workload heavy, time-consuming and laborious, but it may also take too long. Finally, the results obtained often do not meet the requirements of the market. Therefore, shortening the breeding time and speeding up the breeding speed have always been the goals that breeders are striving for. For this reason, many methods have been proposed, such as using anthers or pollen to cultivate haploids, and then doubling them into inbred lines can greatly shorten the breeding time. Time was once regarded as a promising method, but practice has proved that it is difficult to be widely used in practice due to the difficulty of cultivating anthers, complicated technology, and low efficiency.

In 1959, CoeEH of the University of Minnesota in the United States discovered that the maize haploid induction line Stock6 can induce haploids from the maternal material through hybridization, combined with marker selection, it is possible to use the hybridization induction line to induce haploids in offspring for haploid breeding. With the continuous development of corn breeding technology, the method of hybridization-induced haploid breeding has been valued by breeders, and has become the focus and hotspot of research in the international breeding community, and some new induced haploids have been derived from Stock6 through the method of hybridization improvement. Departments, such as SW14 in France (Lashermes, 1988), Krasnodar in the former Soviet Union (Shatskaya, 1994), ZMS in Moldova (ChalykST, 1994), KWS in Germany, etc. China Agricultural University used Stock6 to breed Nongda Gaoyu No. 1 (Liu Zhizeng et al., 2000; Chen Shaojiang et al., 2003,). The utilization value of these inducible lines greatly exceeds that of Stock6.

The patent application "A Method for Breeding New Maize Varieties Using Hybrid Induced Parthenocarpy Materials" (application number: 200610080832.4) discloses that the Jilin Academy of Agricultural Sciences uses the M278×Stock6 combination as the basic population, and adopts the pedigree method to continuously carry out six generations self-cross and test-cross breeding. In each generation, the selection of haploid induction rate and grain Navajo purple spot marker (the aleurone layer at the top of the grain with purple spots) is the main selection, and at the same time, the plant ABP1 (purple plant, controlled by the A1A2C2BP1 complementary gene, for most of the maize materials is expressed as Dominant) markers, pollen count, fruiting and disease resistance were continuously selected. Finally, a single panicle line with an average induction rate of 10.4%, good markers, and excellent comprehensive traits was bred, named Jigaoyuexi No. 3. Jigaoyu Line 3 carries Navajo dominant genetic markers, namely purple aleurone layer and purple germ. After pollinating other maize materials with Jigaoyin Line 3, the normal diploid showed purple aleurone layer and purple germ tip, and the leaf sheath of the plant was purple, while the induced parthenocarpy kernel produced purple aleurone layer and germ tip. Colorless, green leaf sheath of the plant, can be confirmed with this mark. Jigaoyin Line 3 is the maize haploid induction line with the highest induction rate reported in China, and is widely used. However, Jigaoyu Line 3 has various disadvantages to varying degrees: (1) The plants are short and cannot be used for natural pollination in independent fields to produce haploids. In order to overcome this shortcoming, people often cross the induced lines. However, inducible material produced by hybridization may present other problems, such as reduced haploid induction rates and altered expression of marker genes. The R1-nj marker gene (Nanda and Chase, 1966) (purple scutellum and purple aleurone crown) is widely used to select haploids from dry seeds; This effect is large, and sometimes identifying haplotypes can become very difficult, or even impossible, especially in the presence of a suppressor gene (C1-I) in the mother. (2) Grain markings were not consistent with different female parent materials, which made the selection of haploid kernels more difficult. Therefore, breeding new high-frequency induction lines is a prerequisite for this work.

Invention content:

The purpose of the invention is to provide a method for breeding maize inbred lines by using hybridization induced haploid. The breeding procedure of the method is simple, the breeding speed is fast, and the cultivated maize inbred lines are completely homozygous.

