CN116195508A - Creation method of novel yellow pulp crack-resistant high-sugar small watermelon germplasm - Google Patents
Creation method of novel yellow pulp crack-resistant high-sugar small watermelon germplasm Download PDFInfo
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Abstract
The invention discloses a creation method of a new yellow pulp crack-resistant high-sugar small watermelon germplasm, which comprises the following steps: the female parent and the male parent with specific characters are selected for hybridization, then F2 generation after F1 generation selfing is subjected to seedling raising and field planting by means of disease resistance molecular markers, selection of skin toughness, determination of sugar degree and the like, and F3 generation is obtained after specific single melon hybridization is selected, F4 generation to F6 generation selfing is selected for pure breeding, and finally purified yellow pulp crack-resistant high sugar pulp small watermelon new germplasm material is selected from F6 generation. The method screens out the purified yellow pulp crack-resistant high-sugar small watermelons, has steady growth vigor, high resistance to blight, higher sugar degree and good taste, and can be used as a core parent for breeding new varieties of yellow pulp crack-resistant high-sugar small watermelons.
Description
Technical Field
The invention relates to the technical field of small watermelon breeding, in particular to a method for creating a novel yellow pulp crack-resistant high-sugar small watermelon germplasm.
Background
With the continuous development of social production, the living standard of people is increasingly improved, and the consumption structure is also obviously changed. The small watermelons suitable for being planted in the off-season greenhouse protected area have the advantages of good quality, high-grade consumption in accordance with modern small families, and the like, are popular with urban high consumers, have a growing trend year by year, and have 8-10 ten thousand hm in China 2 。
The yellow pulp watermelon is a rare variety, small in size, round, light green in peel, obvious in dark green reticulate patterns, golden in pulp, crispy, fragrant and sweet in pulp, strong in melon fragrance, unique in flavor, only 3kg at maximum, high and uniform in sugar content, and instant in mouth, and is suitable for planting coarse sand and semi-sandy soil with excellent drainage, and matured in 6-8 months, such as 'small blue', 'extra small phoenix' and the like of the watermelon varieties on the market at present.
The production places of small watermelons in China have higher requirements on the quality, are not easy to popularize in a large area, are easy to generate the defects of difficult low-temperature fruit setting in early spring, unstable yield property, easy fruit malformation, low commodity fruit rate, poor disease resistance, easy cracking of the melons and fruits, incapability of tolerating long-distance transportation and the like, and the cracking resistance of the melons and fruits directly determines the storage and transportation resistance of the products, so that the yield loss and the shelf life of the field and logistics links are related. Restricting the sales of small watermelons and affecting the income of melon farmers.
Therefore, aiming at the problems, on the basis of widely introducing domestic and foreign germplasm resources, the Ningbo market academy of agricultural science formulates and breeds the targets of anti-fusarium wilt, anthracnose, high sugar and crack resistance, creates a new crack-resistant high-sugar yellow pulp small watermelon germplasm suitable for long-distance transportation, and lays a foundation for breeding new yellow pulp crack-resistant high-sugar small watermelon varieties.
Disclosure of Invention
The invention aims to provide a method for creating a novel yellow pulp cracking-resistant high-sugar small watermelon germplasm, which is used for breeding the small watermelon germplasm with high resistance to watermelon fusarium wilt and high sugar cracking resistance.
The aim of the invention is achieved by the following technical scheme:
a creation method of a yellow pulp crack-resistant high-sugar small watermelon comprises the following steps:
(1) Parental selection
The female parent selects breeding materials of round fruits, red pulp, tough skin and resistance to wilt, and the male parent selects breeding materials of round fruits, yellow pulp, crisp skin and resistance to disease;
(2) Novel germplasm creation
Hybridizing the female parent and the male parent selected in the step (1) to obtain an F1 generation;
selecting single plants with excellent comprehensive properties of the F1 generation, selfing to obtain the F2 generation, carrying out disease resistance identification on seedling cultivation of F2 generation sowing materials, selecting single plants with pure disease resistance for field planting, screening single melon hybridization with specific properties to obtain the F3 generation, and screening single melon hybridization with specific properties from the F3 generation to obtain the F4 generation;
and F4 generation to F6 generation selfing purification, and screening yellow pulp crack-resistant high-sugar watermelon purebred in F6 generation.
Further, in the step (2), the female parent variety is selected from 'Sumeng No. 6', and the male parent variety is selected from 'Xiaolan'.
Further, the F1 generation in the step (2) has excellent comprehensive properties of round fruits, tough peels, red and yellow pulps and disease resistance;
the special characters of the single melon selected by the F2 generation and the F3 generation are high sugar, round fruits, tough skin, yellow flesh and disease resistance.
