CN107896972B

CN107896972B - Method for breeding perennial feeding coix seeds by distant hybridization

Info

Publication number: CN107896972B
Application number: CN201711096938.8A
Authority: CN
Inventors: 周树峰; 孙福艾; 郭超; 曹墨菊; 刘海岚; 兰海; 荣廷昭; 高世斌; 付凤玲; 卢艳丽
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2017-11-09
Filing date: 2017-11-09
Publication date: 2019-12-31
Anticipated expiration: 2037-11-09
Also published as: CN107896972A

Abstract

The invention discloses a method for breeding perennial coix seeds for feeding by distant hybridization. The method for breeding the perennial coix lacryma through distant hybridization comprises the following steps: and hybridizing the diploid coix seed serving as a female parent and the tetraploid coix seed serving as a male parent to obtain the triploid perennial feeding coix seed. The invention fully combines the heterosis and the characteristics of polyploidy with advanced vegetative growth, and creates a novel forage grass with double advantages; the key for finding the success of the distant hybridization among the coicis with different ploidy is the matching of the parents, the diploid coicis is suitable for being used as a female parent, and the tetraploid coicis is suitable for being used as a male parent.

Description

Method for breeding perennial feeding coix seeds by distant hybridization

Technical Field

The invention belongs to the field of plant variety breeding, and relates to a method for breeding perennial coix for feed by distant hybridization.

Background

Coix Lacryma-jobi L is a plant taxonomic genus of Poaceae Coix, also called as grass beads, Sichuan valley, Coix Lacryma-jobi, six-millet, medicinal corn, Coix Lacryma-jobi L, and the like, native China and southeast Asia such as Laos, Burma and the like, and has at least 6000-10000 years of cultivation and domestication history in China. The traditional utilization mode of the coix is used as a health food and a medicinal plant, and medical famous works such as compendium of materia medica, ben cao jing shu and the like have records that the coix is used for treating diseases such as eczema, rheumatoid arthritis, flat wart and the like. In recent years, coix seed oil and coix seed ester extracted from coix have been used for treating diseases such as cancer, and among them, a conlaite injection and a coix seed dehumidifying and pain-relieving capsule produced from coix as a main raw material have been marketed.

Coix lacryma-jobi is a cross-pollinated crop of hermaphrodite plants, which is 2-fold, has 2 n-20 chromosomes and only a small part of materials are 4-fold, and 2 n-40 chromosomes. The coix seed can be simply divided into cultivated species and wild species according to the form of the coix seed, the cultivated species is mostly provided with black or white membrane general buds outside the seeds, has good water permeability and air permeability, is easy to remove by mechanical processing, can germinate after sowing for generally 7 days, the wild species is mostly provided with black enamel general buds outside the seeds, has hard texture, poor water permeability and air permeability, is difficult to remove, the seeds are not specially treated, and the germination time after sowing can last from 10 days to more than half a year. The cultivation of the coix lacryma-jobi is mainly used for processing the coix lacryma-jobi seeds, the coix lacryma-jobi is mostly grown for 1 year, and can be regenerated after being cut in areas with better photo-thermal conditions, but the regeneration is poorer; the wild species are perennial, can be regenerated after cutting, or produce new tillers from the base in the next spring after winter. China, Yunnan, Sichuan, Guizhou, Guangxi and the like are one of the origins of the coix, and wild coix resources are very rich, are often distributed in humid zones such as river edges, pond edges, marshlands, uncultivated wastelands and the like, have rich forms and genetic variations, and have excellent moisture resistance and good disease resistance. In a natural state, wild coix seeds are propagated in two modes of seeds and roots, the coix seeds are carbon-4 plants, have high-yield biological foundation, are large in tillering, large in plant height and luxuriant in stem and leaf, and are used as excellent forage grass directly by farmers in Guangxi, Yunnan, Hunan and other places. In addition, in the minority regions such as Guangxi Liuzhou and Yunnan Xishuangbanna, the habit of picking wild coix seeds, roots, stems and the like as traditional Chinese medicines still exists so far, but the research of breeding novel pasture by utilizing wild coix is not seen.

