KR102140640B1 - Novel Citrullus vulgaris comprising 2 translocation region in the chromosome - Google Patents

Abstract

The present invention is 1 and 2 of the watermelon chromosome; And chromosomes 3 and 7, each of which comprises a translocation watermelon, a part of the new type watermelon plant, a watermelon regenerated from a part of the new type watermelon plant, and a method for producing the new type watermelon. The new varieties of watermelons provided by the present invention have a significant reduction in the number of seeds in the flesh due to the two translocations, so as to improve the quality of the flesh due to the decrease in the number of seeds, as well as easy cultivation using the seeds, it is possible to grow high quality watermelons. Therefore, it will be able to contribute widely to the increase of farm household income.

Description

New Novel Citrullus vulgaris comprising 2 translocation region in the chromosome}

The present invention relates to a new breed watermelon comprising two translocations in a chromosome, and more specifically, the present invention relates to watermelon chromosomes 1 and 2; And chromosomes 3 and 7, each of which comprises a translocation watermelon, a part of the new type watermelon plant, a watermelon regenerated from a part of the new type watermelon plant, and a method for producing the new type watermelon.

The watermelon cultivation area in Korea is about 34,500ha, of which the field cultivation area is 38.3% and the facility cultivation area is 61.7%, and the facility cultivation area is gradually increasing. With this increase in facility cultivation, consumers can enjoy the sweet and cool taste of watermelon at any time of the year. Various studies have been conducted to improve the quality of such watermelons. Seedless watermelon, developed as part of the above study, was first made in Japan in 1951 by Dr. Kihara in Japan, and has been widely known in the public by Dr. Woo Chang-chun in Korea.

In general, seedless watermelon is easy to eat, and in general watermelon, the seed consumes all the nutrients and the remaining remains accumulate in the flesh, but the seedless watermelon does not absorb the nutrients without the seeds, and all the nutrients accumulate in the flesh, so taste and sugar content It is known to be excellent, and various methods for producing it more effectively have been developed. According to what has been known so far, treatment of hormones and colchicine, irradiation of pollen or seeds with radiation, or cultivation of seedless watermelons by pollinating 3 to 8-fold pollen on female flowers of diploid normal watermelons The method is known, but since it is impossible to produce using seeds, there is a disadvantage that it has to go through a very complicated process compared to the general method of producing watermelon through seeds every time.

In order to solve this shortcoming, a new variety has been developed that contains a small number of seeds in the flesh of a watermelon (Korean Patent Registration No. 1635497). The cultivar is a variety in which the translocation of chromosome 6 and chromosome 7 of watermelon is induced, and it is known that the formation rate of normal spouses is reduced by translocation, and as a result, the number of seeds in the fruit is reduced. It is known that watermelons of the above varieties are varieties in which chromosomal level mutation is induced, and it is impossible to produce them by a planned genetic manipulation known to date.

Against this background, the present inventors have made extensive efforts to develop watermelons of new varieties that contain fewer seeds in the flesh, and as a result, new varieties of watermelons with two independent translocations induced in the chromosome of watermelons have fewer seeds. It was confirmed to include in the pulp, and the present invention was completed.

One object of the present invention is to provide a new variety of watermelon comprising two independent translocations in the chromosome of the watermelon.

Another object of the present invention is to provide a part of the new variety of watermelon plants.

Another object of the present invention is to provide a watermelon regenerated from a part of the new variety of watermelon plants.

Another object of the present invention is to provide a method for producing the new variety of watermelon.

The present inventors sought to develop a new variety of watermelons containing a large number of translocations in a chromosome in order to develop a new variety of watermelons containing fewer seeds in the flesh. To this end, a random mutagenesis is induced on the watermelon seed by irradiation or the like, and by cultivating the mutated watermelon seed, the shape and characteristics of the watermelon plant itself are the same as those of normal individuals, but produced by crossing this with the normal watermelon. The number of seeds in the flesh of the offspring (F1) was about 50% of the normal individuals. Assuming that the selected seed contains one translocation in its chromosome, and the selected seed is self-crossed for several generations to produce a number of varieties (L1 varieties) with one translocation trait fixed in the chromosome.

In order to accumulate various translocations in a chromosome included in each of the produced L1 varieties into a single seed, the L1 varieties are cross-bred, so that the shape and characteristics of the watermelon plant itself are the same as those of normal individuals, but this is crossed with normal watermelons. The seeds produced in the flesh of the progeny breed (F1) produced by incubation were selected for approximately 25% of the normal individuals. It is assumed that the selected seeds contain two translocations in their chromosomes, and the selected seeds are self-crossed for several generations to produce a number of varieties (L2 varieties) with two translocation traits fixed in the chromosome.