The present invention utilizes the method for hybridization induced haploid breeding maize inbred line, comprises the following steps:

(1) Use Jigaoyu Line 31 as the male parent to pollinate the basic population or hybrids that meet the breeding goals, and harvest the female ear; from it, the aleurone layer top is purple, the embryo tip is not purple, and the germ surface is small, Kernels that are triangular in shape and marked with deep depressions;

(2) Artificially or naturally double the material obtained in step (1), remove the leaf sheaths and plants with purple leaves at the jointing stage; perform selfing on plants that can bloom normally after doubling, and harvest self-seed and firm seeds according to individual plants ;

(3) For the grains obtained in the planting step (2), select the ear rows whose progeny traits are uniform and unseparated, which is a new corn inbred line.

The Jigaoyin line No. 31 described in the above method belongs to the maize species of the genus Zeamays (zeamays L.), and is a high-frequency haploid induction line selected by the Jilin Academy of Agricultural Sciences. Culture Collection Center (preservation location: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China), deposit number: CCTCCNo: P201008

The natural doubling in the above method means that under natural conditions, the chromosome doubling in haploid grain or seedling plant cells becomes normal diploid.

The artificial doubling described in the above method refers to the use of colchicine solution to treat haploid grains or seedlings, so that the chromosomes in the cells are doubled to become normal diploid.

The basic population mentioned in the above method refers to the maize population formed by mixed pollination of multiple inbred lines or varieties. This kind of population can aggregate multiple beneficial genes, accumulate excellent traits, have good disease resistance, and excellent comprehensive traits, so it is used as population material for breeding inbred lines.

The material sources of the above-mentioned basic groups can also be various excellent farm varieties and the like.

The hybrid species mentioned in the above method refers to single-cross species, three-cross species or double-cross species.

According to the general understanding of those skilled in the art, the term “meeting the breeding objective” in the above method refers to excellent agronomic traits, high-yield traits, disease resistance traits, stress resistance traits, and quality traits.

The method for cultivating corn hybrids using the corn inbred lines produced in the above method is to use the corn inbred lines cultivated in the above method as one of the parents to cross with other inbred lines to breed new hybrids.

The present invention uses the method of hybridization induction to produce haploid grains to select and breed inbred lines, and uses the self-selected high-frequency hybridization induced by the inventors to induce haploid inducibility --- Jigaoyin Line 31 (the induction rate is as high as 13%) The pollen of the above) is used to pollinate excellent basic populations and hybrids, and the obtained grains are identified through linkage marker traits to obtain haploid grains and plants, which are then doubled artificially or naturally to become diploids, and self-bred into new homozygous inbred line. Realize gametophyte selection, quickly breed new maize inbred lines, create new germplasm resources, and generally shorten the time for inbred line selection by 3-4 years. Jigaoyu Line 31 is bred by improving Jigaoyu Line 3, the hybrid haploid induction rate is 13.67%, which is higher than 10.40% of Jigaoyu Line 3, and the plant height is moderate, which can be used for Haploids were produced by natural pollination in independent fields; grain markings were also more obvious, and the grain markings of Jigaoyin 3 were inconsistent with different female parent materials, which increased the difficulty of haploid grain selection. However, Jigaoyu 31 has strengthened the selection of grain markings, and has good grain markings for different female parent materials.

The source of Jigaoyu No. 31:

In 2003, the hybrids obtained from Jigaoyu Line 3 as the female parent and Stock6/M278-8-9-1-6 as the male parent were used as the basic material. Navajo purple spot markers (the aleurone layer at the top of the grains has purple markings) are mainly selected, and individual plants are tested. After 6 generations of continuous selfing and testcrossing, the ABP1 markers, pollen amount, fruiting and disease resistance of the plants are also taken into consideration. After selection, the high-frequency haploid Jigaoyu Line 31 was finally bred. Jigaoyu No. 31 showed purple plants, purple leaves, purple anthers, large amount of pollen and long flowering time in the field. Especially the Jigao induced line No. 31 has more obvious grain Navajo genetic markers than other induced lines, that is, purple aleurone layer and purple germ markers. After pollinating other maize materials with Jigaoyin Line 31, the normal diploid showed purple aleurone layer and purple germ tip, and the leaf sheath of the plant was purple, while the induced parthenocarpy kernel produced purple aleurone layer and germ tip. Colorless, green leaf sheath of the plant, can be confirmed with this mark.

