Method for breeding new purple-orange Chinese cabbage germplasm

Abstract

A method for breeding new purple-orange Chinese cabbage germplasm crosses the purple head Chinese cabbage inbred line 11S96 as the male parent and the orange Chinese cabbage inbred line 11J11 as the female parent. On the basis of selecting individual plants with good heading property in the F.sub.2 population, molecular markers are used to select individual plants carrying the purple-head gene and the orange gene. Through selfing of individual plants for three consecutive generations, a new Chinese cabbage germplasm with green outer leaves and purple-orange head leaves is bred. This method aggregates the purple-head gene and orange gene which are expressed in the head leaves of Chinese cabbage, creating a new Chinese cabbage germplasm with purple-orange head leaves. It enriches Chinese cabbage breeding materials, lays the foundation for breeding the new purple-orange Chinese cabbage variety, and sets a precedent for the aggregation of the head color traits of Chinese cabbage.

Claims

1. A method for breeding purple-orange Chinese cabbage, comprises the steps: A) crossing an orange Chinese cabbage inbred line 11J11, representative seed of the inbred line having been deposited under China Center for Type Culture Collection (CCTCC) Registration Number P202505, as a female parent and a purple-head Chinese cabbage inbred line 11S96, representative seed of the inbred line having been deposited under China Center for Type Culture Collection (CCTCC) Registration Number P202514, as a male parent to obtain F.sub.1 generation plants; B) selecting individual plants with a stronger disease resistance to a disease selected from the group consisting of viral diseases, soft rot, and downy mildew than that of the parents from the F.sub.1 generation plants for selfing to obtain F.sub.2 generation plants; and C) selecting, from the F.sub.2 generation plants, individual plants carrying both the orange gene and the purple-head gene; wherein the orange gene is a Brcritso gene having a nucleotide sequence shown in SEQ ID NO. 8 of the sequence listing; and wherein the purple-head gene is a BrMYB2 gene having a nucleotide sequence shown in SEQ ID NO. 9 of the sequence listing; and D) performing, with the selected F.sub.2 generation plants, 3 cycles of continuous selfing to obtain the purple-orange Chinese cabbage.

2. The method according to claim 1, further comprising: testing whether or not the plant under test carries the orange gene by: amplifying a genomic DNA of the plant to be tested with primers consisting of a first single-stranded DNA shown in SEQ ID NO. 1 of the sequence listing and a second single-stranded DNA shown in SEQ ID NO. 2 of the sequence listing to obtain a first PCR product; and evaluating the first PCR product for the presence of a first fragment of 536 bp, wherein if the first PCR product contains the first fragment of 536 bp, the plant under test carries the orange gene; and if the first PCR product of the plant under test does not contain the first fragment of 536 bp, the plant under test does not carry the orange gene; and testing whether or not the plant under test carries the purple-head gene by: amplifying the genomic DNA of the plant to be tested with primers consisting of a third single-stranded DNA shown in SEQ ID NO. 3 of the sequence listing and a fourth single-stranded DNA shown in SEQ ID NO. 4 of the sequence listing to obtain a second PCR product; and evaluating the second PCR product for the presence of a second fragment of 185 bp and a third fragment of 178 bp, wherein if the second PCR product contains the second fragment of 185 bp, the plant under test carries the purple-head gene; and if the second PCR product contains only the third fragment of 178 bp, the plant under test does not carry the purple-head gene.