In order to confirm whether the produced L2 varieties contain two independent translocations in the chromosome, genome analysis performed results in the chromosome 1 and 2; And it was confirmed that each translocation was induced between chromosomes 3 and 7.

When the L2 variety produced is self-crossed, the number of seeds in the flesh does not differ from that of the normal individuals, but when the L2 variety is crossed with the general watermelon varieties, about 25 compared to the number of seeds in the flesh of the general watermelon varieties. It is possible to produce watermelons of new varieties with the number of seeds in the flesh, which is only %. In the case of a seedless watermelon that has been developed so far, it has to go through a very complicated process compared to the method of producing a normal watermelon through seed every time, but the new varieties of the present invention can be produced only by a simple crossing method, so it is conventional in terms of convenience of the production method. It was found to be significantly superior to the seedless watermelon varieties of.

As such, varieties containing two independent translocations in the chromosome of watermelon have not been reported at all, and were first developed by the present inventors.

As one embodiment for achieving the above object, the present invention is 1 and 2 of the watermelon chromosome; And, by each translocation between chromosomes 3 and 7, a new variety of watermelon with a reduced number of seeds in the flesh.

The term "translocation" of the present invention means a genetic variation in which parts of different chromosomes (non-homogenous chromosomes) are exchanged or transferred.

The new varieties of watermelons provided by the present invention have a reduced number of seeds in the flesh by two independent translocations in the chromosome, the first of which is the translocation between chromosomes 1 and 2, and the second is chromosome 3 and 7 It is a transposition between.

The translocation between the chromosomes 1 and 2 is not particularly limited, but as an example, some nucleotide sequence of chromosome 2 may be inserted into any part of chromosome 1, and as another example, chromosome 1 The base sequence of the 24,626,084 to 27,627,152b region of chromosome 2 may be inserted into any one of the 30,820,000 to 30,830,000bp regions, and as another example, the 24,626,084 to 27,627,152b region of chromosome 2 at the 30,828,108bp region of chromosome 1 The nucleotide sequence of is inserted, the inserted nucleotide sequence may be a form deleted from chromosome 2.

The translocation between the chromosomes 3 and 7 is not particularly limited, but as an example, some nucleotide sequence of chromosome 7 may be inserted into any part of chromosome 3, and as another example, chromosome 3 The base sequence of the 26,138,780 to 26,694,216bp region of chromosome 7 may be inserted into any one of the 29,170,000 to 29,180,000bp regions, and as another example, the 26,138,780 to 26,694,216bp region of chromosome 7 at 29,179,588bp region of chromosome 3 The nucleotide sequence of is inserted, the inserted nucleotide sequence may be a form deleted from chromosome 7.

The term "normal watermelon" of the present invention means a watermelon plant, watermelon fruit or watermelon seed that does not contain a translocation in the chromosome of the watermelon.

The watermelon of the new variety of the present invention exhibits a characteristic in which the formation rate of a normal spouse is lowered by the above two translocations, and the number of seeds in the fruit is reduced than that of the normal watermelon. It was analyzed that it was because there was no change in traits other than fertility such as plant shape, fruit size, and food taste. However, at the time of meiosis, only a part of the spouse formation process forms a complete genome without deletion or duplication, so only a part of the spouse has immortality.

Eventually, the new watermelon of the present invention, which is a descendant watermelon (F1) generated by cross-mutation of a mutated watermelon variety including two translocations and a normal watermelon variety, has a significantly reduced number of seeds in the flesh compared to the normal watermelon variety. .

The new varieties of watermelons provided in the present invention are characterized in that the number of seeds in the flesh is reduced compared to the number of seeds in the flesh of the watermelon that does not contain a translocation in the chromosome. The level of the number of seeds in the flesh of the new breed watermelon is not particularly limited, but as an example, the number of seeds in the flesh of the watermelon that does not include translocation may be 20 to 35%, and as another example, within the chromosome It may be 23 to 27% of the number of seeds in the flesh of the watermelon that does not contain translocation, and as another example, 25% of the number of seeds of the watermelon that does not include the translocation in the chromosome.

The new variety watermelon exhibits the following characteristics: it contains a chromosome comprising two translocations; Compared to normal varieties (variants without chromosomal translocation) watermelon, the development of female and male flowers shows the same level; The content of embryo and pollen with fertility is 25% or less; The number of seeds in the fruit is 25% or less of the watermelon of normal varieties; Excellent self-cultivation performance; The hyperploid is mono-elliptical; Ground color is green, slightly dark; The skin contains thick, broad skin; Sugar content is 10 to 13 Brix; The middle age is moderate, the nodes are slightly short and stable; Produce fruit that exhibits an excess of 8 to 10 kg in August harvest; During August harvest, maturity is usually around 42 days; Dark flesh and thin skin, but less heat; The flesh is bright red, and the coloration is fast and the sugar content rises early.

