CN110622850B - Sexual reproduction artificial crossbreeding method for fine purified variety and wild variety of sargassum fusiforme - Google Patents

Sexual reproduction artificial crossbreeding method for fine purified variety and wild variety of sargassum fusiforme Download PDF

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CN110622850B
CN110622850B CN201911080563.5A CN201911080563A CN110622850B CN 110622850 B CN110622850 B CN 110622850B CN 201911080563 A CN201911080563 A CN 201911080563A CN 110622850 B CN110622850 B CN 110622850B
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wild
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sargassum fusiforme
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林立东
吴明江
马增岭
陈斌斌
郑影
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Marine And Fishery Development Research Center Of Dongtou District Wenzhou City
Wenzhou University
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Wenzhou Dongtou District Aquatic Product Science And Technology Institute
Wenzhou University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

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  • Marine Sciences & Fisheries (AREA)
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Abstract

The invention provides a sexual reproduction artificial hybridization breeding method for a sargassum fusiforme excellent purified strain and a wild strain, which is characterized by comprising the following steps: the method comprises the steps of carrying out isolated hanging culture on juvenile sporophytes of a sargassum fusiforme purified type and a wild type strain until a breeding support develops and matures, carrying out indoor hybridization breeding on the obtained sargassum fusiforme excellent purified type strain and the wild type strain, carrying out indoor artificial temporary culture on hybrid filial generations of the sargassum fusiforme excellent purified type strain and the wild type strain, carrying out natural sea area culture and daily management on the hybrid filial generations of the sargassum fusiforme excellent purified type strain and the wild type strain, carrying out backcrossing on the hybrid filial generations of the sargassum fusiforme and the wild type parent strain, and carrying out sexual reproduction and selfing on the hybrid strains of the sargassum fusiforme and the wild type parent.

Description

Sexual reproduction artificial crossbreeding method for fine purified variety and wild variety of sargassum fusiforme
Technical Field
The invention relates to a sexual reproduction artificial crossbreeding method for a fine purified variety of sargassum fusiforme and a wild variety, which highlights a new method for fusing a modern crossbreeding technology, an ecological breeding technology and a molecular detection technology.
Background
Hizikia fusiforme (B)Sargassum fusiforme) Is a sea tangle (Saccharina japonica) Shihua vegetable (a)Gelidium amansii) And Undaria pinnatifida (Undaria pinnatifida) Then, the large-scale economic seaweed is artificially cultivated on a large scale. The cave head region of Wenzhou city in Zhejiang province is the only industrial base in China for artificial breeding, cultivation, processing and product export of the sargassum fusiforme with the largest internal sales, the annual cultivation area is more than 1.1 ten thousand mu, the annual sargassum fusiforme primary agricultural product is more than 7000 tons, the annual export crude product is more than 2500 tons, and 90% of the crude product is packaged in small bags and exported to Japan.In recent years, China has remarkable effect on the research and development of the excellent variety of Hizikia fusiforme, and the technological level is ahead of that of the Japanese and Korean countries. At present, the research of cross breeding of terrestrial crops such as rice, wheat, sweet potato and the like in China is the earliest and precedes the world, and particularly in the field of the research of cross breeding of rice, the research of cross breeding of rice is developed to the level of molecular breeding. However, in the case of large brown algae such as sargassum fusiforme, kelp and wakame, the crossbreeding research is in the initial development stage, and a reproducible, generalizable and expandable crossbreeding technology is still lacking. The method absorbs and references the mature technical experience of hybridization breeding of high-grade crops, and develops the technical research of hybridization breeding of the sargassum fusiforme in a rolling way, thereby being beneficial to making up the technical defects of hybridization breeding of macroalgae in China and promoting the quality improvement and the efficiency increase and the sustainable healthy development of the sargassum fusiforme industry.
An artificial cross breeding method for the sexual reproduction of the fine purified Yangxicai strain and wild-type strain includes such steps as ecological isolation of the purified Yangxicai strain from the natural sea region of wild-type strain, artificial culture of the mature sporophyte in air bag and stem, artificial screening and marking of the natural sea region of fine Yangxicai strain, hybridization between said fine purified Yangxicai strain and wild-type strain, purifying and hybridization between said fine purified Yangxicai strain and wild-type strain, artificial culture of the hybridization between them in room, and daily management, backcrossing the filial generation of the sargassum fusiforme with the wild parent with excellent biological character, sexual reproduction and selfing breeding of the hybrid strain of the sargassum fusiforme and the backcrossed strain of the wild parent, evaluating the genetic stability of the excellent purified strain, the wild strain, the hybrid strain and the backcrossed strain of the sargassum fusiforme, evaluating the quality of the excellent purified strain, the wild strain, the hybrid strain and the backcrossed strain of the sargassum fusiforme, comparing the quality with the difference and the like 13. How to implement the normative operation in the above development stages and highlight the systematicness of selection, cultivation, breeding, propagation, promotion and evaluation in the hybridization breeding process of the sargassum fusiforme is the technical problem to be solved by the invention.
Disclosure of Invention
Based on the problems, the invention provides a brown algae crossbreeding method for breeding a new sargassum fusiforme strain with high unit yield, strong resistance, stable character inheritance and excellent quality. The method establishes a scientific sargassum fusiforme crossbreeding method, and provides a solid technical support for the unit yield improvement, the product quality improvement and the sustainable and healthy industrial development of artificially cultured sargassum fusiforme.
In order to solve the above problems, the present invention provides the following technical solutions: a sexual reproduction artificial crossbreeding method for a sargassum fusiforme excellent purified strain and a wild strain is characterized by comprising the following steps:
a. isolated hanging culture of sargassum fusiforme purified type and wild type strain juvenile sporophyte
Selecting a purified strain and a wild strain of the sargassum fusiforme with the same or similar leaf shape characteristics at the juvenile sporophyte stage, placing the purified strain and the wild strain in natural sea areas for ecological isolation artificial cultivation, wherein in order to ensure the purity of the sargassum fusiforme purified strain and the wild strain, each strain is at the beginning of 10 months, the seedling of each strain is clamped on a fiber rope with the diameter of 0.5 cm and the length of 3.3 m, the seedling distance is 20-25 cm, the single rope of a soft raft frame is horizontally hung, the seedling clamping rope distance is 4 m, the purified strain and the wild strain are hung and cultivated in the same sea area in an ecological isolation mode, and the artificial cultivation is carried out for 5-6 months in the next year;
the Hizikia fusiforme is a special large brown alga at northwest coast of the Pacific ocean, and has wild population distribution in the northern sea island south of the North sea road and the southern sea island of the Renzhou peninsula of the Hainan province of China. The artificially cultivated hizikia fusiforme in China has the highest yield and the best quality in the coastal area of Zhejiang. The cave head region of the Wenzhou city is the largest Hizikia fusiforme artificial breeding place in China, 14 existing purified strains are provided, 4 strains with high value-added economic value are provided, the strains are derived from coastal islands in various regions of northeast Asia, and the artificial collection with wild seedlings as seedling sources is started in the early 90 th century. The biological characters of the wild sargassum fusiforme in different producing areas are different, in order to highlight the economic value of the artificially cultured sargassum fusiforme strain, the invention selects the purified strain and the wild strain with excellent biological characters and high economic value to carry out cross breeding, and selects the purified strain and the wild strain with the same or similar leaf shape characteristics in the juvenile sporophyte stage of the sargassum fusiforme to carry out excellent plant screening and collection.
