A method for breeding pepper multi-gene multi-trait aggregate germplasm based on the combination of molecular markers and morphological markers

Abstract

The present invention provides a method for cultivating pepper multi-gene multi-trait aggregated germplasm based on the combination of molecular markers and morphological markers, and belongs to the technical field of plant breeding. The present invention uses two groups of pepper germplasms carrying different target excellent genes or traits as parents for hybridization, respectively, and obtains hybrid first-generation plants having both target excellent genes and traits, and then hybridizes the two groups of hybrid first-generation plants to obtain hybrid first-generation plants, separates the microspores of the hybrid first-generation plants, cultivates haploid plants, screens pepper individual plants with excellent genes and excellent traits, and doubles the chromosomes of the pepper individual plants, so that excellent pepper germplasm with multi-gene multi-trait aggregation can be obtained in a relatively short time. The breeding method of the present invention can significantly improve the breeding efficiency. Compared with the traditional breeding time (more than 10 years), the present invention only takes 3.5 years to obtain homozygous plants with multi-gene multi-trait aggregation, which greatly shortens the breeding time.

Description

Technical Field

[0001] The invention belongs to the technical field of pepper breeding, and in particular relates to a method for cultivating pepper multi-gene and multi-trait aggregate germplasm based on the combination of molecular markers and morphological markers. Background Art

[0002] Pepper (Latin name: Capsicumannuum L.), a herbaceous plant of the genus Capsicum in the Solanaceae family, is native to Mexico and Colombia and is widely cultivated in countries around the world. In the breeding process of pepper, it is usually necessary to obtain pepper varieties or inbred lines with multiple excellent traits. However, since different excellent genes of pepper are usually located in different positions on different chromosomes, it is not easy to create pepper germplasm including multiple excellent genes, and it takes a long time to adopt conventional breeding methods, and it is difficult to solve the technical problem of obtaining excellent varieties of pepper in a short time. At present, although patent CN202210365827.7 has obtained a breeding method for multi-gene polymerized germplasm through molecular markers, it has not achieved the combination of pepper polygenes and traits. Therefore, how to obtain a breeding method for pepper polygene and multi-trait polymerized germplasm to strengthen the creation of excellent breeding materials (lines) is an important problem to be solved in this field. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for cultivating pepper multi-gene and multi-trait aggregated germplasm based on the combination of molecular markers and morphological markers, which can obtain high-quality pepper germplasm with multi-gene and multi-trait aggregation in a relatively short time.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for cultivating pepper multi-gene multi-trait aggregate germplasm based on the combination of molecular markers and morphological markers, comprising the following steps:

[0006] (1) hybridizing pepper parent A and pepper parent B, which have excellent genes or traits, to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plants F carrying both excellent genes and excellent traits. AB ;

[0007] (2) hybridizing pepper parent C and pepper parent D, which have excellent genes or traits, to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plants F carrying both excellent genes and excellent traits. CD ;

[0008] (3) F AB and F CD Perform hybridization to obtain first-generation hybrid seeds, sow the first-generation hybrid seeds to obtain first-generation hybrid plants;

[0009] (4) isolating microspores from the hybrid first-generation plants at the flowering stage, cultivating the microspores, obtaining haploid pepper plants, and screening to obtain pepper plants with excellent genes and excellent traits;

[0010] (5) doubling the chromosomes of pepper plants to obtain diploid pepper plants with excellent genes and excellent traits;

[0011] (6) subjecting the pepper diploid plants with the excellent genes and excellent traits to rooting culture and planting management to obtain pepper multi-gene multi-trait aggregate germplasm;

[0012] The pepper parents in step (1) and step (2) both include at least one excellent gene or one excellent trait.

[0013] Preferably, the pepper parent in steps (1) to (2) is a homozygous pepper plant.

[0014] Preferably, the hybridization is a reciprocal cross.

[0015] Preferably, the two pepper parents in step (1) are pepper with the whole plant hairy trait and pepper carrying a root-knot nematode resistance gene.

[0016] Preferably, the two pepper parents in step (2) are peppers with a whole-plant purple trait and peppers carrying a gene for no spicy taste.

[0017] Preferably, the screening in step (1) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent genes include the root-knot nematode resistance Me gene, and the excellent traits include the whole-plant hairy trait.

[0018] Preferably, the screening in step (2) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent genes include the non-spicy at3 gene, and the excellent traits include the whole plant purple trait.

[0019] Preferably, the screening in step (4) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent genes are the root-knot nematode resistance Me gene and the non-spicy at3 gene, and the excellent traits are the whole plant hairy trait and the whole plant purple trait.