In China, the southwest areas such as Yunnan, Qian, Guizhou, Sichuan and Yu are subtropical zone humid monsoon climate. The area has rich heat resources, except for high and cold mountainous areas, the accumulated temperature of more than 10 ℃ all the year can reach 4500-. The annual precipitation reaches 1000 mm; the solar heat and rain in the same season and early in winter and spring in the same region have the sunshine rate of less than 35 percent in most regions, are nationwide and cloudy, and are particularly suitable for the growth of perennial feeding crops mainly utilizing nutrients. On the other hand, the slope cultivated land area in the southwest region is large, and the total area of the slope cultivated land of which the elevation angle is more than 15 degrees in Yunnan, Qian, Chuan and Yu four provinces (cities) reaches 713.0 kilohm²The ecological slope land area is 48.0 percent of the total area of the slope land in China, has the characteristics of broken terrain, steep slope, shallow soil layer, loose soil, poor water retention and low fertility, and is particularly suitable for planting perennial forage grass in the areas, so that the water and soil loss is reduced, and the ecological environment is protected.

Based on this, the corn research institute of Sichuan agricultural university has pioneered the research of breeding perennial forage grass by using wild coix seed at home and abroad since 2000. The collection, arrangement and storage of wild coix seed resources are widely carried out in the origin places of wild coix, such as Yunnan, Chuan, precious places in China and southeast Asia countries such as Laos, Burma, Vietnam and the like, and the wild coix seed resource garden is established in southern corn breeding base (Gasha Zhen Manyucun in Jinghong city in Yunnan province) of Sichuan agriculture university, 223 coix seed resources are reserved in living bodies, and the researches on identification and evaluation of wild coix agronomic characters, reproductive characteristics, genetic characteristics, stress resistance, feeding characteristics and disease and pest resistance are systematically carried out. A new variety of feeding-type coix, namely Daheishan coix, with excellent comprehensive properties, perennial and high nutritive value is bred, examined by the grass variety examination committee of Sichuan province in 2016 (12 months) and participated in the national pasture grass region test in 2017. By 10 months in 2017, the variety has been popularized in southern provinces and cities (regions) such as Sichuan, Yunnan, Guizhou, Chongqing, Tibet, Hunan, Hubei and the like for more than 1 ten thousand acres in an accumulated way, and is used for breeding herbivorous animals such as cattle, sheep, rabbits, fish, guinea pigs and the like.

Disclosure of Invention

The invention aims to provide a method for breeding perennial coix seeds for feeding by distant hybridization.

The method for breeding the perennial coix lacryma through distant hybridization provided by the invention can comprise the following steps: and hybridizing the diploid coix seed serving as a female parent and the tetraploid coix seed serving as a male parent to obtain the triploid perennial feeding coix seed.

Further, after the hybridization, a step of selecting a line having a high grass yield from the triploid lines obtained by the hybridization is included.

Still further, the method comprises the steps of: in the city of Jinghong of Yunnan province or places with the same climatic conditions, diploid coix seed and tetraploid coix seed are sown in 7 months, the diploid coix seed is used as female parent and the tetraploid coix seed is used as male parent for hybridization in 10 months, the obtained seeds are cultivated, chromosome observation and molecular identification are carried out, the plant identified as triploid (2 n-30) is subjected to asexual propagation, a quality test is carried out, and the line with good resistance and high grass yield is selected. The method can be used for breeding the novel perennial coix seeds for feeding in a short time.

Wherein, the grass yield is obviously improved compared with the parent strain under the same condition.

In the method, in order to meet the florescence, the dipleg coix is preferably sown and raised about 10-15 days (such as 15 days) earlier than the quadruple coix. And transplanting the diploid coix lacryma-jobi and the tetraploid coix lacryma-jobi to a field when the seedlings grow to the stage of five leaves and one heart, wherein the plant spacing is 1.2m, and the row spacing is 1.5 m.

In the method, in order to facilitate hybridization and easy seed setting after hybridization, when the dipleg coix and the quadreg coix grow to tillering to an elongation stage, only 3-5 main stems of each plant are reserved, and the rest tillering is completely removed.

In the method, male inflorescences of male parents (namely the tetraploid coix lacryma-jobi) are tightly sleeved by kraft paper bags 48 hours before hybridization, and each paper bag is suitable for sleeving about 5 male flowers; to prevent selfing, the female parent (i.e., the diploid adlay) was tightly taped over the entire inflorescence 72h before crossing, and the male flowers were removed.