Compared with the prior art, the present invention has advantages and beneficial effects: (1), the induction rate of the haploid induction line used in the method of the present invention is high, and the haploid induction rate of the Jigao induction line No. 31 used in the present invention is The average is more than 13.67%, even if the induction rate increases by 1% in this field, it is a great improvement, let alone an increase of more than 3%. Therefore, the induction rate of Jigao-induced No. 31 in the present invention is much higher than the existing induction rate Lines, such as the haploid induction rate of Jigaoyu Line 3 is 10.40%, and the haploid induction rate of Stock6/M278-8-9-1-6 is 8.58%; (2) Jigao used in the method of the present invention The high-inducing line No. 31 has more obvious grain Navajo genetic markers than other inducing lines, that is, purple aleurone layer and purple germ markers. After pollinating other maize materials with Jigaoyin Line 31, the normal diploid showed purple aleurone layer and purple germ tip, and the leaf sheath of the plant was purple, while the induced parthenocarpy kernel produced purple aleurone layer and germ tip. Colorless, plant leaf sheath green, can be confirmed with this marker, grain marker traits are obvious, haploid grains are easy to identify, and other comprehensive agronomic traits are good; (3), the induction line used in the method of the present invention is Jigaoyin Line 31 The plant height is moderate, the flowering time is long and the pollen amount is large, and natural pollination can be carried out to induce haploids, eliminating the need for artificial pollination, which not only saves labor, but also is suitable for large-scale colony operations; (4), the breeding efficiency of the method of the present invention is high, Because the haploid induction rate of the Jigao lure line No. 31 used in the inventive method is high, the progeny population obtained under the same conditions is large, and the probability of selecting an excellent inbred line increases greatly, and the breeding efficiency is improved; (5 ), the breeding cost of the inventive method is low, because the haploid induction rate is high, compared with utilizing other haploid induction lines, the required unit breeding population is relatively small, which reduces the breeding cost; (6), the inventive method The purity of the inbred line bred is high, and the purity of the hybrids prepared by it is also high, which is conducive to giving full play to the effect of heterosis; (7), the breeding procedure of the inventive method is simple, and saves the traditional method for hybrid progeny. The selfing purification process of at least 4 generations can be directly obtained by doubling the obtained haploid, and the breeding time is also greatly shortened.

detailed description

Example 1: Breeding maize inbred lines using Jigaoyu Line 31 to induce haploid hybridization

Proceed as follows:

(1) In 2010, in the Jilin Provincial Academy of Agricultural Sciences, Jigaoyu Line 31 was used as the male parent to pollinate the foreign hybrid T628394 with high yield and excellent comprehensive traits, and 248 ears were harvested from the female plant, and 42326 grains were obtained after threshing According to the traits of the grain markers, that is, the grains whose top aleurone layer and germ tip are both purple are normal hybrid diploid grains; while the top aleurone layer is purple, the germ tip is colorless, and the germ surface is small, showing a Triangular and deeply depressed marks are the induced parthenocarpic "quasi-haploid" grains of T628394; according to the above marks, a total of 6023 "quasi-haploid" grains were obtained, and the haploid induction rate of the grains = quasi-haploid Haploid kernels/number of hybrid kernels×100%=6023/42326×100%=14.23%.