Further, in the step (2), the single melon screened by the F2 generation and the F3 generation is identified for wilt in the seedling stage.
Further, in the step (2), sugar degree of the watermelon harvested by F2 generation and F3 generation detection is detected, and single watermelon with specific characters is respectively screened from the F2 generation and the F3 generation to obtain 5 single watermelons.
Further, in the step (2), a month before the planting of the pure anti-fusarium wilt single plant is selected, the land preparation of the seed production block is well selected, fertilization and furrow making are carried out, 3-4 leaves and 1 heart planting are selected in spring, and 1-2 leaves and 1 heart planting is carried out in autumn.
Further, the planting density is 1800 plants/667 m2, the plant spacing is 40cm, the row spacing is 90cm, when the female parent and the male parent are hybridized in the step (2), the 1 st female flower is removed, and the female flower pollination melon at the 2 nd or 3 rd node position is selected.
Further, the pollination method comprises the following steps: pollinating the male flowers within 3 hours after the male flowers are opened, lightly pinching the male flower stems by hands, removing the male flower petals, and uniformly coating the pollen on the pistil column heads until obvious yellow pollen grains are on the pistil column heads.
Further, the seed melon can be harvested 35-40 days after pollination
The beneficial effects of the invention are as follows:
the invention provides a method for creating new germplasm of yellow pulp crack-resistant small watermelon, which comprises the steps of hybridizing female parent and male parent with specific characters, then carrying out seedling raising and field planting on F2 generation after F1 generation selfing by means of disease-resistant molecular marker identification, selection of skin toughness, determination of sugar degree and the like, selecting specific single melon from the F2 generation for F3 to be screened, then continuing selfing to F6 generation according to the means, and finally screening purified new germplasm material of yellow pulp crack-resistant high sugar small watermelon from F6 generation. The method screens out the purified yellow pulp crack-resistant high-sugar small watermelon, has steady growth vigor, high resistance to blight and anthracnose, higher sugar degree and good taste, and can be used as a core parent for breeding a new variety of yellow pulp crack-resistant high-sugar small watermelon.
Drawings
FIG. 1 is a schematic diagram of the development of a yellow pulp crack-resistant high-sugar small watermelon variety according to the invention;
FIG. 2 is a PCR detection chart of identification of 5 single melon anti-fusarium wilt molecules in F2 generation of the invention;
FIG. 3 is a PCR detection chart of identification of 5 single melon anti-fusarium wilt molecules in F3 generation of the invention.
Detailed Description
In order to more clearly illustrate the technical scheme of the invention, the specific embodiments are as follows:
example 1
1. The method for creating the yellow pulp crack-resistant high-sugar small watermelon comprises the following steps:
(1) Parent breeding
The female parent is selected from round fruit, red pulp, tough skin and wilt-resistant breeding materials, and the female parent variety is Sumeng No. 6, which is purchased from Jiangsu river vegetable seedling technology Co., ltd; the male parent is selected from round fruits, yellow flesh, crisp skin and disease-sensitive breeding materials, and the male parent variety is 'Xiaolan', and is purchased from Taiwan farmer company;
(2) Novel germplasm creation
The female parent and the male parent are hybridized to obtain an F1 generation, the appearance characters of the F1 generation are round fruits, tough peels (middle type and crisp), red yellow pulp and disease resistance (middle type), a single plant with the tough peels and crisp phenotype in the F1 generation sample is eliminated, and the F2 generation sample after elimination is subjected to selfing.
F2, sowing 2400 plants for seedling culture, detecting the fusarium wilt by using molecular markers at the seedling stage, and selecting single plants with pure resistance to the fusarium wilt for field planting. The fruits are selected from round fruits, tough peels, yellow pulps and high-sugar single melons.
The sugar degree of the watermelon is detected by using a fruit sugar degree meter, and the model of the detection instrument is PAL-1. Screening 5 single melons with high sugar content, round fruits, tough skin, yellow flesh and disease resistance from F2 generation, numbering the single melons, describing specific single melon characteristics as shown in the following table 1 for F3 screening,
TABLE 1
The specific anti-fusarium wilt identification and evaluation test is carried out in vegetable institute of Ningbo municipal agricultural science institute, and the inoculation identification method and the recording standard are carried out by referring to the national standard (industry standard of watermelon anti-fusarium wilt identification technical procedure, standard number: NY/T3626-2020).
The reference substance is a disease-sensitive variety "Xiaolan", and the material is purchased from Xinjiang Hami melon seed industry Limited liability company.