The ecological isolation artificial breeding of the natural sea area of the sargassum fusiforme is beneficial to preventing the natural drift from mixing with each other to influence the purity of the purified strain and the wild strain. Therefore, the purified strain and the wild strain are hung and raised in different sea areas of the same region, and different racks are hung and raised between the purified strain or the wild strain in the same sea area. The good natural habitat is favorable for the healthy growth of the sargassum fusiforme, so that the conditions of the length of seedling clamping ropes, the distance between seedlings, the distance between ropes and the like are in accordance with the ecological effect in the process of hanging and culturing the soft raft frame, and the healthy growth and development of different strains of the sargassum fusiforme are guaranteed.
b. When the hizikia fusiforme excellent purified type and wild type strains grow into mature sporophytes, the characteristics of the air sacs and stems of the mature sporophytes are as follows: the air bag body of the hizikia fusiforme cluster air bag mainly comprises a rod-shaped air bag body and an oval air bag body, wherein the standard of the excellent rod-shaped air bag body (fresh body) in the mature period is as follows: the length of the capsule body is more than or equal to 12.5 mm, and the width of the capsule body is more than or equal to 3.5 mm; the excellent ovate capsule (fresh body) in the mature period has the standard that the capsule length is more than or equal to 10.0 mm, and the capsule width is more than or equal to 4.5 mm; the method comprises the following steps of (1) artificially screening female and male strains of sargassum fusiforme with same or similar biological properties and excellent wild type properties in 3-4 months, carrying out numbering and marking on natural sea areas through floats or beverage bottles, and continuously observing growth and development conditions until a reproductive support is mature;
c. b, hybridizing the excellent purified strains of the sargassum fusiforme obtained in the step b with wild strains indoors
At the beginning of 5 months, namely the mature period of the local sargassum fusiforme in the cave head region of the Wenzhou city, female strains and male strains of the purified strains with good biological characters and wild strains which are marked in advance are respectively collected, and sexual reproduction hybridization is carried out according to the ratio of female: male =1:1 ratio; the single strain of sargassum fusiforme is subjected to sexual reproduction purification by matching of male parent A, male parent B, male parent C and male parent C; the method comprises the following steps of (1) adopting a purification type of male parent and female parent and a purification type of male parent and wild parent for sexual reproduction hybridization among single lines; performing sexual reproduction hybridization among multiple lines by adopting a purified type A ^ wild type A ^ purified type A; a male parent purified type B lonely ±, a wild type B; a male parent purified type C loner |, a male parent wild type C loner lonerpurified type C; a male parent purified type B lonely ±, a wild type A; a male parent purified type C loner |, a male parent wild type C loner |, a purified type a; the male parent purified type B lonely ═ purified type C, and the like.
Indoor cross breeding environment and culture medium selection: the illumination intensity is as follows: 200-230 μm/m2/s, photoperiod: 12:12, seawater salinity: 30-33 per mill, seawater pH value: 7.8-8.3, room temperature: 24-25 ℃, indoor temporary rearing time: 7-10 days, replacing seawater every day: 1-2 times, and assisting aeration to dissolve oxygen; selecting 12-14 pieces of culture medium, a single piece of cotton with the width of 2 cm, 20-30% of cotton and 70-80% of fiber, a seedling attaching curtain or a seedling attaching disc consisting of a plastic frame shaped like a Chinese character 'tian' and two ends of which are respectively supported by a galvanized steel wire, and a cotton rope consisting of 20-30% of cotton and 70-80% of fiber, and numbering and marking to distinguish different strains;
d. indoor artificial temporary breeding of hybrid filial generation of fine purified sargassum fusiforme strain and wild strain
And c, after concentrated release of the sperm and eggs, manually collecting the germ cells by using a 260-280-mesh silk screen, uniformly spreading and spraying the germ cells on a culture medium which is injected with fresh seawater in advance, placing the culture medium on a seedling attaching curtain or a seedling attaching disc, statically culturing for 1 day, then placing the culture medium into a seawater manual aeration device, continuously culturing for 7-10 days, replacing fresh seawater for 1 time every day, and manually removing the germ cells and the silt attached to the surfaces of the germ cells by using auxiliary high-pressure water spraying equipment (a high-pressure spray pot or a water gun). Transferring a seedling attaching curtain or a seedling attaching plate attached with the sargassum fusiforme hybrid strain embryo to a natural sea area for culturing when the sargassum fusiforme hybrid strain embryo grows to 1-2 mm;
e. breeding and daily management of excellent purified Cyrtymenia Sparsa strain and wild type strain hybrid progeny in natural sea area
And (3) hanging seedling attaching curtains or seedling attaching plates attached with the sargassum fusiforme hybrid strain embryos on a centipede frame culture raft frame, wherein the seedling attaching surfaces face the sun, and the sinking water depth is 10-15 cm. Daily management: removing attached sediment by a high-pressure water gun, and manually removing the sessile macroalgae; soaking the seedling attaching curtain or seedling attaching tray for 5-7 min in 8% citric acid solution, cleaning and deacidifying thoroughly, and removing common parasitic algae, namely Japanese multitubular (Polysiphonia japonica, commonly known as red hair or monkey hair); when the typhoon passes through the border, the seedling attaching curtain or the seedling attaching tray is placed in a laboratory or a refrigerating chamber for temporary storage, and the seedling attaching curtain or the seedling attaching tray is hung and placed in a natural sea area again after the typhoon leaves the border. The sargassum fusiforme hybrid strain embryo is cultured for 120-150 days (from the first month to the end of 9 months) through artificial culture in a natural sea area, and can grow and develop into juvenile sporophytes with the length of 10-15 cm;
f. backcrossing of hybrid filial generation of sargassum fusiforme with wild-type parent with excellent biological characteristics
In the process of long-term artificial purification and cultivation, genes such as wind wave resistance, morphological characteristics, reproductive capacity, growth length and the like are easily caused, and the genetic gene on one aspect is closed to express or lack, so that the strain characteristics are gradually degenerated or mutated. In order to obtain the whole genome of the wild type excellent strain, the invention carries out successive purification of the excellent purified strain, the wild type strain and the hybrid strain, and simultaneously carries out backcross of the wild type parent from the third generation of the hybrid strain, and continuously carries out 3-4 life cycles so as to stably obtain the whole genome of the wild type strain. The genetic ratio of the hybridized filial generation of the sargassum fusiforme and the backcross of the wild type parent adopts male parent A ^ wild type; the male parent and wild type B ^ ^ B hybrid progeny B-B ^ B wild type B; backcross combinations of male parent wild type C ^ Rough and male parent filial generation C-C, female parent filial generation C-C ^ Rough and wild type C and the like;
g. sexual reproduction selfing breeding of sargassum fusiforme hybrid line and wild type parent backcross line
After the hybrid line and the wild-type parent backcross line are continuously implemented for 3-5 life cycles, excellent biological character plants (male plants and female plants) are selected, the backcross line is bred through sexual reproduction and selfing, and selfing combinations of backcross filial generation A ≤ backcross filial generation A, backcross filial generation B ≤ backcross filial generation B, backcross filial generation C ≤ backcross filial generation C and the like are adopted to purify and propagate filial generations, so that a large number of excellent plants are obtained.