[0020] Preferably, the detection primer of the anti-root-knot nematode Me gene molecular marker is Me-1, the forward primer sequence of Me-1 is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2;

[0021] The detection primer of the non-spicy at3 gene molecular marker is at3-2, the forward primer sequence of at3-2 is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4;

[0022] The whole-plant hairy trait morphological marker is that the aerial part of the plant is densely covered with hairs;

[0023] The whole plant purple trait morphological marker is manifested in that the above-ground part of the plant appears purple.

[0024] Preferably, the microspores in step (4) are microspores in the uninucleate marginal stage.

[0025] Beneficial effects of the present invention:

[0026] The breeding method of the present invention uses two groups of pepper germplasms carrying different target excellent genes or target excellent traits as parents for hybridization, combined with the screening of target excellent genes and target excellent traits, and can aggregate multiple excellent genes and excellent traits in a relatively short period of time to obtain excellent pepper germplasm with multiple genes and multiple traits, which can significantly shorten the breeding time and improve the breeding efficiency. Compared with the traditional breeding time (more than 10 years), the present invention only takes 3.5 years at least to obtain gene homozygous plants, which greatly shortens the breeding time. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The amplification result of no spicy (at3) on pepper with spicy taste in Example 1;

[0028] Figure 2 : The amplification results of root-knot nematode resistance (Me) in Example 1 on root-knot nematode resistant peppers. DETAILED DESCRIPTION

[0029] The present invention provides a method for cultivating pepper multi-gene multi-trait aggregate germplasm based on the combination of molecular markers and morphological markers, comprising the following steps:

[0030] (1) hybridizing pepper parent A and pepper parent B, which have excellent genes or traits, to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plants F carrying both excellent genes and excellent traits. AB ;

[0031] (2) hybridizing pepper parent C and pepper parent D, which have excellent genes or traits, to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plants F carrying both excellent genes and excellent traits. CD ;

[0032] (3) F AB and FCD Perform hybridization to obtain first-generation hybrid seeds, sow the first-generation hybrid seeds to obtain first-generation hybrid plants;

[0033] (4) isolating microspores from the hybrid first-generation plants at the flowering stage, cultivating the microspores, obtaining haploid pepper plants, and screening to obtain pepper plants with excellent genes and excellent traits;

[0034] (5) doubling the chromosomes of pepper plants to obtain diploid pepper plants with excellent genes and excellent traits;

[0035] (6) subjecting the pepper diploid plants with the excellent genes and excellent traits to rooting culture and planting management to obtain pepper multi-gene multi-trait aggregate germplasm;

[0036] The pepper parents in step (1) and step (2) both include at least one excellent gene or one excellent trait.

[0037] The pepper parents of the present invention all include at least one excellent gene or one excellent trait, and preferably the excellent genes and traits included in the different parents to be hybridized are different. The pepper plant of the present invention can be a heterozygote or a homozygote, and more preferably a homozygote pepper plant, which has better effects and more stable traits. The hybridization of the present invention is preferably a reciprocal cross.

[0038] The present invention divides four pepper parents with excellent genes and traits into two groups for hybridization, wherein the first group (parent A and parent B) comprises peppers with the whole plant hairy trait and peppers carrying the root-knot nematode resistance gene, and obtains first-generation hybrid seeds, which are sown and screened to obtain first-generation hybrid plants (F) carrying both the root-knot nematode resistance gene and the whole plant hairy trait. AB ); the second group (parent C and parent D) is peppers with purple traits throughout the plant and peppers carrying the non-spicy gene, and hybrid first-generation seeds are obtained. The hybrid first-generation seeds are sown and screened to obtain hybrid first-generation plants (F) carrying both the non-spicy gene and the purple traits throughout the plant. CD ); hybridizing the first-generation hybrid plants obtained from the two groups to obtain first-generation hybrid seeds, and sowing the first-generation hybrid seeds to obtain first-generation hybrid plants. The present invention has no special limitation on the sowing method, and conventional sowing methods in the art can be used.

[0039] Microspores are separated from hybrid first-generation plants at full flowering stage, and microspores are cultivated to obtain pepper haploid plants. After screening, pepper individual plants with hairy and purple color, resistance to root-knot nematodes and no spicy taste are obtained. The microspores described in the present invention are microspores in the mononuclear marginal stage.