Inoculation identification method: the experiment is carried out in a greenhouse, the watermelon material to be tested is sown in sterilized sand according to the number, and when the watermelon seedlings grow to 2 leaves and 1 heart, the watermelon material is placed in watermelon fusarium wilt with the spore concentration of 3 multiplied by 106 per mL and watermelon anthracnose germ suspension with the spore concentration of 7 multiplied by 105 per mL for 30min. 3 replicates were set for each variety and disease rates were investigated separately.
DNA extraction: and 7d, sampling a single plant after inoculation, selecting the first true leaf of the robust seedling in a seedling leaf-core period, and extracting genome DNA by adopting a CTAB method.
PCR reaction system:
all were used in 12. Mu.L, including 1.2. Mu.L of 10 XBuffer (containing Mg2+), 1. Mu.L of 2.5mmol/L dNTPs, 1. Mu.L of 10. Mu. Mol/mu.L of primer, 0.2. Mu.L of 5U/. Mu.L Taq DNA polymerase, 2. Mu.L of 30 ng/. Mu.L of template DNA, and made up to 12. Mu.L with deionized water.
PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 40s, 34 cycles total; finally, the mixture is extended for 10min at 72 ℃; preserving at 4 ℃ for standby.
The genomic DNA of watermelon leaf was detected by 2% agarose gel electrophoresis.
Primers were synthesized by Shanghai Biotechnology, inc. using published primer sequences for fusarium wilt (Zhang Yi, progress of watermelon fusarium wilt).
The primer sequences for resisting the fusarium wilt are as follows:
5′-TTAAAAATCATCTCCTCTTTAAAACTATT-3′，
5′ATATATTTGGTCTCCGAGTGTTCAA-3′。
the disease index is calculated as follows:
disease index = total number of dead plants/total number of investigation plants x 100
The resistance ranking criteria were: below 11 (HR, high antibody); 12-33 (R, disease resistance); 34-55 (MR, medium resistance); 56-77 (S, influenza); greater than 78 (HS, high-grade).
The 5 single melon resistance ranking results are shown in table 2 below:
TABLE 2
The numbers in table 2 above are the average of 3 replicates. As can be seen from the above table, the watermelon varieties numbered 21XA-2-1, 21XA-2-2, 21XA-2-12 and 21XA-2-13 all show resistance to watermelon fusarium wilt, the watermelon variety numbered 21XA-2-19 shows resistance to watermelon fusarium wilt, and the control cymbidium shows susceptibility to watermelon fusarium wilt.
Molecular identification results:
the detection results of the fusarium wilt resistance genes of the 5 single melons in F2 generation are shown in figure 2, wherein the infection genotype "A" band type, the homozygous disease resistance genotype "B" band type and the heterozygous disease resistance genotype "AB" type are shown in the following table 3, and the corresponding field numbers and gene detection numbers are as follows:
TABLE 3 Table 3
As can be seen from the final wilt disease resistance detection results in FIG. 2 and Table 2, the disease resistance results are in one-to-one correspondence, and the cymbidium is a wilt-sensitive homozygote, and the 21XA-2-19 is a heterozygote, so that the intermediate wilt resistance is shown, and other single melons are all wilt-resistant homozygotes.
F3 generation is obtained after the hybridization of the 5 single melons of the F2 generation, 5 single melons are selected from the F3 generation, and the serial numbers are respectively: 21XA-2-1-2, 21XA-2-12-2, 21XA-2-13-6, 21XA-2-19-13, 21XA-2-19-14. The specific 5 single melon characteristics selected for the F3 generation are described in Table 4 below.
TABLE 4 Table 4
The results of the grading of the resistance of the 5 single melons selected in the F3 generation are shown in the following table 5:
TABLE 5
The numbers in table 5 above are the average of 3 replicates. As is clear from the above table, the watermelon varieties with the numbers of 21XA-2-1-2, 21XA-2-12-2, 21XA-2-13-6, 21XA-2-19-13, 21XA-2-19-14 are all disease-resistant to watermelon fusarium wilt, and the control cymbidium parvum is disease-resistant to watermelon fusarium wilt.
Molecular identification results:
the detection results of the fusarium wilt resistance genes of the 5 single melons in F3 generation are shown in figure 3, wherein the infection genotype "A" band type, the homozygous disease resistance genotype "B" band type and the heterozygous disease resistance genotype "AB" type are shown in the following table 6, and the corresponding field numbers and gene detection numbers are as follows:
TABLE 6
As can be seen from the final wilt disease resistance detection results shown in FIG. 3 and Table 5, the disease resistance results are in one-to-one correspondence, the cymbidium is a wilt-sensitive homozygote, and the other single melons are all wilt-resistant homozygotes.