Detailed Description
The present invention will be described in detail with reference to specific embodiments in order to make the objects, technical solutions and innovations of the present invention clearer and easier to understand.
Example 1 was carried out: the purified single strain of sargassum fusiforme is artificially crossbred with the sexual reproduction of the wild single strain. This example was carried out by sexual reproduction artificial cross breeding of a fine purified strain of Hizikia fusiforme with a wild-type strain.
As a first embodiment of the invention, the method is suitable for sexual reproduction artificial crossbreeding of the sargassum fusiforme excellent purified type line and the wild type line, and mainly comprises the following steps:
1. sargassum fusiforme purified strain biological character
1.1 juvenile sporophyte leaf shape: the leaf shape of the Yangxicai purified strain at the juvenile sporophyte stage is characterized by flat-edge leaf shape, and manual screening, classification and seedling clamping are carried out;
1.2 mature sporozoite stage air pocket: the shape of the sac body of the clustered air sac at the mature period of the sargassum fusiforme mature sporophyte is rod-shaped or oval, and each clustered air sac comprises 1-2 clustered air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.5 mm, and the width of the capsule body is more than or equal to 3.7 mm; the excellent strain of the oval air cell has the characteristics of mature period: the length of the capsule body is more than or equal to 11.0 mm, and the width of the capsule body is more than or equal to 4.7 mm.
1.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 3.0 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 3.5 mm.
2. Biological character of wild type strain of sargassum fusiforme
2.1 juvenile sporozoite stage leaf shape: the leaf shape of wild type juvenile sporophyte of the sargassum fusiforme is characterized by flat margin, and manual classified screening, collection and seedling clamping are carried out according to the leaf shape characteristics of wild type juvenile sporophyte in different producing areas;
2.2 mature sporozoite stage air sac: the air sac body of the wild type excellent hizikia fusiforme strain is rod-shaped or oval-shaped, and each cluster air sac comprises 1-2 branches of cluster characteristic air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.0 mm, and the width of the capsule body is more than or equal to 3.0 mm; the maturation period of the oval air sac excellent strain is characterized in that: the length of the capsule body is more than or equal to 10.0 mm, and the width of the capsule body is more than or equal to 3.7 mm.
2.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 2.7 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 2.8 mm.
3. Natural sea area ecological isolation artificial breeding of sargassum fusiforme purified strain and wild strain
In order to ensure the purity of the purified strains and the wild strains of the sargassum fusiforme, each strain is at the beginning of 10 months, the separate strains clamp seedlings on fiber ropes with the diameter of 0.5 cm and the length of 3.3 m, the seedling spacing is 20 cm, the flexible raft frames are horizontally hung by single ropes, the seedling clamping rope spacing is 4 m, the purified strains and the wild strains are ecologically isolated, hung and cultured in different sea areas in the same region, and the strains are artificially cultured to the beginning of 5 months next year.
4. Excellent purified and wild type strains of Hizikia fusiforme mature sporophyte air sac and stem characteristics
The air bag body of the hizikia fusiforme cluster air bag mainly comprises a rod-shaped air bag body and an oval air bag body, wherein the standard of the excellent rod-shaped air bag body (fresh body) in the mature period is as follows: the length of the capsule body is more than or equal to 12.5 mm, and the width of the capsule body is more than or equal to 3.5 mm; the excellent ovate capsule (fresh body) in the mature period has the standard that the capsule length is more than or equal to 10.0 mm, and the capsule width is more than or equal to 4.5 mm.
5. Artificial screening and marking of natural sea area of fine strains of sargassum fusiforme
The method comprises the steps of artificially screening female and male strains of the sargassum fusiforme with same or similar biological properties, namely purified and wild excellent properties at the beginning of 3 months, and numbering marks (floats or beverage bottles) in natural sea areas, and continuously observing growth and development conditions until the development of a reproductive support is mature.
6. Cross breeding environment and culture medium condition in excellent purified sargassum fusiforme strain and wild type strain sexual reproduction chamber
The illumination intensity is as follows: 200 μm/m2(s), photoperiod: 12:12, seawater salinity: 30 per mill, seawater pH value: 7.8, room temperature: 24 ℃, indoor temporary rearing time: 7 days, seawater change daily: and (5) carrying out auxiliary aeration for dissolving oxygen for 1 time. The culture medium is 12 strips with single width of 2 cm,20 percent of cotton and 80 percent of fiber, seedling attaching curtains or seedling attaching trays which are respectively formed by two ends of each seedling attaching tray by a galvanized steel wire as a support or cotton ropes formed by 20 percent of cotton and 80 percent of fiber, and different strains are marked and distinguished by numbers.
7. Purification of hizikia fusiforme excellent purification type strain and wild type strain and hybridization sexual reproduction ratio
In the early morning of the fifth month each year, namely the mature period of the local sargassum fusiforme in the cave head region of the Wenzhou city, female strains and male strains of the excellent biological character purified strains and wild strains marked by the marks are respectively collected according to the ratio of female: male =1:1 ratio. Cyrtymenia Sparsa single strain sexual reproduction purificationA⊕♂A、♀B⊕♂BProportioning; sexual reproduction hybridization between two strainsPurification type⊕♂Wild type，♀Wild type⊕♂Purification typeProportioning.
8. Indoor artificial temporary breeding of hybrid filial generation of hizikia fusiforme excellent purified strain and wild strain
The indoor environmental factor conditions are the same as in step 6. After concentrated release of sperm eggs, collecting fertilized eggs by using a 260-mesh silk screen manually, uniformly spreading and sprinkling fresh seawater injected in advance, placing on a culture medium attached with a seedling curtain or a seedling attached plate, statically culturing for 1 day, then placing into a seawater manual aeration device, continuously culturing for 7 days, replacing fresh seawater 1 time every day, assisting a high-pressure water spraying device (a high-pressure spray pot or a water gun), and manually removing silt attached to the surfaces of the fertilized eggs and embryos. And transferring the seedling attaching curtain or the seedling attaching plate attached with the sargassum fusiforme hybrid strain embryo to a natural sea area for culturing when the sargassum fusiforme hybrid strain embryo grows to 1 mm.
9. Artificial cultivation and daily management of natural sea area of hybrid filial generation of fine purified strains and wild strains of sargassum fusiforme
And (3) hanging seedling attaching curtains or seedling attaching plates attached with the sargassum fusiforme hybrid strain embryos on a centipede frame culture raft frame, wherein the seedling attaching surfaces face the sun and the sinking water depth is 10 cm. Daily management: removing attached sediment by a high-pressure water gun, and manually removing the sessile macroalgae; soaking the seedling adhering curtain or disk in 8% citric acid solution for 5 min, cleaning, deacidifying, and removing common parasitic algaeThe main tubes (Polysiphonia japonicaIn common terms, the method is as follows: red hair or monkey hair); when the typhoon passes through the border, the seedling attaching curtain or the seedling attaching tray is placed in a laboratory or a refrigerating chamber for temporary storage, and the seedling attaching curtain or the seedling attaching tray is hung and placed in a natural sea area again after the typhoon leaves the border. The hybrid sargassum fusiforme strain embryo can grow and develop into juvenile sporophyte with the length of about 10 cm after being artificially cultured for 120 days in a natural sea area.