[0040] The root-knot nematode-resistant pepper of the present invention preferably carries the root-knot nematode-resistant gene Me, and the marker of the root-knot nematode-resistant gene Me is the SCAR molecular marker. The pepper with the hairy trait of the whole plant is manifested as the aboveground part of the plant, including stems and leaves, densely covered with hairs, which is dominant inheritance; the non-spicy pepper of the present invention preferably carries the mutated capsaicin synthase gene at3, and the accession number of the mutated capsaicin synthase gene at3 in NCBI is FJ871985. The pepper with the purple trait of the whole plant is manifested as the aboveground part of the plant, including stems, leaves, flowers, and fruits, all of which are purple, which is dominant inheritance, and the purple of roots, stems, leaves, flowers and fruits is linked.

[0041] Preferably, the pepper with the whole plant hairy trait described in the present invention is the pepper resource "2005-15H"; the pepper carrying the root-knot nematode resistance gene is the pepper resource "2018-21H"; the pepper with the whole plant purple trait is the pepper resource "2011-02H"; the pepper carrying the non-spicy gene is the pepper resource "2016-08H".

[0042] The screening in step (1), step (2) and step (4) of the present invention is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent gene in step (1) includes the root-knot nematode resistance Me gene, and the excellent trait includes the whole plant hairy trait; the excellent gene in step (2) includes the non-spicy at3 gene, and the excellent trait includes the whole plant purple trait; the excellent gene in step (4) is the root-knot nematode resistance Me gene and the non-spicy at3 gene, and the excellent traits are the whole plant hairy trait and the whole plant purple trait.

[0043] The screening method of the root-knot nematode resistance Me gene or the non-spicy at3 gene of the present invention is as follows: extracting plant genomic DNA, using the molecular marker of the root-knot nematode resistance Me gene or the non-spicy at3 gene for PCR amplification, and obtaining the pepper plant with the excellent gene polymerization according to the amplification result. The detection primer of the root-knot nematode resistance Me gene molecular marker is Me-1, and the forward primer sequence of Me-1 is shown in SEQ ID NO.1, specifically: 5'-GAAGCTTATGTGGTAMCC-3', and the reverse primer sequence of Me-1 is shown in SEQ ID NO.2, specifically: 5'-GCAAAGTAATTATATGCAAGAGT-3'. The detection primer of the non-spicy at3 gene molecular marker is at3-2, the forward primer sequence of at3-2 is shown in SEQ ID NO.3, specifically: 5'-TGGCAGTTTCCCTTCTCTC-3', and the reverse primer sequence of at3-2 is shown in SEQ ID NO.4, specifically: 5'-GGGAATAGCCATCAGTGTATGCTTTTCG-3'. The present invention has no special limitation on the method of extracting genomic DNA, and the conventional genomic DNA extraction method in the art can be used.

[0044] The present invention does not specifically limit the system and procedure of the PCR amplification, and the conventional system and procedure in the art can be used. After completing the PCR amplification, the present invention preferably further includes electrophoresis of the obtained PCR amplification product to confirm and screen the pepper plants containing the above-mentioned excellent genes. The present invention does not specifically limit the specific process of the electrophoresis, and the conventional electrophoresis process in the art can be used.

[0045] In trait screening, the whole plant purple described in the present invention is manifested as the stems and leaves of the plant are purple visible to the naked eye; the whole plant hairy is manifested as the stems and leaves of the plant are densely covered with hairs visible to the naked eye.

[0046] After obtaining the pepper plant with the excellent genes and traits, the present invention doubles the chromosomes of the pepper plant with the excellent genes to obtain a pepper diploid plant with the excellent genes. The pepper diploid plant is subjected to rooting culture and planting management to obtain pepper multi-gene multi-trait aggregate germplasm.

[0047] The present invention has no special limitation on the chromosome doubling and seedling management, and conventional chromosome doubling and seedling management methods in the art can be used. The pepper multi-gene and trait aggregated germplasm obtained by the present invention is a pepper with hairy and purple plants, resistance to root-knot nematodes and no spicy taste.

[0048] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0049] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0051] Example 1

[0052] The breeding of new pepper lines with hairy and purple color, resistance to root-knot nematodes and no spicy taste consists of the following steps:

[0053] Materials: The pepper resource "2018-21H" carries the root-knot nematode resistance gene (Me), showing resistance to root-knot nematodes; the pepper resource "2005-15H" is hairy and dominant; the pepper resource "2011-02H" is purple throughout the plant, dominant, and the purple traits of the plant's stems, leaves, flowers and fruits are linked, showing that the stems, leaves, flowers and fruits of the whole plant are purple; the pepper resource "2016-08H" carries a mutated capsaicin synthase gene (at3) (FJ871985) and has no spicy taste (the above pepper materials can be obtained from the pepper project team of the College of Landscape and Horticulture, Yunnan Agricultural University).

