Method for creating high-frequency introgression Chinese cabbage-cabbage introgression line

Abstract

The invention discloses a method for creating a high-frequency introgression Chinese cabbage-cabbage introgression line, and relates to the technical field of introgression breeding. According to the method, tetraploid Chinese cabbage and diploid cabbage are subjected to interspecific hybridization to obtain an allotriploid hybrid (AAC), then the AAC is subjected to hybridization and selfing with diploid Chinese cabbage, and a Chinese cabbage-cabbage introgression line with a high-frequency introgression exogenous species genome sequence is created in combination with free microspore culture. According to the invention, the heterotriploid hybrid mediated genetic system is utilized to realize interspecific high introgression, and a new method is provided for breaking the bottleneck of low exogenous species introgression frequency.

Description

Technical Field

[0001] The invention relates to the technical field of introgression breeding, and more particularly to a method for creating a high-frequency introgression Chinese cabbage-cabbage introgression line. Background Art

[0002] Long-term artificial selection and domestication in traditional breeding, coupled with the intensification of climate change, have narrowed the available sources of genetic variation, making it difficult to increase genetic variation within species and cultivate varieties with breakthrough new traits. Introgression is a safe and effective method of interspecific gene transfer in plant breeding, providing a path to enrich the genetic diversity of crops. However, challenges associated with efficient introgression and identification limit the application of introgression breeding.

[0003] At present, excellent foreign fragments and genes have been successfully introduced into various crops through interspecific hybridization. For example, in Brassica, it was reported that the average number of introgressions from the Cn genome to the An genome in natural Brassica napus and artificial Brassica napus (AnAnCnCn) was 3.19 and 9.00 per material, respectively. In other species, such as hybridization between allopolyploid wheat (AABBDD) and wild wheat relative Amblyopyrum muticum (TT), the T genome sequence was gradually introgressed into the D genome chromosome, with an average of 1.24 introgressed fragments per material; hybridization between cotton (Gossypium hirsutum, AADD) and diploid wild species G. anomalum (B1B1), the B1 genome fragment was gradually introgressed into the A genome chromosome, with an average of 1.54 introgressed fragments per line; in tomatoes, the genome fragment of the wild relative Solanum. lycopersicoides was gradually introgressed into the S. lycopersicum genome, with an average of 2.03 introgressed fragments per introgressed line. Due to the restrictions of species kinship and meiotic recombination exchange, the introgression frequency is low, the introduced exogenous fragments are few, and it is difficult to produce breakthrough new traits.

[0004] Therefore, how to significantly increase the frequency of introgression between species is an urgent problem that technicians in this field need to solve. Summary of the invention

[0005] In view of this, the present invention provides a method for creating a high-frequency introgression Chinese cabbage-Cabbage introgression line, which introduces a large number of Cabbage chromosome fragments into the Chinese cabbage genome, greatly enriching the genetic variation of the Chinese cabbage genome.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for creating a high-frequency introgression Chinese cabbage-Cabbage introgression line, comprising: interspecific hybridization of tetraploid Chinese cabbage (AAAA) and diploid Cabbage (CC) to obtain an allotriploid hybrid (AAC), the hybrid is further hybridized with diploid Chinese cabbage (AA), the hybrid progeny are self-pollinated, and free microspore culture is performed on the self-pollinated progeny to create a Chinese cabbage-Cabbage introgression line (DH).

[0008] Among them, the genetic system mediated by allotriploid interspecific hybrids is the key to achieving high-frequency interspecific introgression.

[0009] Basic principle: Chinese cabbage and kale come from a common ancestor. This close genetic relationship provides more opportunities for large-scale and high-frequency occurrence of homologous recombination. In the present invention, tetraploid Chinese cabbage is hybridized with diploid kale to obtain an allotriploid hybrid (AAC) with two A genomes and one C genome. Under the premise that the A genome and the C genome have a strong homology, the presence of a single C genome increases the probability of recombination between A and C, and the presence of two A genomes in the hybrid provides more opportunities for receiving genetic material from the C genome.

[0010] Further, the following steps are included:

[0011] (1) Chromosome doubling of diploid Chinese cabbage (2n=2x=20) to obtain tetraploid Chinese cabbage (2n=4x=40);

[0012] (2) hybridizing tetraploid Chinese cabbage with diploid cabbage (2n=2x=18), taking ovaries 5-7 days after pollination and inoculating them on improved White medium for culture, and peeling out surviving ovules after 28 days of culture and inoculating them on Heller medium for culture to obtain allotriploid hybrids;

[0013] (3) hybridizing an allotriploid hybrid with a diploid Chinese cabbage, and further self-pollinating the hybrid offspring to obtain self-pollinated offspring;

[0014] (4) Flower buds at the mononuclear marginal stage on the self-pollinated offspring plants were selected and free microspore culture was performed using B5 medium and NLN culture medium to obtain the homozygous Chinese cabbage-Cabbage introgression line.