In the method, the hybridization is performed at 10: 00-12: 00, because of the large amount of pollen and high pollen activity.

In the method, when the hybridization is performed, the pollen of the tetraploid coix is pollinated to the stigma of the diploid coix for the first time, and then the pollen is supplemented once every 3 days until the stigma of the diploid coix is dry (generally, the pollen is supplemented 3 times).

In the method, seeds are harvested 40 days after the last pollination. The hybrid seeds and the two parent seeds are simultaneously cultured, for the convenience of identification, the hybrid seeds are planted in the middle, and the parent seeds are planted on the two sides. Removing the selfed seeds according to the phenotype of the parents and the hybrids; and (3) carrying out chromosome number statistics and molecular marker identification on the root tip of the hybrid, and determining successful hybridization if 30 chromosomes are obtained.

In one embodiment of the present invention, the diploid adlay is specifically adlay. The tetraploid coix is specifically tetraploid aquatic coix.

The innovation points of the invention comprise the following aspects:

1. fully combines the heterosis and the characteristics of polyploidy with advanced vegetative growth to create a novel forage grass with dual advantages.

Heterosis is the biological characteristic that the characters of the hybrid progeny such as growth potential, stress resistance, quality and the like exceed those of parents, and is ubiquitous in the plant world. Rice and corn are the most successful crops for utilizing heterosis at present, and the breeding of progeny with strong heterosis is always an important target of cross breeding. Polyploids in plants often grow vigorously and are expressed in the aspects of developed organs such as leaves, stems and the like, and polyploidy breeding is of particular significance to forage grass breeding aiming at harvesting stems and leaves. According to the invention, through distant hybridization among the coix seeds with different ploidy, the created progeny combines the advantages of hybrid and the characteristics of advanced polyploid vegetative growth, has double advantages, and is easier to breed the progeny with high forage grass yield.

2. The key for finding the successful distant hybridization among the coicis with different ploidy is the matching of the parents.

The matching of the male parent and the female parent is one of the key factors influencing the success of distant hybridization, some materials are only suitable for being used as the male parent, and some materials are only suitable for being used as the female parent, and the hybridization is not easy to succeed if the selection of the parents is improper. The research of the invention finds that in the distant hybridization of the coix, the diploid coix is suitable for being used as a female parent, the tetraploid coix is suitable for being used as a male parent, and otherwise, the hybridization fails. The discovery of the rule provides technical guidance for the reasonable matching of parents in the future coix seed distant hybridization.

Since 2014, diploid big black mountain coix lacryma-jobi (2n ═ 20) and tetraploid aquatic coix lacryma-jobi (2n ═ 40) are used as parents for distant hybridization, so that a triploid coix lacryma-jobi (2n ═ 30) with the somatic cell chromosome number of 30 is obtained, the triploid coix lacryma-jobi is expressed as prominent stress resistance, vigorous growth, developed nutrition and better grass yield than parents, the triploid coix lacryma-jobi is qualified to participate in the national pasture regional test in 2018 years and is expected to be examined and determined by the examining and approving committee of the grass variety in Sichuan province at the end of 2017.

Drawings

FIG. 1 shows the phenotype and number of root tip chromosomes of the parent and hybrid F1. In the figure, YY1 is diploid big black mountain coix lacryma-jobi, which is used as a female parent; YY12 is tetraploid aquatic Coicis semen as male parent; f1 is hybrid No. B2.

FIG. 2 is the genomic in situ hybridization identification of distant hybrids. A: performing non-blocking hybridization by taking female parent Daheishan coix as a probe; b: hybridization is carried out by taking female parent Daheishan coix as a probe and male parent tetraploid aquatic coix as a block.

FIG. 3 shows the result of identifying a portion of SSR primers in maize. In the figure, YY1 is female parent Daheishan Job's tears, YY12 is male parent tetraploid aquatic Job's tears, B1, B2 and B3 are hybrids F1.