[0054] Pepper resource "2005-15H" and pepper resource "2018-21H" were used as parents, and reciprocal crosses were carried out in May 2017 to obtain the first-generation seeds of reciprocal crosses, which were sown in December 2017. The individual plants were selected at the seedling stage using the molecular marker of root-knot nematode resistance gene (Me) and the trait of full-plant hairiness, and the individual plants carrying the molecular marker of root-knot nematode resistance and full-plant hairiness were selected;

[0055] Pepper resource "2011-02H" and pepper resource "2016-08H" were used as parents, and reciprocal crosses were carried out in May 2017 to obtain the first-generation seeds of reciprocal crosses, which were sown in December 2017. Individual plants were selected at the seedling stage using the mutated capsaicin synthase gene (at3) molecular marker and the purple trait of the whole plant, and the individual plants carrying the molecular marker of no spicy taste and the purple trait of the whole plant were selected;

[0056] Individual plants carrying molecular markers for resistance to root-knot nematodes and being hairy all over, and individual plants carrying molecular markers for being non-spicy and being purple all over, were hybridized by forward and reverse crosses during the peak flowering period in April 2018 to obtain the first-generation seeds of forward and reverse crosses.

[0057] The seeds were sown in December 2018 and the plants were cultured to the full flowering stage. In March 2019, pollen from the first generation of mononuclear marginal stage of positive and negative cross hybridization was taken respectively and placed on a culture medium of NTh+KT0.5mg / L+2,4-D0.25mg / L+AgNO34mg / L+AC0.2wt.%+sucrose 3wt.%+agar 0.7wt.%, pH5.8. The plants were first treated in the dark at 35℃ for 7d, and then cultured at 26±1℃ during the day and 21±1℃ at night. After the embryoids were formed, the plants were moved to 2000lux light for further culture to obtain haploid plants 3 to 4cm long.

[0058] In September 2019, haploid plants were screened for morphological markers, and haploid plants with purple color and hairy traits visible to the naked eye were selected.

[0059] In September 2019, DNA of haploid plants with purple and hairy whole plants was extracted, and PCR amplification screening was performed using molecular markers closely linked to resistance to root-knot nematodes (Me) (forward primer, SEQ ID NO.1, reverse primer, SEQ ID NO.2) and no pungency (at3) (forward primer, SEQ ID NO.3, reverse primer, SEQ ID NO.4) to screen haploid pepper plants with two genes, resistance to root-knot nematodes (Me) and no pungency (at3). Haploid pepper plants with purple and hairy whole plants, and molecular markers for resistance to root-knot nematodes and no pungency were screened.

[0060] Among them, the root-knot nematode resistance (Me) primer (forward primer, SEQ ID NO.1, reverse primer, SEQ ID NO.2) can amplify a specific product of 250 bp, and the no-spicy (at3) primer (forward primer, SEQ ID NO.3, reverse primer, SEQ ID NO.4) primer is amplified on pepper plants with spicy taste, and the no-spicy (at3) primer can amplify a product of 749 bp (see Figures 1-2 shown).

[0061] In October 2019, the haploid plants containing the above-mentioned whole plant purple, hairy, root-knot nematode-resistant, and non-spicy molecular marker aggregation were soaked in 0.45% colchicine with cotton, and the stem tip was wrapped for 30 hours for chromosome doubling to obtain diploid plants; the above-mentioned diploid plants were placed in 1 / 2MS+NAA0.1mg / L, pH5.8, 26±1℃ during the day, 21±1℃ at night, and 2000lux for rooting. The seedlings were trained and transplanted, and bagged during the flowering period. In June 2020, seeds were harvested by individual plants. After genetic testing (the same method as that for screening the haploid individual plants of peppers with the above-mentioned gene aggregation), a total of 11 individual plants with whole plant purple, hairy, root-knot nematode-resistant, and non-spicy molecular marker aggregation were obtained, and the whole plant purple, hairy, root-knot nematode-resistant, and non-spicy pepper germplasm was obtained. The entire cultivation process took 3.5 years.

[0062] The above-obtained purple, hairy, root-knot nematode-resistant, non-spicy pepper germplasm was planted in a trial, specifically:

[0063] In 2021 and 2022, the plants were planted in plots with root-knot nematodes in Kunming, Wenshan, and Dehong, respectively, with a focus on evaluating resistance to root-knot nematodes, plant color, plant hair, and pungency (capsaicin content). The average data from three different test sites over two years were collected.

