CN112535099B - Breeding and utilizing method of black radish germplasm resources - Google Patents

Abstract

The invention discloses a breeding utilization method of black radish germplasm resources, which is easier to breed a sterile line compared with radish Ogura CMS, an NWB CMS sterile source created by black radish has a wider maintainer line breeding range, almost all radish materials (including Ogura CMS hybrid varieties) which do not contain orf463 specific genes can be bred into corresponding maintainer lines and sterile lines, and a male sterile hybrid variety can be easily bred by the NWB CMS sterile line, so that the intellectual property of a breeder can be better protected. Meanwhile, various restorer lines created by using the NWB CMS restorer gene contained in the black radish can be used for breeding hybrid varieties and can also be used for fixing and utilizing the excellent characters of NWB CMS hybrid sterile varieties in the market, the risk of losing germplasm resources is reduced, and the utilization efficiency of the germplasm resources is improved.

Description

Breeding utilization method of black radish germplasm resources

Technical Field

The invention belongs to the technical field of breeding, and particularly relates to a breeding utilization method of black radish germplasm resources.

Background

China is the first major country of radish production in the world, and the annual average planting area is about 120 million hectares. Radish is a typical cross-pollinated plant, self-pollination declines seriously, and the heterosis is obvious. The hybrid seed production by using Cytoplasmic Male Sterile (CMS) as female parent is the main way of utilizing heterosis of radish, and has the advantages of high purity of first generation of hybrid, low cost of seed production, difficult loss of parent, etc. Cytoplasmic male sterility is sterility caused by a maternally inherited, intramolecular or intermolecular recombination or rearrangement of the cytoplasmic and nuclear genes, which can form chimeric genes or new Open Reading Frames (ORFs), and restoration of fertility by nuclear Restorer genes (Rf).

In 1968, a small storehouse of Japanese scholars discovers a radish cytoplasmic male sterility type Ogura CMS for the first time, the cytoplasmic type is widely applied to breeding of radish hybrid varieties, but restoring genes of the cytoplasmic type widely exist in various radish germplasm resources, and screening and creation of maintaining materials are required to be carried out through mass test cross or by the aid of molecular markers of the restoring genes Rfo, so that the breeding difficulty of the Ogura CMS hybrid radish varieties is increased. In 2005, the korean scholars Nahm reported for the first time that NWB CMS generally lacked the cytoplasm type restoring material in nature, compared to Ogura CMS, indicating that the cytoplasm type is very easy to breed the corresponding sterile line, and can be well applied to radish variety breeding. Hybrid radish varieties prepared by NWB CMS are sterile and can well protect intellectual property rights of breeders, but the lack of recovery materials causes the excellent properties of the varieties to be difficult to breed and utilize, and causes the loss of a large amount of radish germplasm resources for a long time.

Black radish (Raphanus sativus l.var. niger)) is a variety of common radish, is distributed in asia and europe, is not only a root vegetable, but also has certain health care and medical effects due to the rich active substances such as glucosinolate, and can be used for treating various liver, urine, gallbladder, gastrointestinal tract, cardiovascular and respiratory system diseases. Radish consumed in Chinese market is mainly of types such as white radish, red radish, green radish and the like, and the commercial characters of black radish germplasm resources are difficult to utilize in breeding. In recent years, researches of a plurality of scholars show that the NWB CMS mitochondrion specific gene orf463 and a restoring gene thereof are widely distributed in black radish, and provide a theoretical basis for breeding application of black radish germplasm resources.

Disclosure of Invention

Aiming at the prior art, the invention provides a breeding utilization method of black radish germplasm resources, which can obviously improve the breeding efficiency of radish varieties.

In order to achieve the purpose, the invention adopts the technical scheme that: the breeding and utilizing method of the black radish germplasm resources comprises the following steps:

(1) creating sterile source and breeding sterile line

S1: designing an amplification primer by using a specific gene orf463 in an NWB CMS mitochondrial genome;

s2: hybridizing black radish capable of amplifying the orf463 specific gene serving as a female parent and fertile radish material incapable of amplifying the orf463 specific gene serving as a male parent to obtain an F1 generation population; then selfing the F1 generation population to obtain an F2 generation population; separating sterile plants from F2 generation population to obtain sterile source for NWB CMS sterile line breeding;

s3: continuously backcrossing 6 generations by taking the sterile source as a female parent and taking a fertile radish material incapable of amplifying the orf463 specific gene as a male parent, selfing the male parent, and simultaneously evaluating and screening the characters of backcross populations and selfing populations in each generation to breed an NWB CMS sterile line and a maintainer line;