3. A method for breeding purple-orange Chinese cabbage, comprises the steps: 1) crossing the orange Chinese cabbage inbred line 11J11, representative seed of the inbred line having been deposited under China Center for Type Culture Collection (CCTCC) Registration Number P202505, as the female parent and the purple-head Chinese cabbage inbred line 11S96, representative seed of the inbred line having been deposited under China Center for Type Culture Collection (CCTCC) Registration Number P202514, as the male parent to obtain F.sub.1 generation plants; planting the F.sub.1 generation seeds to obtain F.sub.1 generation plants, and selecting a plurality of individual F.sub.1 generation plants with a resistance to a disease selected from the group consisting of viral diseases, soft rot, and downy mildew, and good head firmness; 2) selfing the individual plants obtained in step 1) to obtain the seeds of a F.sub.2 population; planting the seeds of the F.sub.2 population to obtain F.sub.2 generation plants, and selecting a plurality of individual F.sub.2 generation plants carrying both the orange gene and purple-head gene; wherein the orange gene is a Brcritso gene having a nucleotide sequence shown in SEQ ID NO. 8 of the sequence listing; and wherein the purple-head gene is a BrMYB2 gene having a nucleotide sequence shown in SEQ ID NO. 9 of the sequence listing; and 3) selfing the individual F.sub.2 generation plants selected in step 2) to obtain the seeds of a F.sub.2S.sub.1 population; planting the seeds of the F.sub.2S.sub.1 population to obtain F.sub.2S.sub.1 generation plants, and selecting a plurality of individual F.sub.2S.sub.1 generation plants carrying both the orange gene and purple-head gene; 4) selfing the individual F.sub.2S.sub.1 generation plants selected in step 3) to obtain the seeds of a F.sub.2S.sub.2 population; planting the seeds of the F.sub.2S.sub.2 population to obtain F.sub.2S.sub.2 generation plants, and selecting a plurality of individual plants from the F.sub.2S.sub.2 generation plants carrying both the orange gene and purple-head gene; and 5) selfing the individual plants obtained in step 4) to obtain the seeds of a F.sub.2S.sub.3 population; plant the seeds of the F.sub.2S.sub.3 population to obtain F.sub.2S.sub.3 generation plants and both the orange gene and purple-head gene and with uniform horticultural traits from the F.sub.2S.sub.2 generation plants to obtain the purple-orange Chinese cabbage.

4. The method according to claim 1, characterized in that the color of the head leaves of said purple-orange Chinese cabbage are purple-orange.

5. The purple-orange Chinese cabbage plant obtained by utilizing a breeding method according to claim 1.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a flow chart of the breeding method of the present invention.

(2) FIG. 2 is a flow chart of the breeding method of the embodiment.

(3) FIG. 3 is the orange gene molecular marker. Each lane is a 17SF1 single plant.

(4) FIG. 4 is the molecular marker of the purple-head gene. Lanes 1-18 are individual plants of 17SF1, respectively.

(5) FIG. 5 is a picture of the leaf head of the new purple-orange Chinese cabbage germplasm 18SF19.

(6) FIG. 6 is the sectional view of the leaf head of the new purple-orange Chinese cabbage germplasm 18SF19.

EMBODIMENTS

(7) The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores. The quantitative tests in the following examples are all set to repeat the experiment three times, and the results are averaged.

(8) The orange Chinese cabbage inbred line 11J11 in the following examples is described in the document Mo Yunan, Zhang Lugang, Wang Guofang, Extraction and Determination of Total Carotenoids in Orange Heading Chinese Cabbage, Journal of Northwest A&F University (Natural Science Edition), 2014, 42(3): 1-9, is a stable inbred line selected by the Chinese Cabbage Research Group of the College of Horticulture, Northwest A&F University. The plant characteristics are: outer leaves are yellow-green and bright, outer leaves are spreading, closed head with high degree of overlapping leaf, short and round head, and the head leaves are orange.

(9) The purple-head Chinese cabbage inbred line 11S96 in the following examples was described in the document Duan Yanjiao, Zhang Lugang, He Qiong, etc., Expression of Transcriptional Factors and Structural Genes of Anthocyanin Biosynthesis in Purple-heading Chinese Cabbage, Acta Horticulturae Sinica 2012, 39 (11): 2159-2167, and it is a stable inbred line bred by the Chinese cabbage research group of the College of Horticulture, Northwest A&F University, from the hybridization of common Chinese cabbage and purple tsai-tai 95T2-5. The plant characteristics are: outer leaves are green, closed head with high degree of overlapping leaf, tall and narrow elliptic head, and the head leaves are purple, and the purple color is deepened from the outside to the inside.