FIG. 4 shows the application of distant hybrid in production, which is planted in the village before the Huyingzhen county of GuanLing, Guizhou province 3 and 20 days in 2017, and grows 28 days in 8 months.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Adlay: is recorded in the report of animal husbandry in China for 1 month and 8 days in 2017; sichuan rural daily newspaper 2017, 1 month and 12 days; the nutritional value of the whole plant of the large black mountain coix lacryma-jobi sun-dried in Shandong institute, Tiangang, Yubing and the like on the growing meat rabbits is evaluated, the grass science, 2017,34(5):1100 + 1106 ", the public can obtain from the applicant and can only be used for repeated experiments.

Example 1 planting of hybrid parent seedlings

In the city of Jinghong city of Yunnan province, seeds of Job's tears (diploid) of big black mountain are soaked in water for 10 days in 7 months, then sowed in a nursery pot with the size of 10cm multiplied by 10cm, and a small amount of phoxim granules are sprinkled after sowing to prevent and control underground pests such as cutworms and the like. The seeding time of the tetraploid aquatic coix is 15 days later than that of the diploid Daheishan coix. When the seedlings grow to the period of five leaves and one core, the big black-mountain coix and the tetraploid aquatic coix are both transferred to the field (the tetraploid aquatic coix is transplanted 10-15 days earlier than the big black-mountain coix because the seed is sowed 10-15 days earlier), the plant spacing is 1.2m, and the row spacing is 1.5 m.

Example 2 field management of hybrid parents before flowering

0.1kg of compound fertilizer is applied to each plant in a hole at the 6-leaf stage, 0.1kg of urea is sprayed before and after rainwater at the jointing stage, redundant tillers are removed for several times from tillering to the jointing stage, and each plant only keeps 3-5 growing strong main stems. Depending on the soil condition, intertillage and soil loosening are carried out once, and omethoate is sprayed before blooming to prevent and control aphids and stem borers.

Example 3 remote hybridization

During hybridization, the Daheishan coix seed is used as a female parent, and the tetraploid aquatic coix seed is used as a male parent. In order to prevent the pollution of the mixed pollen, the male inflorescences of the male parents are tightly sleeved by kraft paper bags 48 hours before hybridization, and each paper bag is suitable for sleeving about 5 male flowers; to prevent selfing, the female parent was tightly taped throughout the inflorescence 72h before crossing and the male flowers were strictly removed. Hybridization to 10: 00-12: 00, the powder dispersing amount is large and the pollen activity is high. Gently shaking the kraft bag of the male parent plant, taking fresh pollen, pollinating the stigma of the female parent, allowing the pollen to be excessive, and tightly covering the kraft bag of the female parent plant after pollination. Supplementing pollen once every 3 days for 3 times until the stigma of the female parent is dry, and removing the newly grown male flowers in the cow skin bag during each pollination.

Example 4 statistics of hybrid seed set percentage and hybrid seedling acquisition

And harvesting the seeds 40 days after the last pollination. The buds of wild coix seeds become black after fertilization, while the buds of unfertilized seeds become white, so that whether the seeds are fertilized or not can be identified according to the characteristic. As shown in Table 1, when diploid Heijishan Job's tears and tetraploid aquatic Job's tears are hybridized in a distant source, the tetraploid aquatic Job's tears can only be used as male parents, and the hybridization is not successful when the tetraploid aquatic Job's tears are used as female parents. In this test, when tetraploid coix lacryma-jobi is used as male parent, the number of pollinated flowers is 1123, the number of total bud blackened seeds is 10, and the distant hybridization seed setting rate is 0.89%. As the parents of the hybrid are far away in genetic relationship, the problems that the mutual identification of pollen of the male parent and stigma of the female parent is difficult, pollen cannot germinate, pollen tube growth is malformed, male and female gametes cannot normally combine, and the early development stage of an embryo after combination is easy to die exist, so that the setting rate of the distant hybridization is very low, and the research result conforms to the general rule of the distant hybridization.

Soaking the parent seeds and the hybrid seeds in water for 10 days, sowing the seeds into a seedling pot with the size of 10cm multiplied by 10cm, and spraying a small amount of phoxim granules after sowing to prevent and control underground pests such as cutworms and the like. The parent seeds can germinate, while only 5 seeds of 10 hybrid seeds germinate, and 1 seed of the 10 hybrid seeds dies after emergence of seedlings, which is probably caused by physiological incoordination of early development stages of distant hybrids (the 4 hybrid seedlings are numbered as B1, B2, B3 and B4 respectively). When the parents and the 4 hybrid seedlings grow to the period of five leaves and one heart, the hybrid seedlings are moved to the field, the plant spacing is 1.2m, and the row spacing is 1.5 m.