10. Backcrossing of hybrid filial generation of sargassum fusiforme with wild-type parent with excellent biological characteristics
From the third generation of the hybrid strain, 2, 3 and 4 steps are repeatedly adopted to screen excellent biological character plants of the excellent wild type strain parent and the hybrid strain parent (third generation, fourth generation and fifth generation … …), and the best biological character plants are implementedWild type A⊕♂Hybrid progeny A-A，♀Hybrid progeny A-A⊕♂Wild type ABackcross combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the excellent wild-type strain and the hybrid strain is synchronously performed.
11. Sexual reproduction selfing breeding of sargassum fusiforme hybrid line and wild type parent backcross line
Backcrossing 3 generations of excellent wild-type lines, new lines and hybridized lines, sexual reproduction, selfing, and collecting their male and female parentBackcross progeny A⊕♂Backcross progeny AAnd (4) selfing combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the wild-type strain and the hybrid strain is synchronously performed.
12. Evaluation of genetic stability of hizikia fusiforme excellent purification type strain, wild type strain, hybrid type strain and backcross type strain
And (3) carrying out artificial purification on the purified strains, the wild strains and the hybrid strains for three consecutive generations, and monitoring and comparing the characteristics of biological characters (leaf shapes, air sac shapes, lengths, yields and the like) among the three strains. Selecting excellent plants of purified, wild and hybrid strains with the same or similar biological characters in each generation, implementing simplified genome sequence (DNA) and SSR molecular markers, and comparing genetic characteristics. And comprehensively judging the genetic stability of the hybrid strain according to the biological character characteristics, simplified genome (DNA) sequence differences and SSR molecular marker results. Of hybrid strains of Cyrtymenia SparsaAnd (3) carrying out backcrossing of the parental lines after the fourth generation starts for three consecutive generations, namely: female parentHybrid lines⊕♂Parental strain，♀Parental strain⊕♂Hybrid linesAnd matching, performing biological character characteristics, simplifying genome (DNA) sequences and monitoring molecular markers on selfed filial generations, and comprehensively judging the genetic stability of the backcross strain.
13. Quality evaluation and difference comparison of hizikia fusiforme excellent purified strains, wild strains, hybrid strains and backcross strains
13.1 yield per unit: the hybrid seeds of the sargassum fusiforme are 2700 jin/mu, 200 jin/mu higher than 2500 jin/mu of the parent seeds, 1600 jin/mu and 1100 jin/mu higher than the traditional seedlings;
13.2 product processing ratio: raw materials: the crude product is less than or equal to 1: 0.45;
13.3 typical residual pesticides: prometryn (Prometryn), Terbutryn (Terbutryn) and Propamocarb hydrochloride (Propamocarb hydrochloride), wherein the content of the Prometryn and the Terbutryn is less than 0.01 mg/kg (GB 23200.113-2018 national standard), and the content of the Propamocarb hydrochloride is less than 0.01 mg/kg (GB/T20769-2008 national standard);
13.4 typical heavy metals: the heavy metal types are lead content which is less than or equal to 1.0 mg/kg (dry weight of Pb, GB 2762-2017 national standard);
13.5 carbohydrate: adopting a carbohydrate method;
13.6 dietary fiber: adopting an enzyme weight method (GB 5009.88-2014 national standard);
13.7 Total protein: adopting a Kjeldahl method (GB 5009.5-2016 national standard);
13.8 vitamins: the vitamin A detection method adopts reversed phase high performance liquid chromatography (GB 5009.82-2016 national standard) as national standard, and vitamin B2The detection method adopts high performance liquid chromatography (GB 5009.85-2016 national standard), vitamin B6The detection method adopts high performance liquid chromatography (GB 5009.154-2016 national standard), the vitamin C detection method adopts high performance liquid chromatography (GB 5009.86-2016 national standard), and the vitamin E detection method adopts reversed-phase high performance liquid chromatography (GB 5009.82-2016 national standard)Home standard), etc.;
13.9 total fat: adopting a Soxhlet extraction method or an acid hydrolysis method or (GB 5009.6-2016 national standard);
13.10 amino acids: an amino acid analyzer (Ninhydrin post-column derivative ion exchange chromatograph) is selected for determination (GB 5009.124-2016 national standard);
13.11 trace elements: the sodium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the potassium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the calcium detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.92-2016 national standard), the magnesium content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.214-2017 national standard), and the zinc content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry, and inductively coupled plasma emission spectrometry, The method comprises an inductively coupled plasma mass spectrometry or a dithizone colorimetric method (GB 5009.14-2017 national standard), an iron content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.90-2016), a manganese content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.242-2017), a phosphorus content detection method refers to a molybdenum blue spectrophotometry and an inductively coupled plasma emission spectrometry (GB 5009.87-2016), and a selenium content detection method refers to a hydride atomic fluorescence spectrometry, a fluorescence spectrophotometry and an inductively coupled plasma mass spectrometry (GB 5009.93-2017).
Example 2 was carried out: two purified strains of sargassum fusiforme and two wild strains of sargassum fusiforme are bred by sexual reproduction artificial hybridization. This example was carried out by sexual reproduction artificial cross breeding of two fine purified strains of Cyrtymenia Sparsa with two wild-type strains, respectively.
As a second embodiment of the invention, the method is suitable for the sexual reproduction artificial crossbreeding of the sargassum fusiforme excellent purified type line and the wild type line, and mainly comprises the following steps:
1. sargassum fusiforme purified strain biological character
1.1 juvenile sporophyte leaf shape: the leaf shape of the Yangxicai purified strain juvenile sporophyte stage is characterized by two leaf shapes, namely a plain edge leaf and a odontoblast leaf, and manual screening, classification and seedling clamping are respectively carried out;
1.2 mature sporozoite stage air pocket: the shape of the sac body of the clustered air sac at the mature period of the sargassum fusiforme mature sporophyte is rod-shaped or oval, and each clustered air sac comprises 1-2 clustered air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.6 mm, and the width of the capsule body is more than or equal to 3.8 mm; the excellent strain of the oval air cell has the characteristics of mature period: the length of the capsule body is more than or equal to 11.2 mm, and the width of the capsule body is more than or equal to 4.8 mm.
1.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 3.2 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 3.6 mm.
2. Biological character of wild type strain of sargassum fusiforme
2.1 juvenile sporozoite stage leaf shape: the leaf shapes of wild type juvenile sporophyte of the sargassum fusiforme are characterized by flat margin leaves and odontobutis leaves, and manual classified screening, collection and seedling clamping are respectively carried out according to the leaf shape characteristics of the wild type juvenile sporophyte in different producing areas;
2.2 mature sporozoite stage air sac: the air sac body of the wild type excellent hizikia fusiforme strain is rod-shaped or oval-shaped, and each cluster air sac comprises 1-2 branches of cluster characteristic air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.2 mm, and the width of the capsule body is more than or equal to 3.2 mm; the maturation period of the oval air sac excellent strain is characterized in that: the length of the capsule body is more than or equal to 10.2 mm, and the width of the capsule body is more than or equal to 3.8 mm.
2.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 2.8 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 2.9 mm.
3. Natural sea area ecological isolation artificial breeding of sargassum fusiforme purified strain and wild strain
In order to ensure the purity of the purified strains and the wild strains of the sargassum fusiforme, the strains are planted in 10 middle days of the month, the branch strains are clamped on fiber ropes with the diameter of 0.5 cm and the length of 3.3 m, the seedling spacing is 22 cm, the soft raft frame is hung on the flat single rope, the seedling clamping rope spacing is 4 m, the purified strains and the wild strains are planted in different sea areas of the same region in an ecological isolation way, and the strains are artificially cultured to the next 5 middle days of the year.