[0064] Among them, the plant villi were initially screened by multi-person visual inspection and then counted by microscope, referring to Chang J, Yu T, Yang Q, Li C, et al. Hair, encoding a single C2H2 zinc-fingerprotein, regulates multicellular trichome formation in tomato. Plant J. 2018, 96(1): 90-102.

[0065] The plant color was initially screened by multi-person visual inspection and then quantitatively detected by HPLC, referring to DB64 / T1578-2018 Determination of anthocyanin content in black wolfberry.

[0066] Resistance to root-knot nematodes shall be identified in accordance with NY / T2060.1-2011, Technical Specifications for Identification of Disease Resistance of Pepper, Part 5: Technical Specifications for Identification of Resistance to Southern Root-knot Nematode Disease of Pepper;

[0067] Whether there is spiciness or not: Capsaicin and dihydrocapsaicin are detected according to GB / T21266-2007 Method for Determination of Capsaicinoids and Expression of Spiciness in Peppers and Pepper Products. If one of them can be detected, it is spicy. If neither of them can be detected, it is not spicy. The results are shown in Table 1. The capsaicin content in Table 1 is the total amount of capsaicin and dihydrocapsaicin.

[0068] Table 1 Quality and yield index results of whole-plant purple, hairy, root-knot nematode-resistant, and non-spicy pepper germplasms

[0069]

[0070]

[0071] It can be concluded from the above embodiments that the breeding method provided by the present invention can aggregate multiple excellent genes and traits in a shorter period of time, obtain gene homozygous plants, and obtain excellent pepper germplasm with multiple genes and traits, which shortens the breeding time by more than 6 years compared with traditional breeding methods.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for cultivating pepper multi-gene multi-trait aggregate germplasm based on the combination of molecular markers and morphological markers, characterized in that: The steps include: (1) hybridizing pepper parent A and pepper parent B to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plant F carrying both the root-knot nematode resistance gene and the whole-plant hairy trait. AB ; The two pepper parents are pepper resource "2018-21H" and pepper resource "2005-15H"; (2) hybridizing pepper parent C and pepper parent D to obtain first-generation hybrid seeds, sowing the first-generation hybrid seeds to obtain first-generation hybrid plants, and screening to obtain plant F carrying both the capsaicin synthase gene and the whole plant purple trait. CD ; The two pepper parents are pepper resource "2011-02H" and pepper resource "2016-08H"; (3) F AB and F CD Perform hybridization to obtain first-generation hybrid seeds, sow the first-generation hybrid seeds to obtain first-generation hybrid plants; (4) isolating microspores from the hybrid first-generation plants at the flowering stage, cultivating the microspores, obtaining haploid pepper plants, and, after screening, obtaining pepper plants with root-knot nematode resistance genes, capsaicin synthase genes, whole-plant hairiness traits, and whole-plant purple traits; (5) Chromosome doubling of pepper plants to obtain pepper diploid plants with root-knot nematode resistance genes, capsaicin synthase genes, whole-plant hairiness traits, and whole-plant purple traits; (6) subjecting the pepper diploid plants having the root-knot nematode resistance gene, capsaicin synthase gene, whole-plant hairiness trait, and whole-plant purple trait to rooting culture and planting management to obtain pepper multi-gene multi-trait aggregate germplasm; The hybridization is a reciprocal cross.

2. The cultivation method according to claim 1, characterized in that The screening in step (1) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent gene is the root-knot nematode resistance Me gene, and the excellent trait is the whole-plant hairy trait.

3. The cultivation method according to claim 1, characterized in that The screening in step (2) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent gene is the non-spicy at3 gene, and the excellent trait is the purple trait of the whole plant.

4. The cultivation method according to claim 1, characterized in that The screening in step (4) is assisted by excellent gene molecular markers and excellent trait morphological markers; the excellent genes are the root-knot nematode resistance Me gene and the non-spicy at3 gene, and the excellent traits are the whole plant hairy trait and the whole plant purple trait.

5. The cultivation method according to any one of claims 2 to 4, characterized in that: The detection primer of the anti-root-knot nematode Me gene molecular marker is Me-1, the forward primer sequence of Me-1 is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The detection primer of the non-spicy at3 gene molecular marker is at3-2, the forward primer sequence of at3-2 is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The whole-plant hairy trait morphological marker is that the aerial part of the plant is densely covered with hairs; The whole plant purple trait morphological marker is manifested in that the above-ground part of the plant appears purple.

6. The cultivation method according to claim 1, characterized in that: The microspores in step (4) are microspores in the uninucleate marginal stage.

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