Example 1. A Breeding Method of a New Germplasm of Purple-Orange Chinese Cabbage

(10) In 2014, the applicant of the present invention started to create a new germplasm of purple-orange Chinese cabbage according to the flowchart shown in FIG. 1. Through the hybridization of orange Chinese cabbage and purple-head Chinese cabbage, molecular marker selection and continuous selfing of individual plants, after 4 generations of selection, a new Chinese cabbage germplasm with green rosette leaves and outer leaves of leaf head and purple-orange head leaves was obtained. The flow chart of the specific breeding operation is shown in FIG. 2. The specific breeding operation steps were as follows:

(11) 1. In the spring of 2014, the orange Chinese cabbage inbred line 11J11 was used as the female parent and the purple-head Chinese cabbage inbred line 11S96 was used as the male parent for single plant to plant pairing crossing, 5 F.sub.1 plants of hybrid generation seeds (F.sub.1 generation) were obtained, and numbered from 14ZF1 to 14ZF5. Planted F.sub.1 seeds in the autumn of 2014 (30 plants for each F.sub.1 of 14ZF1 to 14ZF5, 150 plants in total), and observed the horticultural traits of individual plants of F.sub.1 generation. In the 14ZF2 population, 10 individual plants with strong disease resistance and good heading property (strong disease resistance means that its disease resistance is stronger than that of the parents, and the disease resistance is specifically resistance to virus disease, soft rot and downy mildew disease; good heading property means that its heading property is better than that of the parents, and the heading property is specifically the heading firmness) were selected, and let them got vernalization naturally.

(12) 2. In the spring of 2015, individual plants selected from 14ZF2 were cultivated and selfed (10 plants in total), and 5 strains of F.sub.2 population seeds were obtained, and numbered from 14ZF2-1 to 14ZF2-5. Planted seeds of each F.sub.2 population strains (100 plants for each from 14ZF2-1 to 14ZF2-5, 500 plants in total) in the autumn of 2015, and observed the segregation of traits of the plants. In the 14ZF2-2 population, 20 individual plants with good heading property and carrying orange gene and purple-head gene were selected, and let them got vernalization naturally. The orange gene referred to a mutant (Brcritso) of the carotenoid isomerase gene (BrCRITSO); Due to the insertion mutation (Brcritso) of BrCRITSO, its function was lost, resulting in the accumulation of prolycopene to produce orange; The nucleotide sequence of the orange gene Brcritso was shown as SEQ ID NO.8. The purple-head gene referred to the mutant BrMYB2 of the Brmyb2 gene of Chinese cabbage; Due to the deletion mutation(BrMYB2) of Brmyb2, the original non-expressing Brmyb2 gene, highly expresses BrMYB2, thereby regulating the anthocyanin synthesis gene and making Chinese cabbage leaf head accumulating anthocyanins and showing purple-head trait; The nucleotide sequence of the purple-head gene BrMYB2 was shown as SEQ ID NO.9. 1) The method for identifying whether the plant to be tested carries the orange gene Brcritso was as follows: using the genomic DNA of the plant to be tested as a template, the following C-F and C-R primers were used for PCR amplification to obtain a PCR product. The primers sequences were as follows:

(13) TABLE-US-00001 C-F: (SEQIDNO.1) CAGAAACATCAGGGTTGAAATC; C-R: (SEQIDNO.2) TTACTGCCGAAAGCGAAA.

(14) The PCR amplification procedure was as follows: 95 C. pre-denaturation for 3 min; 95 C. denaturation for 30 s, 57 C. annealing for 30 s, 72 C. extension for 1 min, 38 cycles; 72 C. extension for 10 min.

(15) The PCR amplification system was as follows: primer C-F (10 mol) 1 L, primer C-R (10 mol) 1 L, DNA template (50 ng/L) 2 L, Genstar mix 10 L, and made up to 20 L with RNase-Free ddH.sub.2O.

(16) The PCR products were electrophoresed on a 9% non-denaturing PAGE gel and separated at a constant pressure of 180V for 80-110 min. Finally, a 0.1% silver nitrate solution was used for silver staining. Determined whether the plant to be tested carried the orange gene Brcritso according to the presence and size of the PCR product: If the PCR product of the plant to be tested contains a fragment with a size of 536 bp, the plant to be tested carries the orange gene Brcritso; If the PCR product of the plant to be tested does not contain a 536 bp fragment, the plant to be tested does not carry the orange gene Brcritso. The nucleotide sequence of the 536 bp fragment was shown in SEQ ID NO.5. 2) The method for identifying whether the plant to be tested carried the purple-head gene BrMYB2 was as follows: Using the genomic DNA of the plant to be tested as a template, PCR amplification was performed using the following Z-F and Z-R primers to obtain a PCR product. The primers sequences were as follows:

(17) TABLE-US-00002 Z-F: (SEQIDNO.3) TGTCTATCTGACTTGTGGTGT; Z-R: (SEQIDNO.4) ATACCTCATAACACTGCATCCATAG.