According to an embodiment of the present invention, after processing high-level gamma rays on watermelon seeds, they are sown and cultivated to primaryly select seeds having normal watermelon characteristics, and to watermelon watermelon plants grown from primaryly selected seeds. By crossing the plants, the seeds with the number of seeds in the flesh showing about 50% of the normal watermelon were second-selected, and the second-selected watermelon was self-crossed for several generations, so that a single translocation trait was fixed in the chromosome (L1 variety). Many were produced.

In order to develop a variety in which two translocation traits were fixed to one seed from the plurality of produced L1 varieties, each of the L1 varieties was sown, cultivated, and cross-crossed to obtain a number of other seeds. By sowing and cultivating each of the obtained other seed seeds, the seeds having normal watermelon characteristics are first sorted, and the watermelon plants grown from the first sorted seeds are crossed with normal watermelon plants, so that the number of seeds in the flesh is about the amount of normal watermelon. Seeds representing 25% were second-selected, and second-selected watermelons were self-crossed for several generations to produce varieties with two translocation traits fixed in the chromosome (L2 varieties).

As a result of analyzing the genome of the produced L2 varieties, regions 24,626,084 to 27,627,152b of chromosome 2 were inserted into the 30,828,108bp region of chromosome 1, and the same region was deleted from chromosome 2; And 26,138,780 to 26,694,216bp of chromosome 7 was inserted into the 29,179,588bp region of chromosome 3, and it was confirmed that the same region includes all translocations deleted in chromosome 7.

Thus, the present inventor is produced by crossing the watermelon plant grown from the seed of the L2 variety and the finished watermelon plant, and the number of seeds in the flesh is about 25% of the normal watermelon. Seeds of watermelon fruit " Citrullus lanatus var . lanatus A5282", and deposited with the deposit number KCTC18462P on April 14, 2016, at the Bio-Resources Center, Jeonbuk Branch, Korea Research Institute of Bioscience and Biotechnology, 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea.

In another embodiment, the present invention provides a watermelon regenerated from a portion of the new species of watermelon plant and a portion of the watermelon plant.

In the present invention, a part of the new-type watermelon plant is not particularly limited as long as it can be used for production or regeneration of the new-type watermelon, but as an example, seeds, pollen, egg cells or nutrient tissue derived from the new-type watermelon may be Can.

In addition, according to a known method using a part of the new varieties of watermelon plants, the new varieties of watermelon can be produced or regenerated.

As an example, after sowing and cultivating the seeds of the new varieties of watermelons, self-crossing may produce new varieties of watermelons, and as another example, the pollen of the new varieties of watermelons may be processed into female flowers of other watermelon varieties to produce new varieties of watermelons. As another example, a new flower of watermelon can be produced by treating a pollen of another watermelon variety on a female flower containing an egg cell of the new variety of watermelon, and as another example, another watermelon of the nutritional tissue obtained by cultivating the new variety of watermelon By grafting to the plants of the variety, it is also possible to produce new varieties of watermelon.

As another embodiment, the present invention provides a method for producing the new variety of watermelon.

Specifically, the method for producing a new variety of watermelon provided by the present invention is, for example, a watermelon plant containing one translocation in the chromosome as a parent, and a watermelon plant containing another translocation in the chromosome as a parent to each other By mating, it may include a step of obtaining a watermelon of its offspring, and as another example, cross-crossing a watermelon plant containing two translocations in a chromosome as a parent, and a watermelon plant not containing a translocation in a chromosome as a parent In other words, it may include a step of obtaining a watermelon of its offspring, and as another example, cross-crossing a watermelon plant containing no translocation in the chromosome as a parent, and a watermelon plant containing two translocations in the chromosome as a parent To obtain a progeny watermelon thereof.

In each of the above methods, a watermelon plant including one translocation is not particularly limited, but may be obtained by sowing and cultivating a watermelon seed containing one of the two translocations described above, and the two translocations The watermelon plant containing is not particularly limited, but may be obtained by sowing and cultivating watermelon seeds containing both the two translocations described above.

The watermelon seed comprising the one translocation can be obtained by mutating a normal watermelon seed, but the mutation is not particularly limited thereto, and may be performed by irradiating gamma rays to the watermelon seed, as another example. , High-level gamma ray using Co-60 Gamma-ray on watermelon seeds can be performed, and as another example, high-level gamma ray using Co-60 Gamma-ray on watermelon seeds is used for 24 hours at a total dose of 1,000 Gy. You can do it by investigating.