[0015] Furthermore, the chromosome doubling method in step (1) is to treat the growing point with 0.2% colchicine for 48 hours.

[0016] Furthermore, in step (2), the tetraploid Chinese cabbage is the female parent, and the diploid cabbage is the male parent.

[0017] Furthermore, in step (3), the allotriploid hybrid is the female parent, and the diploid Chinese cabbage is the male parent.

[0018] Furthermore, the free microspore culture method in step (4) is:

[0019] About 35 flower buds were taken each time, and the microspores were purified in B5 medium after surface disinfection and sterilization; the purified microspores were sealed and cultured with NLN culture medium, and then cultured in the dark at 35°C for 24 hours before being transferred to a dark culture box; when the embryoid developed to the cotyledon stage, the cotyledon embryo was inoculated on MS medium, and the plant was regenerated at 25°C to obtain the Chinese cabbage-cabbage introgression line.

[0020] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0021] The present invention utilizes a genetic system mediated by allotriploid hybrids to achieve high interspecific introgression, providing a new method for breaking the bottleneck of low frequency of exogenous species introgression.

[0022] At the genome level, the Chinese cabbage-cabbage introgression system created by the introgression system infiltrates a large number of donor cabbage genome fragments into the Chinese cabbage genome through frequent partial homologous recombination between the A genome and the C genome. In a single introgression line, it can be achieved that all A chromosomes of Chinese cabbage have cabbage fragments infiltrated, and the introgressed cabbage fragments can involve all C chromosomes in the cabbage genome; at the trait level, the created Chinese cabbage-cabbage introgression line produces rich trait variation, including the generation of new traits that are not found in Chinese cabbage and cabbage; the generation of new traits that are not found in Chinese cabbage but are unique to cabbage; and the expansion of the variation range of the original traits within the Chinese cabbage species. The introgression system established by the present invention provides a new path for rapidly improving species genetic diversity and cross-species aggregate improvement breeding, and provides technical support for realizing the leap from the utilization of intraspecific hybrid vigor to the utilization of interspecific hybrid vigor.

[0023] Using tetraploid Chinese cabbage as the female parent and hybridizing it with diploid cabbage, an allotriploid hybrid was obtained, and then the DH (Double Hyploid) introgression line was created through hybridization, selfing, and free microspore culture. A large number of exogenous cabbage chromosome fragments were introgressed into the Chinese cabbage genome at a high frequency. It was identified that the maximum number of introgressed cabbage fragments / introgression line was 67, and the average number of introgressed cabbage fragments / introgression line was 28.28. It was significantly higher than the average recombination frequency of 3.19 and 9.00 between the natural cabbage-type rapeseed and the artificial synthetic rapeseed AC of the Brassica genus. In addition, it is much higher than other species, such as wheat introgression lines with an average of 1.24 introgression fragments per material, cotton chromosome fragment substitution lines with an average of 1.54 introgression fragments per line, and tomato introgression lines with an average of 2.03 introgression fragments per line. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 Flow chart for the construction and genome identification of the Chinese cabbage-Brassica oleracea introgression system; Figure 1 a is the construction diagram of the introgression line from Brassica oleracea to Chinese cabbage; Figure 1 b is the phenotypic characteristics of the tetraploid Chinese cabbage parent 9403Br;

[0026] Figure 1 c is the phenotypic characteristics of the diploid cabbage parent 9501Bo; Figure 1 d is the phenotypic characteristics of the diploid Chinese cabbage parent 01-4-11Br; Figure 1 e is a typical phenotypic characteristic diagram of the Chinese cabbage-Brassica oleracea introgression line; Figure 1 f is the cytological analysis of the Chinese cabbage-Brassica oleracea introgression line at different meiotic stages; Figure 1 g is a schematic diagram of identifying the introgression fragments of Brassica oleracea based on SNP tracking between parents and introgression lines;