4. Excellent purified and wild type strains of Hizikia fusiforme mature sporophyte air sac and stem characteristics
The air bag body of the hizikia fusiforme cluster air bag mainly comprises a rod-shaped air bag body and an oval air bag body, wherein the standard of the excellent rod-shaped air bag body (fresh body) in the mature period is as follows: the length of the capsule body is more than or equal to 12.6 mm, and the width of the capsule body is more than or equal to 3.6 mm; the excellent ovate capsule (fresh body) in the mature period has the standard that the capsule length is more than or equal to 10.2 mm, and the capsule width is more than or equal to 4.6 mm.
5. Artificial screening and marking of natural sea area of fine strains of sargassum fusiforme
The method comprises the steps of selecting female and male Cyrtymenia Sparsa plants, artificially selecting purified and wild female and male plants with same or similar biological properties in 3 middle ten days, and numbering marks (floats or beverage bottles) in natural sea areas, and continuously observing growth and development conditions until the development of a reproductive support is mature.
6. Cross breeding environment and culture medium condition in excellent purified sargassum fusiforme strain and wild type strain sexual reproduction chamber
The illumination intensity is as follows: 220 μm/m2(s), photoperiod: 12:12, seawater salinity: 31 per mill, seawater pH value: 8.0, room temperature: 24.5 ℃, indoor temporary culture time: 8 days, seawater change daily: and 2 times, assisting aeration and oxygen dissolution. The culture medium is characterized in that 13 pieces of single seedling attaching curtains or seedling attaching trays which are 2 cm in width, 25% of cotton and 75% of fibers, are supported by galvanized steel wires at two ends respectively, and are composed of cotton ropes consisting of 25% of cotton and 75% of fibers are selected, and different strains are distinguished by numbering marks.
7. Purification of hizikia fusiforme excellent purification type strain and wild type strain and hybridization sexual reproduction ratio
In the beginning of the fifth month of the year (Wenzhou city cave-head region sheep)The maturity period of the sedum aizoon), respectively collecting female strains and male strains of the excellent biological character purified strains and wild strains marked by the seeds, and according to the ratio of female: male =1:1 ratio. Cyrtymenia Sparsa single strain sexual reproduction purificationA⊕♂A、♀B⊕♂B、♀C⊕♂CAnd (B)D⊕♂DProportioning; male and female for sexual reproduction cross breeding of four strainsPurified form A⊕♂Wild type A，♀Wild type A⊕♂Purified form A；♀Purified form B⊕♂Wild type B，♀Wild type B⊕♂Purified form BProportioning.
8. Indoor artificial temporary breeding of hybrid filial generation of hizikia fusiforme excellent purified strain and wild strain
The indoor environmental factor conditions are the same as in step 6. After concentrated release of the sperms, collecting the fertilized eggs by utilizing a 270-mesh silk screen, uniformly spreading and sprinkling fresh seawater injected in advance, placing on a culture medium attached with a seedling curtain or a seedling attached plate, statically culturing for 1 day, then placing into a seawater artificial aeration device, continuously culturing for 8 days, replacing the fresh seawater 1 time every day, and assisting a high-pressure water spraying device (a high-pressure spray pot or a water gun) to manually remove the silt attached to the surfaces of the fertilized eggs and the embryos. And transferring the seedling attaching curtain or the seedling attaching plate attached with the sargassum fusiforme hybrid strain embryo to a natural sea area for culturing when the sargassum fusiforme hybrid strain embryo grows to 1.5 mm.
9. Artificial cultivation and daily management of natural sea area of hybrid filial generation of fine purified strains and wild strains of sargassum fusiforme
And (3) hanging seedling attaching curtains or seedling attaching plates attached with the sargassum fusiforme hybrid strain embryos on a 'centipede frame' raft frame for cultivation, wherein the seedling attaching surfaces face the sun and the sinking water depth is 12 cm. Daily management: removing attached sediment by a high-pressure water gun, and manually removing the sessile macroalgae; soaking the seedling adhering curtain or seedling adhering tray in 8% citric acid solution for 6 min, cleaning, deacidifying, and removing common parasitic algae (Japanese multitubular: (or)Polysiphonia japonicaIn common terms, the method is as follows: red hair or monkey hair); when the typhoon passes through the border, the seedling attaching curtain or the seedling attaching tray is placed in a laboratory or a refrigerating chamber for temporary storage, and the seedling attaching curtain or the seedling attaching tray is hung and placed in a natural sea area again after the typhoon leaves the border. Sargassum fusiforme hybridThe embryo is cultured artificially in natural sea area for 130 days, and can grow and develop into juvenile sporophyte with length of about 12 cm.
10. Backcrossing of hybrid filial generation of sargassum fusiforme with wild-type parent with excellent biological characteristics
From the third generation of the hybrid strain, 2, 3 and 4 steps are repeatedly adopted to screen excellent biological character plants of the excellent wild type strain parent and the hybrid strain parent (third generation, fourth generation and fifth generation … …), and the best biological character plants are implementedWild type A⊕♂Hybrid progeny A-A，♀Hybrid progeny A-A⊕♂Wild type A；♀Wild type B⊕♂Hybrid progeny B-B，♀Hybrid progeny B-B⊕♂Wild type BBackcross combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the excellent wild-type strain and the hybrid strain is synchronously performed.
11. Sexual reproduction selfing breeding of sargassum fusiforme hybrid line and wild type parent backcross line
Backcrossing 4 generations of excellent wild-type lines, new lines and hybridized lines, sexual reproduction, selfing, and collecting their male and female parentBackcross progeny A⊕♂Backcross progeny A，♀Backcross progeny B⊕♂Backcross progeny BAnd (4) selfing combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the wild-type strain and the hybrid strain is synchronously performed.
12. Evaluation of genetic stability of hizikia fusiforme excellent purification type strain, wild type strain, hybrid type strain and backcross type strain
And (3) carrying out artificial purification on the purified strains, the wild strains and the hybrid strains for three consecutive generations, and monitoring and comparing the characteristics of biological characters (leaf shapes, air sac shapes, lengths, yields and the like) among the three strains. Selecting excellent plants of purified, wild and hybrid strains with the same or similar biological characters in each generation, implementing simplified genome sequence (DNA) and SSR molecular markers, and comparing genetic characteristics. And comprehensively judging the genetic stability of the hybrid strain according to the biological character characteristics, simplified genome (DNA) sequence differences and SSR molecular marker results. The fourth generation of the sargassum fusiforme hybrid strain is continued for three generations, and the parents are implementedBackcrossing of strains, namely: female parentHybrid lines⊕♂Parental strain，♀New product system⊕♂Hybrid linesAnd matching, performing biological character characteristics, simplifying genome (DNA) sequences and monitoring molecular markers on selfed filial generations, and comprehensively judging the genetic stability of the backcross strain.