The watermelon seeds containing the two translocations can be obtained by cross-referencing the watermelon seeds containing the one translocation, for example, obtained by sowing and cultivating the watermelon seeds containing the one translocation. Among the offspring seeds obtained by cross-breeding each of the watermelon plants, it can be obtained by selecting the offspring seeds with about 25% of the fertility of the normal watermelon. The method for measuring the fertility is not particularly limited, but as an example, by observing the shape of the pollen obtained from the watermelon flowers of watermelon plants obtained by sowing and cultivating watermelon seeds to measure fertility, normal and abnormal forms A method of comparing the ratios and calculating the ratio of the pollen in a normal form can be used. When the ratio of the pot in the normal form to the total number of pots is 25%, it can be determined that the fertility of the offspring seeds is 25%. As another example, a method of crossing a watermelon plant obtained by sowing and cultivating a watermelon seed to measure fertility with a normal watermelon plant and counting the number of seeds in the flesh of the progeny watermelon obtained therefrom may be used. When the number of seeds in the flesh of the offspring watermelon is 25% compared to the number of seeds in the flesh of the normal watermelon, it can be determined that the fertility of the offspring seeds is 25%.

On the other hand, the method for producing a new variety of watermelon may further include a step of verifying whether the number of seeds obtained in the flesh is reduced by the two translocations of the obtained offspring watermelon.

The verification step is not particularly limited thereto, but may be performed by hybridizing a plant of a descendant watermelon and a parent or sub copy used for its production.

As an example, a watermelon plant (AB/AB) containing two translocations is a parent, and a progeny watermelon produced by cross-crossing a watermelon plant (ab/ab) that does not contain a translocation in a chromosome (new variety watermelon) In the case of ), a method of hybridizing females using a watermelon plant obtained by sowing and cultivating the offspring watermelon as a parent and a watermelon plant including two translocations as a parent can be used.

First, when the offspring watermelon is a new breed watermelon provided by the present invention, the plants of the offspring watermelon include a genotype (AB/ab) of a heterozygote comprising two translocations, and a watermelon plant comprising two translocations Since the genotype of the homozygote (AB/AB) containing two translocations is included, the F1 generation generated by the cross-hybridization is the genotype of the heterozygote (AB/ab) with the genotype (AB/ab) of the heterozygote AB). In addition, when comparing fertility, the F1 generation generated by cross-breeding is about 100% compared to the fertility of a seed with a genotype (AB/ab) of heterozygotes having a fertility of about 25% compared to that of normal watermelon Seeds having a genotype (AB/AB) of the homozygote having the fertility of 1 may be included. At this time, the method of measuring the fertility is the same as described above.

On the other hand, when the offspring watermelon is not a new breed watermelon provided by the present invention, the F1 generation generated by the crossing is 1:1 with the seed having the genotype of the heterozygous and the seed having the genotype of the homozygous. Seeds that do not contain or have a genotype of a heterozygote having a gonorrhea of about 25% compared to that of a normal watermelon and a seed having a genotype of a homozygote having a gonorrhea of about 100% compared to that of a normal watermelon 1: May not be included as 1.

The term "backcross" of the present invention means a method of crossing the first generation (F1) of hybrids obtained by breeding A and B breeds with either of the parents A and B, that is, (A× B)×A or (A×B)×B.

As another example, a watermelon plant (Ab/Ab) containing one translocation is a parent, and a watermelon plant (a new variety watermelon) produced by cross-crossing a watermelon plant (aB/aB) containing another translocation as a parent. In the case of ), a method of hybridizing females using a watermelon plant obtained by sowing and cultivating the offspring watermelon as a parent and a watermelon plant containing one translocation as a parent can be used.

First, when the offspring watermelon is a new breed watermelon provided by the present invention, the plant of the offspring watermelon includes a genotype (Ab/aB) of a heterozygote comprising two translocations, and a watermelon plant comprising one translocation is Since it contains the genotype (Ab/Ab) of the homozygote containing one translocation, the F1 generation generated by the crossover is the genotype (Ab/) of the homozygote with the seed having the genotype (Ab/aB) of the heterozygous Ab) with 1:1. In addition, when comparing fertility, the F1 generation generated by the crossing is about 100% compared to the fertility of the seed with the genotype (Ab/aB) of the heterozygote having a fertility of about 25% compared to the fertility of the normal watermelon. Seeds having a genotype (Ab/Ab) of the homozygote having the fertility of 1 may be included. At this time, the method of measuring the fertility is the same as described above.