[0027] Figure 2 This is a characteristic diagram of the distribution of introgression fragments in the Chinese cabbage-Brassica oleracea introgression system; Figure 2 a shows the introgression distribution of Brassica oleracea in the Chinese cabbage genome; Figure 2 a The serial numbers AE in the lower right corner represent: the introgression rates of 10 chromosomes (A), 9501Bo (B) and 01-4-11Br (C) of Chinese cabbage, the densities of genes (D) and transposons (E) in the 500 Kb-50 Kb sliding window; Figure 2 b is the genome proportion map of 9403Br, 9501Bo and 01-4-11Br in 46 introgression lines; Figure 2 c is a violin plot comparing the length and density distribution of the introgressed cabbage fragment and the corresponding substituted 9403Br fragment; Figure 2 d is the frequency diagram of introgressed cabbage fragments of different lengths;

[0028] Figure 3 This is the molecular marker identification map of the Chinese cabbage-Brassica oleracea introgression system;

[0029] Figure 4 This is a map of the rich trait variation in the Chinese cabbage-Brassica oleracea introgression system; Figure 4 a is the leaf color diagram of the three parents and the introgression lines; Figure 4 b is the plant type diagram of the three parents and the introgression lines; Figure 4 c is the leaf ball shape diagram of the three parents and the introgression line; Figure 4 d is the internal color map of the bulbs of the three parents and the introgression lines; Figure 4 e is the root swelling trait diagram of the three parents and the introgression lines; Figure 4 f is the leaf shrinkage diagram of the three parents and the introgression line; Figure 4 g is the diagram of shortened stem shape of three parents and introgression lines; Figure 4 h is the leaf rib color diagram of the three parents and the introgression line; Figure 4 i is the midrib shape diagram of the three parents and the introgression line;

[0030] Figure 5 This is the characteristic diagram of trait variation in the Chinese cabbage-Brassica oleracea introgression system; Figure 5 a is the frequency distribution diagram of introgression traits; Figure 5 b is the heat map of trait variation among the three parents and the introgression lines. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Experimental Materials

[0033] Table 1: Components of modified White medium

[0034]

[0035]

[0036] Table 2: Heller medium composition

[0037]

[0038] Table 3: Composition of B5 medium

[0039]

[0040]

[0041] Table 4: NLN medium composition

[0042]

[0043]

[0044] Table 5: MS medium composition

[0045]

[0046] Example 1

[0047] (1) The chromosomes of diploid Chinese cabbage (2n=2x=20) were doubled (the growing point was treated with 0.2% colchicine for 48 hours) to obtain tetraploid Chinese cabbage 9403Br (2n=4x=40).

[0048] (2) Using tetraploid Chinese cabbage 9403Br as the female parent, the buds were removed and emasculated 1-2 days before flowering, and it was hybridized with diploid cabbage 9501Bo (2n=2x=18). The ovaries were taken 5-7 days after pollination, and the surface was disinfected and inoculated on the improved White medium for culture.

[0049] After 28 days of culture, the surviving ovules were removed and inoculated on Heller medium to grow seedlings. Allotriploid hybrids (AAC) were obtained through morphological and cytological identification.

[0050] (3) The allotriploid hybrid was crossed with the diploid Chinese cabbage 01-4-11Br and then self-pollinated.

[0051] (4) Select flower buds at the mononuclear marginal stage on the self-pollinated offspring plants for free microspore culture. Take about 35 flower buds each time, disinfect and sterilize the surface, and then purify the microspores in B5 medium (pH 5.8).

[0052] The purified microspores were sealed and cultured with NLN culture medium (pH 5.8), and then cultured in the dark at 35°C for 24 hours before being transferred to a dark incubator. When the embryoids developed to the cotyledon stage, the cotyledon embryos were inoculated on MS medium and plant regeneration was performed at 25°C to obtain DH lines.

[0053] (5) The obtained DH lines were subjected to whole-genome resequencing, and the genome sequence of Brassica oleracea was identified on a genome-wide basis based on the SNP similarity between the introgression lines and their parents;

[0054] Clean reads were aligned to the Chinese cabbage Brara_Chiifu_V3.0 genome to obtain SNPs of all materials ( Figure 1 ag).

[0055] The SNP variation similarity between the introgression line and the parent was calculated along the Chinese cabbage genome chromosome with a sliding window of 500Kb-50Kb. The similarity of SNP variation between the introgression line and the parent in the same window of the genome was compared to determine the source of the chromosome fragment. The window with the highest similarity to the cabbage parent was defined as the candidate cabbage introgression region. The reads in the candidate region were aligned to the cabbage genome to determine the position of the introgression fragment on the Braol_JZS_V1.1 cabbage genome. A total of 1,301 cabbage fragments were identified in the 46 introgression lines, with an average of 28.28 cabbage fragments / introgression line, and a maximum of 67 cabbage fragments / introgression line ( Figure 2 ad).