Hybridizing the 5 single melons screened by the F3 generation to obtain an F4 generation, sowing and raising seedlings of watermelon seeds obtained from the F4 generation, and selfing after flowering to obtain an F5 generation; sowing and raising seedlings of the watermelon seeds obtained in the F5 generation, carrying out selfing after flowering to obtain the F6 generation, carrying out selfing screening on the F4 to F6 generation to obtain two generations, and finally obtaining the pure-bred yellow-pulp crack-resistant high-sugar blight-resistant small watermelon germplasm from the F6 generation.
2. Cultivation and field planting of small watermelons
(1) Seeding and seedling raising
Sowing in the middle and late 1 month to middle 2 months in spring; autumn is from 7 months to 8 months. The seedling can be cultivated by adopting a 32-hole tray or a nutrition pot. The seedling age in spring is 3-4 leaves 1 heart, generally 30-40 d; autumn seedling age is 1-2 leaves 1 heart, generally 10-15 d.
(2) Soil preparation and furrow making
The base fertilizer is generally about 1000kg of farmyard manure per mu, 25kg of Norway compound fertilizer, 15kg of calcium superphosphate and 0.5kg of borax.
And (3) vertical cultivation: and (3) cultivating in a greenhouse by using a vertical frame, wherein the width of a furrow is 1.2m, the height of the furrow is 25cm, and the width of the furrow is about 50 cm.
(3) Planting
When the seedlings grow to 10-12cm, the seedlings are planted in time, and the base fertilizer application, soil preparation (deep ploughing for 15-20 cm) and furrow making work are performed before planting. The planting density is 1800 plants/667 m < 2 >, the plant spacing is 40cm, the row spacing is 90cm, 3-4 leaves are selected for 1-heart field planting in spring, 1-2 leaves are selected for 1-heart field planting in autumn, and the grafting interface is higher than the soil surface by more than 1cm during field planting.
The row spacing of the vertical frame cultivation and field planting of the small fruit type watermelons is 0.9m, the plant spacing is 0.40m, and each 667m 2 1800 plants are planted.
(4) Field management
1) Temperature, light and humidity are controlled. The temperature in the daytime is kept at 28-32 ℃ and the temperature at night is kept above 15 ℃ during the seedling reviving period so as to promote the seedling reviving. The temperature of the facility in the daytime in the tendril stretching period is controlled at 25-28 ℃, the temperature at night is controlled at 13-15 ℃, the illumination is increased by proper ventilation, the temperature is controlled, the humidity is reduced, and the overgrowth is prevented. The fruit expansion period should be kept at a higher temperature of 30deg.C to promote fruit development, and the melon should be properly cooled when expanding to commercial melon size, and the ventilation and gradual cooling should be increased beyond 35deg.C, especially the night temperature. The cultivation in summer and autumn is carried out by taking care of ventilation and cooling in the early stage and heat preservation in the later stage.
2) Pruning and vine leaving. Hanging vine cultivation is adopted, when the main vine of a watermelon plant is 50-60 cm long, pruning is carried out, 2 vines are selected and reserved, other vines are scraped off in time except the reserved melon vine, and nutrition supply of the reserved vine is ensured. And after the vines are put on the shelf, the vines are bound for 1 time by a vine binding machine every 3-4 sections.
After the melon is sitting for 7 days, the melon handle is hung by a nylon rope and the young melon is hung on the rope by a net bag net. Pruning is generally not performed after fruit setting.
3) And (5) fruit setting. Normally, the 2 nd to 3 rd female flower sections of the main vines begin to leave the melon. And (3) pollinating by bees, and setting the watermelon when the watermelon grows to the egg size, and timely thinning the abnormal melon. 1 melon is reserved for each vine of the small fruit type watermelon variety, 2 melons are planted in each plant of 1 batch, and 1-2 melons are planted in each plant of 2 batches.
4) And (5) fertilizer and water management. Every 667m of the fruit swelling period 2 And applying 15kg of compound fertilizer and 7.5kg of potassium sulfate.
5) And (5) pest control. The main diseases in the field are anthracnose, gummy stem blight, epidemic disease and virus disease; the main insect pests include thrips, aphids, red spiders, liriomyza sativae, bemisia tabaci, and the like. The method is based on the principle of prevention and comprehensive prevention, mainly agricultural prevention, physical prevention and biological prevention and chemical prevention and control, and is assisted by harmless prevention and control principles.