(2), in the winter of 2010, the "quasi-haploid" grains screened out were sown in a single seed at the Hainan base of Jilin Academy of Agricultural Sciences, and 5847 plants emerged (haploid breeding is the selection of gametophytes, each gamete has 1 Set of chromosomes, each gene exists in a single form, and must accept environmental challenges and natural selection pressures, so many haploids cannot develop and degenerate into embryoless seeds; some haploid seeds are due to dysplasia and cannot germinate; or even if the germination is too weak and dies). At the jointing stage, haploid plants were further confirmed based on seedling growth, plant height, leaf length, leaf sheath (sheet) color, and leaf insertion angle. The haploid seedlings grow slowly, the plant height is low, the plants are thin, the leaves are short and relatively upward, and the leaf color is light; the diploid seedlings grow strong, the plants are tall and the leaves are hypertrophic, and the leaf sheaths (sheets) are purple. The haploid plant 5663 was confirmed strains, 184 diploid (hybrid) strains. Since the hybrid plants are normal diploids, their germination potential and growth potential are strong, and they can grow into normal plants through fine on-demand sowing and fine management in the field, so the number of hybrid plants also represents the number of pseudo-haploid seeds . Therefore, the haploid induction rate = the number of quasi-haploid seeds - the number of hybrid plants / the number of hybrid seeds × 100% = (6023-184) / 42326 × 100% = 13.80%. After natural doubling in the field, 455 plants with normal silking and normal flowering were obtained, and 389 selfed ears were obtained through selfing, and the fruit size of a single ear ranged from 1 to 154. The other 66 plants did not obtain selfed fruit ears due to uncoordinated flowering period.

(3) In the spring of 2011, the self-fertilized fruit ears obtained in Hainan in the winter of 2010 were planted in Jilin Academy of Agricultural Sciences into ear rows, and self-crossed, and the self-bred offspring were selected to be uniform and have good comprehensive traits. There are 24 new maize inbred lines, named Ji DH628394-1, Ji DH628394-2, Ji DH628394-3, Ji DH628394-4, DH628394-5, Ji DH628394-6, Ji DH628394-7, Ji DH628394-8, DH628394-9, DH628394-10, DH628394-11, DH628394-12, DH628394-13, DH628394-14, DH628394-15, DH628394-16, DH628394-17, DH628394-683 19. Ji DH628394-20, DH628394-21, Ji DH628394-22, Ji DH628394-23, Ji DH628394-24.

The above results show that the haploid induction rate of the method of the present invention is high, the grain Navajo genetic marker is obvious, and it is relatively easy to screen and identify haploid grains; the breeding procedure is short, the breeding speed is fast, the breeding efficiency is high, and the breeding cost is low.

Example 2: Breeding maize inbred lines using Jigaoyu Line 31 to induce haploid hybridization

Proceed as follows:

(1) In 2007, a total of 10 people with the blood of Huang Zao Si were Ji 853, Ji 854, Chang 7-2, Si 287, Jing 24, 773-2, Huang C, Huang Zao Si, Si 444, and Zhe 461. The population synthesized by artificial mixing and pollination of inbred lines (referred to as "Huang Zao Si population"). In 2010, "Huangzao Four Populations" was used as the basic material, and Jigaoyu Line 31 was used as the male parent to pollinate the "Huangzao Four Populations", and 315 spikes on the female plant were harvested, with a total of 53,897 grains. The aleurone layer was purple, the germ tip was not purple, and the germ surface was small, triangular, and deeply depressed, and other marking traits were identified, and 7589 "quasi-haploid" grains were obtained.

(2) In the spring of 2011, the selected "quasi-haploid" seeds were sown in a single seed in Jilin Academy of Agricultural Sciences, and 7431 plants emerged. The jointing stage was further confirmed according to the growth of seedlings, plant height, leaf length, and leaf color. There were 6983 ploidy plants; 247 hybrid plants with strong growth, tall plants, hypertrophic leaves and purple leaves. Since the hybrid plants are normal diploids, their germination potential and growth potential are strong, and they can grow into normal plants through fine on-demand sowing and fine management in the field, so the number of hybrid plants also represents the number of pseudo-haploid seeds. Therefore, the haploid induction rate in this experiment = the number of quasi-haploid seeds - the number of hybrid plants / the number of hybrid seeds × 100% = (7589-247) / 53897 × 100% = 13.62%. After natural doubling in the field, 636 plants with normal silking and normal flowering were obtained, and 463 selfed ears were obtained through selfing, and the fruit size of a single ear ranged from 1 to 202. The other 173 plants did not obtain selfed fruit ears due to uncoordinated flowering period and other reasons.