[0056] Finally, the cabbage segment introgressed in the introgression line was verified using the InDel markers that were specific to the position of cabbage relative to Chinese cabbage and polymorphic between Chinese cabbage and cabbage. InDel markers distributed in the C05 chromosome of cabbage from 26,863,688bp to 32,208,950bp were selected for verification. The introgression line was able to amplify the cabbage-specific product corresponding to the InDel marker, indicating that the cabbage segment where the marker was located was introgressed in the introgression line ( Figure 3 ).

[0057] (6) New traits were produced in the introgression lines. Ⅰ New traits that were not found in Chinese cabbage or kale appeared. For example, we found that the taproot and lateral roots of the introgression lines were enlarged, and the roots were rich in starch ( Figure 4 e); Ⅱ produced new traits that are not found in Chinese cabbage but exist in Brassica oleracea. The most notable example is the presence of beneficial glucosinolate Glucoraphanin (GRA) in multiple introgression lines, which has significant anti-cancer and anti-tumor effects on the human body. GRA is widely present in the Cruciferae family, especially in cabbage vegetables, where its content is the highest, but it is almost non-existent in Chinese cabbage. The introgression lines in this study accumulated a relatively high content of GRA (higher than the parent cabbage 9501Bo), which greatly improved the nutritional value of cabbage; Ⅲ broadened the variation range of the original traits in Chinese cabbage. For example, the vitamin C (VC) content of cabbage varieties bred by conventional hybrid breeding is usually less than 40 mg / 100 g FW, while the VC content of the introgression lines in this study can reach more than 70 mg / 100 g FW, far exceeding the current ordinary cabbage ( Figure 5 a). In addition, we also found rare traits in Chinese cabbage, such as special leaf color. The parent cabbage has green leaves, the cabbage has gray-green leaves, and the introgression line has a rare creamy yellow color and an abnormal green and white color ( Figure 4 a). In addition, the plant type, ball shape, leaf ball color, leaf wrinkling, midrib and stem shortening all showed a rich variety of variations ( Figure 4bi). At the same time, it was observed that a single introgression line could produce multiple trait variations, and from these lines, strains with multiple advantageous traits were screened out ( Figure 5 b).

[0058] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for creating a high-frequency infiltration Chinese cabbage-Cabbage infiltration system, characterized in that: Allotriploid hybrids were obtained by interspecific hybridization of tetraploid Chinese cabbage and diploid Cabbage, which were further hybridized with diploid Chinese cabbage. The hybrid offspring were self-pollinated and free microspore culture was performed on the self-pollinated offspring to create Chinese cabbage-Cabbage introgression lines.

2. The method according to claim 1, characterized in that The following steps are involved: (1) doubling the chromosomes of diploid Chinese cabbage to obtain tetraploid Chinese cabbage; (2) hybridizing tetraploid Chinese cabbage with diploid cabbage, taking ovaries 5-7 days after pollination and inoculating them on a modified White medium for culture, and after 28 days of culture, peeling out the surviving ovules and inoculating them on a Heller medium for culture to obtain allotriploid hybrids; (3) hybridizing an allotriploid hybrid with a diploid Chinese cabbage, and further self-pollinating the hybrid offspring to obtain self-pollinated offspring; (4) Flower buds at the mononuclear marginal stage on the self-pollinated offspring plants were selected and free microspore culture was performed using B5 medium and NLN culture medium to obtain the homozygous Chinese cabbage-Cabbage introgression line.

3. The method according to claim 2, characterized in that The chromosome doubling method in step (1) is to treat the growing point with 0.2% colchicine for 48 hours.

4. The method according to claim 2, characterized in that: In step (2), the tetraploid Chinese cabbage is the female parent, and the diploid cabbage is the male parent.

5. The method according to claim 2, characterized in that: In step (3), the allotriploid hybrid is the female parent, and the diploid Chinese cabbage is the male parent.

6. The method according to claim 2, characterized in that The free microspore culture method in step (4) is: About 35 flower buds were taken each time, and the microspores were purified in B5 medium after surface disinfection and sterilization; the purified microspores were sealed and cultured with NLN culture medium, and then cultured in the dark at 35°C for 24 hours before being transferred to a dark culture box; when the embryoid developed to the cotyledon stage, the cotyledon embryo was inoculated on MS medium, and the plant was regenerated at 25°C to obtain the Chinese cabbage-cabbage introgression line.

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