On the other hand, when the offspring watermelon is not a new breed watermelon provided by the present invention, the F1 generation generated by the crossing is 1:1 with the seed having the genotype of the heterozygous and the seed having the genotype of the homozygous. Seeds that do not contain or have a genotype of a heterozygote having a gonorrhea of about 25% compared to that of a normal watermelon and a seed having a genotype of a homozygote having a gonorrhea of about 100% compared to that of a normal watermelon 1: May not be included as 1.

This result was researched with the support of the Golden Seed Project of the Ministry of Food, Agriculture, Forestry and Fisheries Technology Planning and Evaluation (Task No. 213006-05-1-SBQ10).

The new varieties of watermelons provided by the present invention have a significant reduction in the number of seeds in the flesh due to the two translocations, so as to improve the quality of the flesh due to the decrease in the number of seeds, as well as easy cultivation using the seeds, it is possible to grow high quality watermelons. Therefore, it will be able to contribute widely to the increase of farm household income.

1 is a photograph showing a cross section of each fruit produced by cultivating a number of L2 varieties produced in the present invention.
2 is a chart showing translocation results between chromosomes 1 and 2.
3 is a chart showing translocation results between chromosomes 3 and 7.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: 1 Translocation To Production of containing varieties (L1 varieties)

A plastic container (28 x 20 x 8 cm3) contains 12,000 grains of watermelon seeds (approximately 500 g), and a high-level gamma ray using Co-60 Gamma-ray is total dose of 1,000 Gy. Obtained.

The obtained radiation treated seeds (generation TR0) were sown in a 200-neck tray containing a medium for sowing.

Then, cultivating for 48 hours at 28° C. while irrigating the tray with a sufficient amount of water, sprouting it, cultivating it according to the well-known seed germination and grafting seedling method, and as a result, 4,000 seedlings that can be planted. Obtained.

Each seedling obtained above was mulched with a green film in a mockup of 3.5 m in a vinyl house and set at intervals of 45 cm during the day. Subsequently, 5 stems were soaked to maintain 3 stems per abandon, and were cultivated according to a known watermelon cultivation method. Then, about 20% of the mutants different from the characteristics of the untreated line were generated in each cultivated watermelon plant, and the mutants thus generated were first removed to select individuals exhibiting normal growth characteristics.

Self-pollination was performed on the third female flower in the individual showing the selected normal growth characteristics.

The characteristics of the untreated system are checked for the fruits that have been harvested from the individual who performed the self-pollination, and the individuals showing other fruit characteristics are removed secondarily, and 1,214 fruits showing normal fruit characteristics are harvested. Seeds (TR1 generation) were obtained from the fruit and stored.

Except for sowing the obtained TR1 generation seeds for 40 weeks for each individual, the same method as described above was performed to obtain seedlings for each individual, and the obtained seedlings were formulated for 10 weeks for each individual, and known watermelon It was cultivated according to the cultivation method. Then, each pollen was obtained from the male flowers blooming in each cultivated individual, and the obtained pollen was observed under a microscope (x100), and the number of pots in a normal form and the number of pots in a crushed form were 1:1. 125 weeks were selected. Fruits were harvested by self-pollination in the selected individuals, and seeds (TR2 generation) were obtained from these harvested fruits and stored.

Except for sowing the obtained TR2 generation seeds for 40 weeks for each individual, the same method as described above was performed to obtain seedlings for each individual, and the obtained seedlings were formulated for 8 weeks for each individual, and known watermelon It was cultivated according to the cultivation method. Then, each pollen was obtained from the male flowers blooming in each cultivated individual, and the obtained pollen was observed under a microscope (x100), and the number of pots in a normal form and the number of pots in a crushed form were 1:1. Was selected first, and 12 weeks of the individual who showed 100% fertility among the selected individuals were selected second. Fruits were harvested by self-pollination in the selected individuals, and seeds (TR3 generation) were obtained from these harvested fruits and stored.

Except for sowing the obtained TR3 generation seeds for 30 weeks for each individual, the same method as described above was performed to obtain seedlings for each individual, and the obtained seedlings were formulated for 6 weeks for each individual, and known watermelon It was cultivated according to the cultivation method. Then, each pollen was obtained from a male flower blooming in each cultivated individual, and the obtained pollen was observed under a microscope (x100), and it was confirmed that all pots exhibited a normal shape. Fruits were harvested by self-pollination in the cultivated individuals, and seeds were obtained and stored from these harvested fruits.