TABLE 1 influence of hybridization patterns on seed setting rate of Coix lacryma-jobi distant hybridization

Hybridization method	Number of pollinated flowers	Number of seeds turned black	Setting percentage
				YY1×YY12	1123	10	0.89％
YY12×YY1	1023	0	0

Note: in the table, YY1 is diploid Daheishan Job's tears, YY12 is tetraploid aquatic Job's tears.

Example 5 phenotypic identification of distant hybrids

In the research, the parents of the hybridization are euploid, wherein the male parent tetraploid aquatic coix has 40 chromosomes, the female parent Daheishan coix has 20 chromosomes, both can normally disperse powder and fruit, and the number of the chromosomes of the hybrid of the two is 30, the hybrid is aneuploid and is represented as sterility and sterility, and the male flower glume cannot be opened and does not disperse powder. Therefore, whether the hybridization is successful or not can be preliminarily judged through the pollen scattering property of the male flowers of the hybrid offspring. As can be seen from Table 2, the male parent and the female parent can normally loose pollen, the number of root tip chromosomes of the male parent and the female parent respectively conforms to the actual expectation, in 4 hybrids, B1, B2 and B3 glumes cannot open and loose pollen, microscopic examination shows that the chemicals are abnormally developed, the identification result of the number of the root tip chromosomes is 30, the hybrids are real hybrids, B4 loose pollen is normal, the number of the root tip chromosomes is 20, the phenotypes of the root tip chromosomes are basically consistent with that of the female parent, and the pollen of the female parent is presumed to be caused by selfing in the pollination process.

TABLE 2 pollen Scattering Properties and root tip chromosome numbers of parents and hybrid progeny

Note: in the table, YY1 is diploid Daheishan Job's tears, YY12 is tetraploid aquatic Job's tears, and B1, B2, B3 and B4 are F1 generation of cross

The phenotype of parents and progeny of the hybrid at the seedling stage is shown in FIG. 1, and it can be seen that hybrid F1 (which is a B2 single plant) shows the phenotype of parents. Meanwhile, the agronomic traits of the three are compared (Table 3), the plant height, tillering number, leaf length, stem thickness and exposed node number of the hybrid F1 exceed those of parents, and the leaf width, leaf length, leaf angle and high point length are between those of the parents.

TABLE 3 comparison of partial agronomic traits for the parent and hybrid F1

Note: in the table, YY1 is diploid adlay; YY12 is tetraploid aquatic Coicis semen; f1 is the average value of B1, B2 and B3 single plants, wherein the capital letters represent that P is less than 0.01 significant, and the lowercase letters represent that P is less than 0.05 significant.

Example 6 genomic in situ hybridization identification of distant hybrids

We used the method of in situ hybridization of genome to identify the genetic composition of hybrid. In the test, the genome of the big black coix lacryma-jobi marked by digoxin is taken as a probe (fluorescence is green signal), and the polyploid offspring F1 is subjected to genome in-situ hybridization without blocking. The result is shown in A in FIG. 2, almost all green dense hybridization signals are provided on 30 chromosomes in the metaphase of mitosis of the polyploid offspring F1, and the signal intensity is uniform, which indicates that F1 contains the genetic material of the female parent Daheishan Coix lacryma-jobi, and the chromosomes are all full of hybridization signals, and probably the parental genomes have higher homology. In order to further explore the source situation of polyploid F1 genetic materials, the female parent large black mountain coix seed total genome is used as a probe, the male parent tetraploid aquatic coix seed total genome is used as blocking DNA, genome in-situ hybridization is carried out on F1 metaphase chromosomes again, and as a result, shown as B in figure 2, the fragmented male parent tetraploid aquatic coix seed total DNA is preferentially hybridized with a target chromosome and then is hybridized with the female parent large black mountain coix seed total DNA, 20 chromosomes of polyploid offspring F1 are covered by intense and dense green hybridization signals, and 10 chromosomes almost have no green hybridization signals, which indicates that F1 contains the genetic materials of parents.