13. Quality evaluation and difference comparison of hizikia fusiforme excellent purified strains, wild strains, hybrid strains and backcross strains
13.1 yield per unit: the hybrid seeds of the sargassum fusiforme 2750 jin/mu, 250 jin/mu higher than 2500 jin/mu of the parent seeds, 1650 jin/mu and 1100 jin/mu higher than the traditional seeds;
13.2 product processing ratio: and (3) crude product: the raw material is less than or equal to 1: 0.42;
13.3 typical residual pesticides: prometryn (Prometryn), Terbutryn (Terbutryn) and Propamocarb hydrochloride (Propamocarb hydrochloride), wherein the content of the Prometryn and the Terbutryn is less than 0.01 mg/kg (GB 23200.113-2018 national standard), and the content of the Propamocarb hydrochloride is less than 0.01 mg/kg (GB/T20769-2008 national standard);
13.4 typical heavy metals: the heavy metal types are lead content which is less than or equal to 1.0 mg/kg (dry weight of Pb, GB 2762-2017 national standard);
13.5 carbohydrate: adopting a carbohydrate method;
13.6 dietary fiber: adopting an enzyme weight method (GB 5009.88-2014 national standard);
13.7 Total protein: adopting a Kjeldahl method (GB 5009.5-2016 national standard);
13.8 vitamins: the vitamin A detection method adopts reversed phase high performance liquid chromatography (GB 5009.82-2016 national standard) as national standard, and vitamin B2The detection method adopts high performance liquid chromatography (GB 5009.85-2016 national standard), vitamin B6The detection method adopts high performance liquid chromatography (GB 5009.154-2016 national standard), the vitamin C detection method adopts high performance liquid chromatography (GB 5009.86-2016 national standard), the vitamin E detection method adopts reversed-phase high performance liquid chromatography (GB 5009.82-2016 national standard), and the like;
13.9 total fat: adopting a Soxhlet extraction method or an acid hydrolysis method or (GB 5009.6-2016 national standard);
13.10 amino acids: an amino acid analyzer (Ninhydrin post-column derivative ion exchange chromatograph) is selected for determination (GB 5009.124-2016 national standard);
13.11 trace elements: the sodium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the potassium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the calcium detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.92-2016 national standard), the magnesium content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.214-2017 national standard), and the zinc content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry, and inductively coupled plasma emission spectrometry, The method comprises an inductively coupled plasma mass spectrometry or a dithizone colorimetric method (GB 5009.14-2017 national standard), an iron content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.90-2016), a manganese content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.242-2017), a phosphorus content detection method refers to a molybdenum blue spectrophotometry and an inductively coupled plasma emission spectrometry (GB 5009.87-2016), and a selenium content detection method refers to a hydride atomic fluorescence spectrometry, a fluorescence spectrophotometry and an inductively coupled plasma mass spectrometry (GB 5009.93-2017).
Example 3 was carried out: the three purified strains of sargassum fusiforme and the three wild strains are artificially crossbred with sexual reproduction. In this example, sexual reproduction artificial crossbreeding of three fine purified strains of Cyrtymenia Sparsa with three wild strains is performed.
As a third embodiment of the invention, the method is suitable for the sexual reproduction artificial crossbreeding of the sargassum fusiforme excellent purified line and the wild type line, and mainly comprises the following steps:
1. sargassum fusiforme purified strain biological character
1.1 juvenile sporophyte leaf shape: the leaf shape of the Yangxicai purified strain juvenile sporophyte stage is characterized by three leaf shapes, namely a plain leaf, a odontoblast or a clavate leaf, and manual screening, classification and seedling clamping are respectively carried out according to different leaf shapes;
1.2 mature sporozoite stage air pocket: the shape of the sac body of the clustered air sac at the mature period of the sargassum fusiforme mature sporophyte is rod-shaped or oval, and each clustered air sac comprises 1-2 clustered air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.7 mm, and the width of the capsule body is more than or equal to 3.9 mm; the excellent strain of the oval air cell has the characteristics of mature period: the length of the capsule body is more than or equal to 11.4 mm, and the width of the capsule body is more than or equal to 4.9 mm.
1.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 3.4 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 3.7 mm.
2. Biological character of wild type strain of sargassum fusiforme
2.1 juvenile sporozoite stage leaf shape: the leaf shape of wild type juvenile sporophyte of the sargassum fusiforme is characterized by flat margin leaves, odontobutis leaves or rod-shaped leaves, and manual classification screening, collection and seedling clamping are carried out according to the leaf shape characteristics of the wild type juvenile sporophyte in different producing areas;
2.2 mature sporozoite stage air sac: the air sac body of the wild type excellent hizikia fusiforme strain is rod-shaped or oval-shaped, and each cluster air sac comprises 1-2 branches of cluster characteristic air sacs. The excellent strain maturation period of the rod-shaped air bag is characterized in that the cluster characteristic air bag is used as a strain classification standard: the length of the capsule body is more than or equal to 12.5 mm, and the width of the capsule body is more than or equal to 3.4 mm; the maturation period of the oval air sac excellent strain is characterized in that: the length of the capsule body is more than or equal to 10.4 mm, and the width of the capsule body is more than or equal to 3.9 mm.
2.3 mature sporophytic stems: the diameter of the stem of the mature algae of the rod-shaped air sac is more than or equal to 2.9 mm, and the diameter of the stem of the mature algae of the oval air sac is more than or equal to 3.0 mm.
3. Natural sea area ecological isolation artificial breeding of sargassum fusiforme purified strain and wild strain
In order to ensure the purity of the purified strains and the wild strains of the sargassum fusiforme, the strains are arranged at the end of 10 months, the seedlings are clamped on fiber ropes with the diameter of 0.5 cm and the length of 3.3 m by different strains, the seedling spacing is 25 cm, the single rope of the soft raft frame is horizontally hung, the seedling clamping rope spacing is 4 m, the purified strains and the wild strains are ecologically isolated and hung in the same region and different sea areas, and are artificially cultured to 5 th days in the next year.
4. Excellent purified and wild type strains of Hizikia fusiforme mature sporophyte air sac and stem characteristics
The air bag body of the hizikia fusiforme cluster air bag mainly comprises a rod-shaped air bag body and an oval air bag body, wherein the standard of the excellent rod-shaped air bag body (fresh body) in the mature period is as follows: the length of the capsule body is more than or equal to 12.7 mm, and the width of the capsule body is more than or equal to 3.7 mm; the excellent ovate capsule (fresh body) in the mature period has the standard that the capsule length is more than or equal to 10.4 mm, and the capsule width is more than or equal to 4.7 mm.
5. Artificial screening and marking of natural sea area of fine strains of sargassum fusiforme
The method comprises the steps of breeding male and female Cyrtymenia Sparsa plants, manually screening purified and wild female and male plants with the same or similar biological properties in month 4, and continuously observing growth and development conditions until the development of a reproductive support is mature.
6. Cross breeding environment and culture medium condition in excellent purified sargassum fusiforme strain and wild type strain sexual reproduction chamber
The illumination intensity is as follows: 230 μm/m2(s), photoperiod: 12:12, seawater salinity: 33 per mill, seawater pH value: 8.3, room temperature: 25 ℃, indoor temporary rearing time: 10 days, seawater change daily: and 2 times, assisting aeration and oxygen dissolution. The culture medium is selected from 14 seedling attaching curtains or seedling attaching discs which are formed by single cotton with the width of 2 cm, 30 percent of cotton and 70 percent of fiber, two ends of each seedling attaching curtain are respectively supported by a galvanized steel wire, and the seedling attaching discs are formed by cotton ropes formed by 30 percent of cotton and 70 percent of fiber, and the different strains are distinguished by numbering marks.