(5) Harvesting, judging the fruit harvesting time according to the variety characteristics and pollination time of the small fruit type watermelons, wherein the small fruit type watermelons are ripe about 30d (days) after pollination. Mature fruits can fully represent the characteristics of the variety: the surface patterns of the fruits are clear, the fruit peel has luster, the fruit surface is smooth, the fruit umbilicus is concave inwards, and the base part of the fruit stalk is slightly contracted; the pulp is bright in color, high in sugar content and good in taste; the fruits were tapped by hand, making a voiced sound as ripe melon and a crisp sound as raw melon.
3. Watermelon seeds
The melon seeds are immediately washed after being cut, melon pulp and seeds are separated, and the seeds are dried in the sun and stored in time after being washed for screening for standby.
4. Hybrid pollination melon-retaining operation item
4.1 paper cap and marker preparation: a number of thumb-sized paper caps were prepared.
4.2 male parent male flower bagging:
male flowers of male parents to be opened are covered by paper caps before pollination operation, and are covered by paper caps after pollination is completed in the next day.
Picking male flowers of male parent plants bagging yesterday in the morning of pollination operation; the male flowers which are not used up on the same day cannot be pollinated and utilized the next day or later;
4.3 pollination: pollination is generally preferred for pollination with the second female flower; pollinating the male flowers within 3 hours after the male flowers are opened, lightly pinching the male flower stems by hands, removing the male flower petals, and uniformly coating the pollen on the pistil column heads until obvious yellow pollen grains are on the pistil column heads.
4.4 fruit retention: the fruit retaining sections are suitable for retaining 2 nd and 3 rd female flowers on the main vines or the side vines, and the fruit growth occupies more leaf area, so that the fruit shape can be increased.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (10)
1. A method for creating a novel yellow pulp crack-resistant high-sugar small watermelon germplasm is characterized by comprising the following steps:
(1) Parental selection
The female parent selects round fruit, red pulp, ductile skin and anti-fusarium wilt hybrid, and the male parent selects round fruit, yellow pulp, brittle skin and infected hybrid;
(2) Novel germplasm creation
Hybridizing the female parent and the male parent selected in the step (1) to obtain an F1 generation;
selecting single plants with excellent comprehensive properties of the F1 generation, selfing to obtain the F2 generation, carrying out disease resistance identification on seedling cultivation of F2 generation sowing materials, selecting single plants with pure disease resistance for field planting, screening single melon hybridization with specific properties to obtain the F3 generation, and screening single melon hybridization with specific properties from the F3 generation to obtain the F4 generation;
and F4 generation to F6 generation selfing purification, and screening yellow pulp crack-resistant high-sugar watermelon purebred in F6 generation.
2. The method according to claim 1, wherein the female parent variety in the step (1) is selected from "Sumeng No. 6" and the male parent variety is selected from "Xiaolan".
3. The method according to claim 1, wherein the single plant with excellent comprehensive properties of the F1 generation in the step (2) is round fruits, tough peels, red yellow pulps and disease resistance;
the special characters of the single melon screened by the F2 generation are high sugar, round fruits, tough skin, yellow flesh and disease resistance;
the special characters of the single melon screened in the F3 generation are high sugar, round fruits, tough skin, yellow flesh and disease resistance.
4. The creating method as claimed in claim 1, wherein said step (2) is characterized in that said single melon selected from the F2 generation and the F3 generation is identified in the seedling stage.
5. The method according to claim 1, wherein in the step (2), the sugar degree of the watermelon harvested by the F2 generation and the F3 generation is detected, and the number of single watermelon with specific characteristics is 5, which are respectively selected from the F2 generation and the F3 generation.
6. The creating method as claimed in claim 1, wherein in the step (2), a single plant with pure resistance to wilt is selected for one month before planting, a seed production land is selected for soil preparation, fertilization and furrow making, 3-4 leaves for 1 heart planting in spring and 1-2 leaves for 1 heart planting in autumn are selected.
7. The method according to claim 6, wherein the planting density is 1800 plants/667 m in general 2 Plant spacing 40cm, row spacing 90cm.
8. The method according to claim 1, wherein the female flowers 1 are removed and the female flowers 2 or 3 are selected for pollination and melon keeping when the female parent and the male parent are hybridized in the step (2).
9. The method of creating of claim 8, wherein the pollination method is: pollinating the male flowers within 3 hours after the male flowers are opened, lightly pinching the male flower stems by hands, removing the male flower petals, and uniformly coating the pollen on the pistil column heads until obvious yellow pollen grains are on the pistil column heads.
10. The method of claim 9, wherein the seed melon is harvested 35 to 40 days after pollination.
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