(18) The PCR amplification system was as follows: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 30 s, annealing at 55 C. for 30 s, extension at 72 C. for 30 s, 30 cycles; extension at 72 C. for 5 min.

(19) The PCR amplification system was as follows: genomic DNA 50 ng, 2Taq Master (Vazyme) 5.0 L, primer Z-F 0.5 mol, primer Z-R 0.5 mol, and finally diluted to 10.0 L with enzyme-free water.

(20) The PCR products were electrophoresed on a 9% non-denaturing PAGE gel and separated at a constant pressure of 180V for 80-110 min. Finally, a 0.1% silver nitrate solution was used for silver staining and statistical analysis. Determined whether the tested plant carried the purple gene BrMYB2 according to the presence and size of the PCR product: If the PCR product of the tested plant only contains a fragment of 185 bp or both fragments of 185 bp and 178 bp, the plant to be tested carries purple-head gene BrMYB2 (When the PCR product contains only a fragment of 185 bp, the plant to be tested is a homozygote carrying the purple-head gene BrMYB2; when the PCR product contains both fragments of 185 bp and 178 bp, the plant to be tested is a heterozygote carrying the purple-head gene BrMYB2); If the PCR product of the plant to be tested only contains a fragment of 178 bp, the plant to be tested does not carry the purple-head gene BrMYB2. The nucleotide sequence of the 185 bp fragment is shown in SEQ ID NO.6. The nucleotide sequence of the 178 bp fragment is shown in SEQ ID NO.7. 3. In the spring of 2016, cultivated individual plants selected from 14ZF2-2 and selfed the individual plants to obtain 20 strains of the F.sub.2S.sub.1 population seeds, numbered from 16SF1 to 165F20. Planted the seeds of the F.sub.2S.sub.1 population in the autumn of 2016 (30 plants for each stain of 16SF1 to 16SF20, 600 plants in total), observed the segregation of traits, selected 10 individual plants with good heading property and carrying the orange gene and the purple-head gene among the 16SF1 population (the selection method was the same as step 2), and let them got vernalization naturally. 4. In the spring of 2017, individual plants selected from 16SF1 were cultivated, and the individual plants were selfed to obtain 10 strains of seeds of the F.sub.2S.sub.2 population, numbered from 17SF1 to 17SF10. Planted the seeds of the F.sub.2S.sub.2 population in the autumn of 2017 (30 plants for each stain of 17SF1 to 17SF10, 300 plants in total). Observed the segregation of traits. Selected 10 individual plants with good heading property and carrying the orange gene and the purple-head gene respectively in each stain of 17SF1 to 17SF10 (the selection method was the same as step 2), and let them got vernalization naturally. 5. In the spring of 2018, individual plants selected from 17SF1 to 17SF10 were cultivated, and the individual plants (100 plants) were selfed to obtain 60 strains of seeds of the F.sub.2S.sub.3 population, numbered from 18SF1 to 185F60. In the autumn of 2018, the seeds of the F.sub.2S.sub.3 population (30 plants each from 18SF1 to 18SF60, 1800 plants in total) were planted in the field. Observed the segregation of traits, the horticultural traits of the 18SF19 line were found to be consistent(the horticultural traits included: plant type, leaf color, petiole color, shape and size of leaf head, head type, color of leaf head), also good heading, the rosette leaves and the outer leaves of the leaf head were green, and the head leaves were purple-orange. The purple-orange Chinese cabbage was identified carrying the orange gene and the purple-head gene by molecular markers, and it was named as a new germplasm of purple-orange Chinese cabbage 18SF19, its phenotype was shown in FIG. 5 and FIG. 6.