7. Purification of hizikia fusiforme excellent purification type strain and wild type strain and hybridization sexual reproduction ratio
In the beginning of the fifth month of the year (Wenzhou city cave-head region sheep)The maturity period of the sedum aizoon), respectively collecting female strains and male strains of the excellent biological character purified strains and wild strains marked by the seeds, and according to the ratio of female: male =1:1 ratio. Cyrtymenia Sparsa single strain sexual reproduction purificationA⊕♂A、♀B⊕♂B、♀C⊕♂CProportioning; adopting male parent and female parent for sexual reproduction hybridization among six strainsPurified form A⊕♂Wild type A，♀Wild type A⊕♂Purified form A；♀Purified form B⊕♂Wild type B，♀Wild type B⊕♂Purified form B；♀Purified form C⊕♂Wild type C，♀Wild type C⊕♂Purified form C；♀Purified form B⊕♂Wild type A，♀Wild type B⊕♂Purified form A；♀Purified form C⊕♂Wild type A，♀Wild type C⊕♂Purified form A；♀Purified form B⊕♂Wild type C，♀Wild type B⊕♂Purified form CProportioning.
8. Indoor artificial temporary breeding of hybrid filial generation of hizikia fusiforme excellent purified strain and wild strain
The indoor environmental factor conditions are the same as in step 6. After concentrated release of sperm eggs, collecting fertilized eggs by utilizing a 280-mesh silk screen manually, uniformly spreading and sprinkling fresh seawater injected in advance, placing on a culture medium attached with a seedling curtain or a seedling attached plate, statically culturing for 1 day, then placing into a seawater manual aeration device, continuously culturing for 10 days, replacing fresh seawater 2 times every day, and manually removing silt attached to the surfaces of the fertilized eggs and embryos by using auxiliary high-pressure water spraying equipment (a high-pressure spray pot or a water gun). And transferring the seedling attaching curtain or the seedling attaching plate attached with the sargassum fusiforme hybrid strain embryo to a natural sea area for culturing when the sargassum fusiforme hybrid strain embryo grows to 2 mm.
9. Artificial cultivation and daily management of natural sea area of hybrid filial generation of fine purified strains and wild strains of sargassum fusiforme
And (3) hanging seedling attaching curtains or seedling attaching plates attached with the sargassum fusiforme hybrid strain embryos on a 'centipede frame' raft frame for cultivation, wherein the seedling attaching surfaces face the sun and the sinking water depth is 15 cm. Daily management: high pressureRemoving attached sediment by a water gun, and manually removing the sessile macroalgae; soaking the seedling adhering curtain or seedling adhering tray in 8% citric acid solution for 7 min, cleaning, deacidifying, and removing common parasitic algae (Japanese multitubular: (or)Polysiphonia japonicaIn common terms, the method is as follows: red hair or monkey hair); when the typhoon passes through the border, the seedling attaching curtain or the seedling attaching tray is placed in a laboratory or a refrigerating chamber for temporary storage, and the seedling attaching curtain or the seedling attaching tray is hung and placed in a natural sea area again after the typhoon leaves the border. The hybrid sargassum fusiforme strain embryo can grow and develop into juvenile sporophyte with the length of about 15 cm after being cultured artificially for 150 days in a natural sea area.
10. Backcrossing of hybrid filial generation of sargassum fusiforme with wild-type parent with excellent biological characteristics
From the third generation of the hybrid strain, 2, 3 and 4 steps are repeatedly adopted to screen excellent biological character plants of the excellent wild type strain parent and the hybrid strain parent (third generation, fourth generation and fifth generation … …), and the best biological character plants are implementedWild type A⊕♂Hybrid progeny A-A，♀Hybrid progeny A-A⊕♂Wild type A；♀Wild type B⊕♂Hybrid progeny B-B，♀Hybrid progeny B-B⊕♂Wild type B；♀Wild type C⊕♂Hybrid progeny C-C，♀Hybrid progeny C-C⊕♂Wild type CBackcross combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the excellent wild-type strain and the hybrid strain is synchronously performed.
11. Sexual reproduction selfing breeding of sargassum fusiforme hybrid line and wild type parent backcross line
Backcrossing 3-4 generations of excellent wild-type lines, new lines and hybrid lines, performing sexual reproduction selfing breeding on the backcrossed lines, and selecting the male parentBackcross progeny A⊕♂Backcross progeny A，♀Backcross progeny B⊕♂Backcross progeny B，♀Backcross progeny C⊕♂Backcross progeny CAnd (4) selfing combination. Meanwhile, sexual reproduction selfing purification of the excellent purified strain, the wild-type strain and the hybrid strain is synchronously performed.
12. Evaluation of genetic stability of hizikia fusiforme excellent purification type strain, wild type strain, hybrid type strain and backcross type strain
And (3) carrying out artificial purification on the purified strains, the wild strains and the hybrid strains for three consecutive generations, and monitoring and comparing the characteristics of biological characters (leaf shapes, air sac shapes, lengths, yields and the like) among the three strains. Selecting excellent plants of purified, wild and hybrid strains with the same or similar biological characters in each generation, implementing simplified genome sequence (DNA) and SSR molecular markers, and comparing genetic characteristics. And comprehensively judging the genetic stability of the hybrid strain according to the biological character characteristics, simplified genome (DNA) sequence differences and SSR molecular marker results. The fourth generation of the sargassum fusiforme hybrid strain starts for three continuous generations, and the backcross of the parent strain is implemented, namely: female parentHybrid lines⊕♂Parental strain，♀New product system⊕♂Hybrid linesAnd matching, performing biological character characteristics, simplifying genome (DNA) sequences and monitoring molecular markers on selfed filial generations, and comprehensively judging the genetic stability of the backcross strain.