Example 7 SSR molecular identification of distant hybrids

Searching for corn SSR primers published on the mail GDB (www.maizegdb.org) website, and selecting primers of chromosome 2 part for screening, wherein the primers umc1542 and umc2247 have good amplification effect, umc2251 has no band, and the rest 17 pairs of primers are seriously dragged. Polymorphism is amplified by two pairs of polymorphic primers umc1284 and umc1335 on coix, primer umc2247 amplifies two bands in both female parent and male parent, primer umc1284 amplifies one band in female parent big black mountain coix, three bands are amplified in male parent tetraploid aquatic coix, three hybrids B1, B2 and B3 integrate the band types of the female parent and the male parent, and the result shows that the hybrids contain genetic materials of both parents, and the amplification result is shown in figure 3.

Example 8 comparison of the yields of parent and hybrid grass

Identifying female parent Daheishan Coix lacryma-jobi, male parent tetraploid aquatic Coix lacryma-jobi and 3 plants as real hybrid F1, performing asexual propagation by cuttage method, and performing quality test. The test is designed according to random block, 3 times of repetition and 72m of cell area²The row spacing of the plants is 1.2 m. The grass yield is the cumulative fresh grass yield after 3 mows.

As can be seen from Table 4, hybrids B1, B2 and B3 all show strong vegetative hybrid vigor, the fresh grass yield is remarkably higher than that of parents, the maximum fresh grass yield of B3 is 11.21 t/mu, and the yield is increased by 35.9% compared with that of female parent large black mountain coix seeds, which indicates that the breeding of high-yield forage grass by distant hybridization among different ploidy coix seeds is feasible. At present, B3 is already applied to production, and has the characteristics of high yield and high quality, no pest and disease damage needs to be prevented and controlled in the whole growth period, and FIG. 4 shows the large-area demonstration of the village before the Huiying town of Guanling county of Guizhou province.

TABLE 4 comparison of grass yield (unit: t/mu) for parents and hybrid F1

	I	II	III	Average grass yield
					YY1	8.33	7.97	8.45	8.25Cd
YY12	6.23	5.44	7.08	6.25De
					B1	9.34	9.76	10.4	9.83Bc
B2	8.99	10.23	10.79	10.00Bb
					B3	10.43	11.40	11.79	11.21Aa

Note: in the table, YY1 is female parent adlay, YY12 is male parent tetraploid aquatic adlay, B1, B2 and B3 are hybrids F1, the capital letter represents that P is less than 0.01 obviously, and the lowercase letter represents that P is less than 0.05 obviously.

Claims

1. A method for breeding perennial feeding coix by distant hybridization comprises the following steps: hybridizing diploid coix seeds serving as female parents and tetraploid coix seeds serving as male parents to obtain triploid perennial feeding coix seeds;

the diploid coix is big black coix;

the tetraploid coix is tetraploid aquatic coix.

2. The method of claim 1, wherein: after the hybridization, a step of selecting a line having a high grass yield from the triploid lines obtained by the hybridization is further included.

3. The method of claim 1, wherein: the method comprises the following steps: in the city of Jinghong city of Yunnan province or places with the same climatic conditions, the diploid coix seed and the tetraploid coix seed are sown in 7 months, the diploid coix seed is used as a female parent and the tetraploid coix seed is used as a male parent for hybridization in 10 months, the obtained seeds are bred, identified as triploid plants are subjected to asexual propagation, a quality ratio test is carried out, and a strain with high grass yield is selected.

4. The method of claim 1, wherein: the double-time coix seeds are sown 10-15 days earlier than the quadruple-time coix seeds.

5. The method of claim 1, wherein: when the dipleg coix and the quadruploid coix grow to tillering to an elongation stage, only 3-5 main stems of each plant are reserved, and the rest tillering is completely removed.

6. The method of claim 1, wherein: the hybridization was performed at 10: 00-12: 00 is carried out.

7. The method of claim 1, wherein: when the hybridization is carried out, the pollen of the tetraploid coix is firstly pollinated to the stigma of the diploid coix, and the method also comprises the following step of supplementing the pollen once every 3 days until the stigma of the diploid coix is dried.