(21) The new purple-orange Chinese cabbage germplasm 18SF19 selected and bred by the invention had the following characteristics: good heading, closed head with high degree of overlapping leaf, high-narrow obovate head, the rosette leaves and outer leaves of the leaf head were green, the head leaves were purple-orange, the purple color of the inner leaves of leaf head gradually deepened from outside to inside (from light purple to dark purple of the heart leaves). The new purple-orange Chinese cabbage germplasm 18SF19 of the present invention completes the perfect combination of the orange character and the purple-head character, enriches the Chinese cabbage germplasm resources, and lays the foundation for cultivating the new purple-orange Chinese cabbage variety.

Example 2: Detection of Carotenoid and Anthocyanin Content in 18SF19, a New Germplasm of Purple-Orange Chinese Cabbage

(22) The carotenoid content and the anthocyanin content of the new purple-orange Chinese cabbage germplasm 18SF19 obtained in Example 1 were detected respectively.

(23) The specific detection procedure of the carotenoid content of the new germplasm 18SF19 of purple-orange Chinese cabbage referred to the method in the literature Mo Yunan, Zhang Lugang, Wang Guofang, Extraction and Determination of Total Carotenoids in Orange Heading Chinese Cabbage, Journal of Northwest A&F University (Natural Science Edition), 2014, 42(3): 1-9.

(24) The specific detection procedure of the anthocyanin content of the new germplasm 18SF19 of purple-orange Chinese cabbage referred to the method in the literature Duan Yanjiao, Zhang Lugang, He Qiong, etc., Expression of Transcriptional Factors and Structural Genes of Anthocyanin Biosynthesis in Purple-heading Chinese Cabbage, Acta Horticulturae Sinica 2012, 39 (11): 2159-2167.

(25) The test results showed that the carotenoid content of the new purple-orange Chinese cabbage germplasm 18SF19 was 32.847 g/gFW; the anthocyanin content of the new purple-orange Chinese cabbage germplasm 18SF19 was 171.03 g/gFW. The new purple-orange Chinese cabbage germplasm 18SF19 of present invention not only had beautiful color, but was also rich in carotenoids and anthocyanins, and rich in nutrients.

(26) The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the field, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and modifications should also be regarded as the protection scope of the present invention.

INDUSTRIAL APPLICATION

(27) The invention provides a method for breeding new purple-orange Chinese cabbage germplasm, and successfully selected and bred new purple-orange Chinese cabbage germplasm by using the breeding method provided by the invention.

(28) The new purple-orange Chinese cabbage germplasm has the following characteristics: good heading property, closed head with high degree of overlapping leaf, high-narrow obovate head, the rosette leaves and outer leaves of the leaf head are green, the head leaves are purple-orange, the purple color of the inner leaves of leaf head gradually deepens from outside to inside. It has beautiful color, is rich in carotenoids and anthocyanins, and rich in nutrients. The new purple-orange Chinese cabbage germplasm bred by the present invention completes the perfect combination of the orange character and the purple-head character, and not only enriches Chinese cabbage breeding materials, lays the foundation for breeding the new purple-orange Chinese cabbage variety, but also set a precedent for the aggregation of the head color traits of Chinese cabbage.

DEPOSIT INFORMATION

(29) Applicant has made a deposit of representative seeds of orange Chinese cabbage inbred line 11J11 with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The deposit of the orange Chinese cabbage inbred line 11J11 was assigned Registration No. CCTCC No: P202505. The date of deposit of the seeds of the inbred line 11J11 with the CCTCC was Apr. 16, 2025. Applicant has made a deposit of representative seeds of purple-head Chinese cabbage inbred line 11S96 with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The deposit for the purple-head Chinese cabbage inbred line 11S96 was assigned Registration No. CCTCC No: P202514. The date of deposit of the seeds of the inbred line 11S96 with the CCTCC was Apr. 16, 2025. Access to both seed stocks will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant will make the deposits available to the public pursuant to 37 C.F.R. 1.808. These deposits of the orange Chinese cabbage inbred line 11J11 and the purple-head Chinese cabbage inbred line 11S96 will be maintained in the CCTCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has or will satisfy all the requirements of 37 C.F.R. 1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant has no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant does not waive any infringement of their rights granted under this patent or under the Plant Variety Protection Act (7USC2321 et seq.).