On the other hand, by crossing the pollen by adding it to the female flower of a common watermelon, a seed for black mating is obtained, and after sowing and cultivating it to bloom the water flower, the pollen of the water flower is observed under a microscope (x100) to count the number of pots in a normal form. When and the number of crushed pollen is included 1:1, the TR3 generation was determined to be a lineage in which the mutation includes a fixed genotype.

Subsequently, by repeating the above-described process several times, various L1 varieties including genotypes in which various mutations were immobilized were produced.

Example 2: 2 Translocation To Production of containing varieties (L2 varieties)

After sowing and cultivating the seeds of the plurality of L1 varieties prepared in Example 1 by the method of Example 1, and crossing them with each other, after harvesting the fruit, various other seeds were obtained therefrom.

Individuals showing normal growth characteristics were first selected by sowing and cultivating the obtained other seeds.

A pollen was obtained from the male flower of the first selected individual, and the pollen was added to a female flower of a common watermelon and crossed to obtain seeds for black mating. After sowing and cultivating the obtained seeds for black mating to bloom the male flower, the pollen of the male flower was observed under a microscope (x100) to show the result that the number of pots in the normal form and the number of pots in the crushed form were 25:75, Second seed was selected.

Subsequently, seeding, cultivation and self-pollination of the second-selected other seed seeds to obtain a child seed, and sowing and cultivation to obtain a pollen from a male flower, and crossing the pollen by adding it to a female flower of a common watermelon, for black mating Seeds were obtained. After sowing and cultivating the obtained seeds for black mating to cultivate male flowers, the pollen of male flowers was observed under a microscope (x100) to select the other type of seed, which was the third time, showing the result of all pots being in a normal form.

The process of sowing and cultivating the third-selected other seeds, and self-crossing them to obtain a child seed was repeated twice, thereby producing a plurality of L2 varieties with a fixed genotype.

The seeds of the produced L2 varieties were sown, cultivated, and self-pollinated, respectively, to produce fruits, and the types of seeds included in each of the produced fruits were analyzed (FIG. 1).

1 is a photograph showing a cross section of each fruit produced by cultivating a number of L2 varieties produced in the present invention.

As shown in FIG. 1, among the fruits produced from three different L2 varieties, a number of black seeds were observed in the left fruit, and black seeds were not observed in the central fruit, and a few black seeds were observed in the right fruit. Was observed.

As a result of analyzing the black seeds, it was confirmed that only the seed coat was present, and there was no abnormal seed inside.

As a result, it was found that even in the case of L2 varieties produced by the same method, different traits can be exhibited from the viewpoint of fruit produced.

Accordingly, the present inventors finally selected L2 varieties in which no abnormal seeds exist in the fruit.

Example 3: Fruit Characterization

After seeding and cultivating the seeds of each of the varieties prepared in Examples 1 and 2 in the manner of Example 1 to bloom each male flower, the pollen of each male flower is added to female flowers of a common watermelon variety and crossed to make fruit. After harvesting, the average weight of each fruit, the average sugar, and the average number of seeds contained therein were analyzed using a statistical analysis package R package (ver 3.2.3) (Table 1). At this time, a normal watermelon without radiation was used as a control.

Fruit characteristics of each variety watermelon Watermelon fruit Average fruit weight (kg) Average sugar content (brix) Average number of seeds Control 5.4±0.8 11.5±1.0 296.3±133.9 L1 varieties 4.8±0.4 11.7±0.9 134.9±54.8 L2 varieties 5.8±0.9 10.8±0.9 95.6±20.2

As shown in Table 1, among the fruit characteristics of each variety, the fruit weight and the sugar content did not show a particularly significant difference between each fruit, but the number of seeds contained in the fruit was about 46% of the L1 variety, and the L2 variety was weak compared to the control group. It was confirmed that it was 32%.

Example 4: Chromosome analysis of L2 varieties

From the results of Example 3, since it was confirmed that the fruit obtained by crossing the L2 variety and the general variety contains the smallest number of seeds, the genetic variation of the L2 variety was analyzed at the chromosome level.

In general, the watermelon reference genome utilizes genome sequences (Nature Genetics, 2012) registered to the International Cucurbit Genomics Initiative (ICuGI; www.icugi.org) worldwide. In addition, with the recent advancement in science and technology, I/S instrument and IrysChip® of BioNano Genomics company (www.bionanogenomics.com) that can read chromosomes linearly and very long, can be used to solve the structural variation of chromosomes (Structural Variation). (Single-molecule resolution) can be measured at the level. Therefore, it can be used as an important tool in the analysis of structural changes of chromosomes, such as translocation. However, in order to confirm the translocation position of the translocation lines targeted in the present invention, as a result of a comparative analysis using the Irys system with reference dielectrics registered in ICuGI, the exact location could not be confirmed. As a result, it was inferred that the scaffold order of the existing reference genome was inaccurate and analysis was impossible. In particular, it was confirmed that the more accurate position can be confirmed by comparing with the reference genome created in the lineage having the same genomic sequence as the translocated lineage. Did.