13. Quality evaluation and difference comparison of hizikia fusiforme excellent purified strains, wild strains, hybrid strains and backcross strains
13.1 yield per unit: the hybrid seeds of the sargassum fusiforme are 2800 jin/mu, 300 jin/mu higher than 2500 jin/mu of the parent seeds, and 1200 jin/mu higher than 1600 jin/mu of the traditional seedlings;
13.2 product processing ratio: and (3) crude product: the raw material is less than or equal to 1: 0.40;
13.3 typical residual pesticides: prometryn (Prometryn), Terbutryn (Terbutryn) and Propamocarb hydrochloride (Propamocarb hydrochloride), wherein the content of the Prometryn and the Terbutryn is less than 0.01 mg/kg (GB 23200.113-2018 national standard), and the content of the Propamocarb hydrochloride is less than 0.01 mg/kg (GB/T20769-2008 national standard);
13.4 typical heavy metals: the heavy metal types are lead content which is less than or equal to 1.0 mg/kg (dry weight of Pb, GB 2762-2017 national standard);
13.5 carbohydrate: adopting a carbohydrate method;
13.6 dietary fiber: adopting an enzyme weight method (GB 5009.88-2014 national standard);
13.7 Total protein: adopting a Kjeldahl method (GB 5009.5-2016 national standard);
13.8 vitamins: the vitamin A detection method adopts reversed phase high performance liquid chromatography (GB 5009.82-2016 national standard) as national standard, and vitamin B2The detection method adopts high performance liquid chromatography (GB 5009.85-2016 national standard), vitamin B6The detection method adopts high performance liquid chromatography (GB 5009.154-2016 national standard), the vitamin C detection method adopts high performance liquid chromatography (GB 5009.86-2016 national standard), the vitamin E detection method adopts reversed-phase high performance liquid chromatography (GB 5009.82-2016 national standard), and the like;
13.9 total fat: adopting a Soxhlet extraction method or an acid hydrolysis method or (GB 5009.6-2016 national standard);
13.10 amino acids: an amino acid analyzer (Ninhydrin post-column derivative ion exchange chromatograph) is selected for determination (GB 5009.124-2016 national standard);
13.11 trace elements: the sodium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the potassium content detection method refers to flame atomic absorption spectrometry, flame atomic emission spectrometry or inductively coupled plasma emission spectrometry (GB 5009.91-2017 national standard), the calcium detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.92-2016 national standard), the magnesium content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry or inductively coupled plasma mass spectrometry (GB 5009.214-2017 national standard), and the zinc content detection method refers to flame atomic absorption spectrometry, inductively coupled plasma emission spectrometry, and inductively coupled plasma emission spectrometry, The method comprises an inductively coupled plasma mass spectrometry or a dithizone colorimetric method (GB 5009.14-2017 national standard), an iron content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.90-2016), a manganese content detection method refers to a flame atomic absorption spectrometry, an inductively coupled plasma emission spectrometry and an inductively coupled plasma mass spectrometry (GB 5009.242-2017), a phosphorus content detection method refers to a molybdenum blue spectrophotometry and an inductively coupled plasma emission spectrometry (GB 5009.87-2016), and a selenium content detection method refers to a hydride atomic fluorescence spectrometry, a fluorescence spectrophotometry and an inductively coupled plasma mass spectrometry (GB 5009.93-2017).
Those skilled in the art will understand that: sargassum fusiforme and Sargassum thunbergii (Sargassum thunbergii)Sargassum thunbergii) Sargassum horneri (sargassum horneri) (II)Sargassumhorneri) Sargassum muticum (sargassum muticum) (II)Sargassummuticum) And Sargassum henryi ((sargassum henryi))Sargassumhenslowianum) Phaeophyta of the same genus (A), (B), (C)Phaeophyta) Sargassaceae (Sargassaceae) (III)Sargassaceae) They all have a sexual reproduction mode. The illustrated examples and discussion of the present invention are exemplary only and are not intended to imply that the scope of the present disclosure, including the claims, is limited to the hizikia fusiforme sexual reproduction artificial hybridization examples. In the operational procedures of the present invention, alternatives and combinations between the technical procedures in the above-described embodiments or different embodiments may also be possible, and there are different variations of the above-described embodiments, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, and substitutions that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (1)

1. A sexual reproduction artificial crossbreeding method for a sargassum fusiforme excellent purified strain and a wild strain is characterized by comprising the following steps:
a. isolated hanging culture of sargassum fusiforme purified type and wild type strain juvenile sporophyte
Selecting a purified strain and a wild strain of the sargassum fusiforme with the same or similar leaf shape characteristics at the juvenile sporophyte stage, placing the purified strain and the wild strain in natural sea areas for ecological isolation artificial cultivation, wherein in order to ensure the purity of the sargassum fusiforme purified strain and the wild strain, each strain is at the beginning of 10 months, the seedling of each strain is clamped on a fiber rope with the diameter of 0.5 cm and the length of 3.3 m, the seedling distance is 20-25 cm, the single rope of a soft raft frame is horizontally hung, the seedling clamping rope distance is 4 m, the purified strain and the wild strain are hung and cultivated in the same sea area in an ecological isolation mode, and the artificial cultivation is carried out for 5-6 months in the next year;
b. after the excellent purified type and wild type strains of the sargassum fusiforme grow into mature sporophytes, the female and male strains of the sargassum fusiforme are manually screened in 3-4 months for the purified type and wild type excellent property female strains and male strains with the same or similar biological properties, numbering and marking are carried out in a natural sea area through a floater or a beverage bottle, the growth and development conditions are continuously observed until the reproductive support grows mature;
c. b, hybridizing the excellent purified strains of the sargassum fusiforme obtained in the step b with wild strains indoors
At the beginning of 5 months, namely the mature period of the local sargassum fusiforme in the cave head region of the Wenzhou city, female strains and male strains of the purified strains with good biological characters and wild strains which are marked in advance are respectively collected, and sexual reproduction hybridization is carried out according to the ratio of female: male =1:1 ratio;
indoor cross breeding environment and culture medium selection: the illumination intensity is as follows: 200 to 230 μm/m2(s), photoperiod: 12:12, seawater salinity: 30-33 per mill, seawater pH value: 7.8-8.3, room temperature: 24-25 ℃, indoor temporary rearing time: 7-10 days, replacing seawater every day: 1-2 times, and assisting aeration to dissolve oxygen; selecting 12-14 pieces of culture medium, a single piece of cotton with the width of 2 cm, 20-30% of cotton and 70-80% of fiber, a seedling attaching curtain or a seedling attaching disc consisting of a plastic frame shaped like a Chinese character 'tian' and two ends of which are respectively supported by a galvanized steel wire, and a cotton rope consisting of 20-30% of cotton and 70-80% of fiber, and numbering and marking to distinguish different strains;
d. indoor artificial temporary breeding of hybrid filial generation of fine purified sargassum fusiforme strain and wild strain
C, after concentrated release of the sperm eggs, manually collecting the fertilized eggs by using a 260-280-mesh silk screen, uniformly spreading and sprinkling fresh seawater which is injected in advance, placing on a culture medium with a seedling attaching curtain or a seedling attaching plate, statically culturing for 1 day, then placing into a seawater manual aeration device, continuously culturing for 7-10 days, replacing fresh seawater for 1 time every day, assisting a high-pressure water spraying device, manually removing the silt attached to the surfaces of the fertilized eggs and the embryos, and allowing the hybrid line embryos of the sargassum fusiforme to grow and develop to 1-2 mm;
e. breeding and daily management of excellent purified Cyrtymenia Sparsa strain and wild type strain hybrid progeny in natural sea area
Transferring the embryo-attached seedling curtain or seedling-attached disc to a natural sea area, hanging and culturing the embryo-attached seedling curtain or seedling-attached disc on a 'centipede frame' raft frame for culturing, enabling the seedling-attached surface to face the sun, sinking to a depth of 10-15 cm, assisting in removing enemy algae, removing enemy animals and daily managing typhoon damage, and culturing the hijiki hybrid strain embryos from the beginning of 5 months to the end of 9 months, namely 120-150 days, so that juvenile sporophytes with the length of 10-15 cm can grow and develop;
f. backcrossing of hybrid filial generation of sargassum fusiforme with wild-type parent with excellent biological characteristics
Performing secondary purification on the excellent purified strain, the wild-type strain and the hybrid strain, performing wild-type parent backcross from the third generation of the hybrid strain, and continuously performing 3-4 life cycles to stably obtain the whole genome of the wild-type strain;
g. sexual reproduction selfing breeding of sargassum fusiforme hybrid line and wild type parent backcross line
After the hybrid strain and the wild-type parent backcross strain are continuously implemented for 3-5 life cycles, male plants and female plants with excellent biological properties are selected, and the backcross strain sexual reproduction inbreeding breeding is adopted to realize the purification and propagation of filial generation so as to obtain a large number of excellent plants.
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