(3) In the spring of 2012, the self-fertilized fruit ears obtained in 2011 were planted in Jilin Academy of Agricultural Sciences into panicle rows, carefully managed in the field and self-fertilized, and the self-fertilized offspring were selected to be uniform and have good comprehensive traits. 20 new maize inbred lines, respectively named Ji DH Huang 1, Ji DH Huang 2, Ji DH Huang 3, Ji DH Huang 4, Ji DH Huang 5, Ji DH Huang 6, Ji DH Huang 7, Ji DH Huang 8. DH yellow 9, DH yellow 10, DH yellow 11, DH yellow 12, DH yellow 13, DH yellow 14, DH yellow 15, DH yellow 16, DH yellow 17, DH yellow 18, Ji DH Huang 19, Ji DH Huang 20.

The above results show that the haploid induction rate of the method of the present invention is high, the grain Navajo genetic marker is obvious, and it is relatively easy to screen and identify haploid grains; the breeding procedure is short, the breeding speed is fast, the breeding efficiency is high, and the breeding cost is low.

references

1. Coe E H. Amer Mat, 1959, 93: 381-382

2. Lashermes, P, MBeckert. Theor Appl Genet, 1988, 76: 405-410

3. ShatskayaOA, ERZabirova, VSScherbaketal. MaizeGenetCoopNewsLett, 1994, 68:51

4. Chalyk ST. Euphytica, 1994, 79: 13-18

5. Chase SS. Production of homozygous diploids of maize from monoploids [J]. Agron. J., 1952(44): 263-267

6. GayenP, JK Maden, RKumar, et al. Chromosome doubling in haploids through colchicines [J].

7. ChalykST, VVOstrovsky and VVOstrovakii. Comparison of haploidmaize plants without identical notypes [J]. J. Genet. Breed, 1993, (47): 77-80

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Claims (6)

1. utilize the method for hybridized induction monoploid selecting and breeding corn inbred line, it is characterized in that comprising the steps:

(1), with high the luring of Ji be No. 31 and make male parent to meeting the basic population of breeding objective or crossbreed pollination, gather in the crops maternal fruit ear; Therefrom screen aleurone layer top purple, plumule point is without purple, and embryo face is less, triangular in shape, the seed of the comparatively dark mark of depression;

(2), to step (1) obtain material and carry out artificial doubling or Natural double, the shooting stage removes leaf sheath and blade is the plant of purple; Selfing is carried out to the plant that can normally bloom after doubling, according to individual plant results self-fertility seed;

(3), plant step (2) gained seed, select characters of progenies performance neat and consistent, do not have separative head progeny row, be new corn inbred line.

2. in accordance with the method for claim 1, it is characterized in that described Natural double refers under field conditions (factors), monoploid seed or the intracellular chromosome doubling of seedling plants become normal dliploid.

3. in accordance with the method for claim 1, it is characterized in that described artificial doubling refers to utilize colchicine solution process monoploid seed or seedling, make its endocellular chromosome double to become normal dliploid.

4. in accordance with the method for claim 1, it is characterized in that described basic population refers to by multiple inbred line or kind after mixed pollination, the maize population of formation.

5. in accordance with the method for claim 1, it is characterized in that described crossbreed refers to single cross hybrid, triple hybrid or double cross hybrid.

6. utilize the corn inbred line produced in the method described in claim 1 to cultivate the method for corn hybrid seed, it is characterized in that with the corn inbred line cultivated in claim 1 for one of parent, with other hybridizations between selfed lines, be bred as new crossbreed.