Accordingly, the present inventors tried to assemble a reference genome for the same line as the translocation line presented in the present invention, and then tried to confirm the final chromosomal structural variation by comparing the Irys results of the translocation line again. The Bionano Irys Dataset Stats and Bionano Irys Assembly Stats used are shown in Tables 2 and 3, respectively.

Bionano Irys Dataset Stats sample A529 (translocation) SBA Enzyme
Quantity >150Kb (Gbp)
Quantity per Scan (Mbp)
Molecule N50 >150Kb (Kbp)
# of Molecules
Empirical Label Density(per 100Kb) BspQI
96.5
1,636
285.9
350,755
7.4 BssSI
96.3
1,604.7
288.7
346,107
10.0 BspQI
70.9
1,182.2
265.4
270,508
8.1 BssSI
30.5
1,015.5
241.6
126,848
10.9

Bionano Irys Assembly Stats sample A529 (translocation) SBA Enzyme
# of Consensus Genome Map
Consensus Genome Maps Size (Mbp)
consensus Genome Maps N50 (Mbp)
Average Depth of Mol Coverage BspQI
148
397.521
4.587
84.5 BssSI
172
388.818
3.318
85.1 BspQI
167
392.947
4.159
74.9 BssSI
431
361.877
1.141
46.5

Example 4-1: SMRT sequencing and Irys Platform SBA system Reference dielectric complete

In the present invention, for assembly and completion of reference genome sequences for translocation identification, rather than contig of short-read of existing Illumina company and scaffolding through mate-pair of various sizes, PacBio's single molecule real- time) We tried to utilize the results of the study that sequencing can read and assemble longer DNA strands. The analysis was commissioned to a domestic DnLink company, and as a result, 513 scaffolds and N50s were able to obtain 12.5M levels of results (Table 4).

Results of scaffolding through SMRT sequencing of PacBio and hybrid scaffolding (BsQI and BssSI restriction enzyme site sequence comparison) using BioNano Genomics' Irys platform PacBio BspQl BssSl No. contigs/scaffolds
Total size of contigs/scaffolds
Longest contigs/scaffolds
No. contigs/scaffolds> 1K nt
No. contigs/scaffolds> 10K nt
No. contigs/scaffolds> 100K nt
No. contigs/scaffolds> 1M nt
No. contigs/scaffolds> 10M nt
N50 contigs/scaffolds length
N's (%)
GC contents(%) 513
373,537,612
27,985,524
510
427
58
43
14
12,545,686
0
34.02 18
364,378,392
36,398,214
18
18
18
18
12
31,040,585
0.33
33.47 15
365,666,204
37,950,524
15
15
15
14
12
31,040,585
0.68
33.36

As shown in Table 4, it was confirmed that the size of the entire genome is 373.5M.

In addition, in order to reduce 513 scaffolds obtained from PacBio's SMRT sequencing results, the BioNano Genomics company's Irys platform was used. After processing BspQI and BssSI restriction enzymes, each sequence variation was sequentially compared with SMRT sequencing results. Through hybrid scaffolding, the final scaffold was reduced to 15 scaffolds, and the N50 resulted in 31M. In addition, the final assembly of 11 chromosomes was completed through a blast targeting the genome (Table 5).

Reference genome chromosome sequence assembly results and size for each chromosome for SBA strains at the level of watermelon 2n=2x=22 (n=11) Chromosome number size One
2
3
4
5
6
7
8
9
10
11
Total
unscaffolded+mitochondria 37,023,786
37,974,157
37,560,498
27,052,703
35,983,385
29,626,062
32,016,966
23,850,001
37,950,524
35,311,591
31,040,585
365,390,258
7,625,934

Example 4-2: Translocation Analysis of chromosome mutations in the lineage

In comparison with the SBA reference dielectric obtained in Example 4-1, Irys analysis was performed in the translocation line to analyze chromosomal variations of the translocation line derived from the same species.

Example 4-2-1: Analysis of chromosome 1

As a result of analyzing the chromosome mutation of chromosome 1, it was confirmed that translocation occurred between chromosomes 1 and 2 (FIG. 2).

2 is a chart showing translocation results between chromosomes 1 and 2.

As shown in Figure 2, the 30,828,108bp region of chromosome 1 was inserted by translocation of 24,626,084 to 27,627,152bp (total 3,001,068bp) of chromosome 2, and it was confirmed that the same site was deleted in chromosome 2.

Example 4-2-2: Analysis of chromosome 3

As a result of analyzing the chromosomal variation of chromosome 3, it was confirmed that translocation occurred between chromosomes 3 and 7 (FIG. 3).

3 is a chart showing translocation results between chromosomes 3 and 7.

As shown in FIG. 3, 26,138,780 to 26,694,216bp (total 555,436bp) of chromosome 7 was translocated and inserted into the 29,179,588bp site of chromosome 3, and the same site was deleted in chromosome 7.

Summarizing the results of the above Examples 4-2-1 and 4-2-2, the L2 varieties provided in the present invention have two translocations, each translocation occurring between chromosomes 1/2 and 3/7. It was found that it was a mutant variety.

Thus, the present inventors produced the seeds of watermelon fruit with the reduced number of seeds produced by cross-breeding the L2 varieties with the general varieties " Citrullus lanatus var . lanatus A5282", and deposited with the deposit number KCTC18462P on April 14, 2016, at the Bio-Resources Center, Jeonbuk Branch, Korea Research Institute of Bioscience and Biotechnology, 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea.

Biological Resource Center KCTC18462P 20160414

Claims (20)

1 and 2 of watermelon chromosomes; And, by each translocation between chromosomes 3 and 7, a new variety watermelon with a reduced number of seeds in the flesh, wherein the new variety watermelon is derived from the seed of deposit number KCTC18462P, and a new variety watermelon with a reduced number of seeds in the flesh.
According to claim 1,
The translocation between chromosome 1 and 2 is the base sequence of the 24,626,084 to 27,627,152b region of chromosome 2 inserted into any one of the 30,820,000 to 30,830,000bp regions of chromosome 1, and the inserted base sequence is deleted from chromosome 2 A new type of watermelon with a reduced number of seeds in the flesh.
According to claim 1,
The translocation between chromosome 3 and 7 is inserted into any of the 29,170,000 to 29,180,000bp regions of chromosome 3, and the nucleotide sequence of 26,138,780 to 26,694,216bp of chromosome 7 is inserted, and the inserted nucleotide sequence is deleted from chromosome 7 A new type of watermelon with a reduced number of seeds in the flesh.
According to claim 1,
The number of seeds in the flesh contained in the new variety watermelon is 20 to 35% of the number of seeds in the flesh compared to the number of seeds in the flesh that does not contain a translocation in the chromosome, a new variety watermelon with a reduced number of seeds in the flesh.
delete A part of the new-type watermelon plant derived from the new-type watermelon according to any one of claims 1 to 4.
The method of claim 6,
Part of the new variety of watermelon plants, seeds, pollen, egg cells or trophic tissue, part of the new variety of watermelon plants.
A watermelon regenerated from a part of the new-type watermelon plant of claim 6.
Claims 1 to 1, comprising the steps of obtaining a watermelon of its progeny by cross-linking a watermelon plant containing one translocation in a chromosome as a parent, and a watermelon plant containing another translocation in a chromosome as a parent. A method for producing a watermelon of a new variety according to any one of the four clauses.
The method of claim 9,
The watermelon plant including the one translocation includes translocations of chromosomes 1 and 2 among watermelon chromosomes; Or a method for producing watermelon of new varieties, which is derived from watermelon seeds including translocations of chromosomes 3 and 7.
The method of claim 10,
The translocation between chromosome 1 and 2 is the base sequence of the 24,626,084 to 27,627,152b region of chromosome 2 inserted into any one of the 30,820,000 to 30,830,000bp regions of chromosome 1, and the inserted base sequence is deleted from chromosome 2 Method of producing new varieties of watermelon, which is in the old form.
The method of claim 10,
The translocation between chromosome 3 and 7 is inserted into any of the 29,170,000 to 29,180,000bp regions of chromosome 3, and the nucleotide sequence of 26,138,780 to 26,694,216bp of chromosome 7 is inserted, and the inserted nucleotide sequence is deleted from chromosome 7 Method of producing new varieties of watermelon, which is in the old form.
The method of claim 9,
The translocation is produced by mutation, the production method of new varieties of watermelon.
The method of claim 9,
The method further comprises the step of verifying the genotype or fertility of the offspring watermelon, a method for producing a new breed watermelon.
The method of claim 14,
The verification step is performed by hybridizing the plants of the offspring watermelon and the parent or sub-bone used for its production, a method for producing a new variety of watermelon.
The method of claim 15,
When the seed having the genotype of the heterozygote and the seed having the genotype of the homozygous are produced in 1:1 by the hybridization, the progeny watermelon is judged to be normally produced, and the method for producing a new breed watermelon.
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