(2) creation and breeding recovery line

S1: hybridizing black radish serving as a female parent and radish materials to be transferred serving as backcross male parents to obtain an F1 generation population; backcrossing with backcross male parent with F1 generation material as female parent, and continuous backcrossing for 3 generations with each generation of screened fertile backcrossed generation as female parent; continuously selfing the backcross population for 5 generations to obtain an NWB CMS restoring line containing orf463 specific genes;

s2: hybridizing black radish serving as a male parent and a fertile radish material to be transferred serving as a female parent to obtain an F1 generation group; taking an F1 generation material as a female parent, taking a radish material to be transferred as a backcross male parent for backcross, carrying out test cross on backcross progeny and an NWB CMS sterile line, screening backcross progeny capable of restoring the fertility of the NWB CMS sterile line as the female parent, carrying out continuous backcross for 3 generations, simultaneously carrying out test cross fertility identification, carrying out continuous selfing for 5 generations, carrying out test cross identification on the selfed progeny and the NWB CMS sterile line, and breeding into an NWB CMS restorer line without orf463 specific genes;

(3) NWB CMS sterile variety good character fixation

Taking NWB CMS sterile varieties containing excellent properties in the market as female parents, hybridizing the NWB CMS recovery line created in the step (2) as male parents, selfing the F1 generation, selecting fertile materials in the F2 generation for selfing, simultaneously screening and evaluating the properties, selecting materials containing more than one excellent properties of the NWB CMS sterile varieties for selfing, selfing continuously for 5 generations, and breeding a series of selfing lines containing the excellent properties of the NWB CMS sterile varieties for variety breeding.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the forward primer sequence of the amplification primer is shown as SEQ ID NO.1, and the reverse primer sequence is shown as SEQ ID NO. 2.

The invention has the beneficial effects that: the invention discloses a breeding utilization method of radish NWB CMS sterile line and a restorer line thereof by utilizing black radish germplasm resources. Compared with the radish Ogura CMS, the breeding of the sterile line is easier, the NWB CMS sterile source created by black radish has wider maintainer line breeding range, almost all radish materials (including Ogura CMS hybrid varieties) which do not contain orf463 specific genes can be bred into corresponding maintainer lines and sterile lines, and the NWB CMS sterile line is easy to prepare a male sterile hybrid variety, so that the intellectual property of breeders can be better protected. Meanwhile, various restorer lines created by using the NWB CMS restorer gene contained in the black radish can be used for breeding hybrid varieties and can also be used for fixing and utilizing the excellent characters of NWB CMS hybrid sterile varieties in the market, the risk of losing germplasm resources is reduced, and the utilization efficiency of the germplasm resources is improved.

Drawings

FIG. 1 shows a material of black radish; wherein, 3: white radish with long root, flower leaf and black skin, introduced from Italy; 4: a radish having a black skin and a white meat cone, which is introduced from Germany; 5: radish with black skin and white meat round root shape, imported from hungary;

FIG. 2 is a PCR electrophoresis chart of 11 parts of material orf463 specific molecular markers (M: DL 2000; 1-11: test material);

FIG. 3 shows the different types of radish NWB CMS sterile lines, wherein 6-11 represent maintainer line sources;

FIG. 4 shows the transfer of NWB CMS restorer line; wherein, a: the round black radish and the white long radish are hybridized to have the phenotype of F1; b, transforming into a white long radish NWB CMS restorer; c: the transformed round white radish NWB CMS restoring line.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Examples

A breeding method of black radish germplasm resources comprises the following steps.

(I) test materials

The relevant studies were carried out using 3 parts of black radish germplasm resources (FIG. 1) introduced from Europe and 8 parts of different types of radishes as test materials, and the details of the materials are shown in Table 1.

Table 1 details of the materials tested

(II) extraction of genomic DNA from materials and PCR amplification

Selecting young and tender radish leaves, and extracting DNA by adopting a general plant DNA kit of Tiangen biology (Beijing) Co.

Primers were designed based on the published sequence of the specific gene orf463 in the radish NWB CMS mitochondrial genome (Genebank ID: MN056360) at the NCBI website, with the forward primer orf463-F sequence as follows:

GTGCCATTGCTGGAGTGAT(5′→3′)(SEQ ID NO.1)；

the reverse primer orf463-R has the following sequence:

ATTGTCCTGACGGTTCTGA(5′→3′)(SEQ ID NO.2)；

the length of the amplified specific fragment is 1106 bp. The amplification system was 25. mu.L containing 40ng DNA, 2.5. mu.L 10 XTaq Plus Buffer (Mg-containing)²⁺) 2 μ L of 2.5mM Super pure dNTPs, 0.5 μ L of 10 μ M forward primer, 0.5 μ L of 10 μ M reverse primer, 0.5 μ L of 2.5U/. mu.L Taq Plus, using ddH₂And the content of O is filled up to 25 mu L. The PCR reaction program is: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 90s, and circulating for 32 times; finally, extension is carried out for 10min at 72 ℃. PCR products were detected electrophoretically on a 1.2% agarose gel. As shown in FIG. 2, it was confirmed that three black radish plants all contained NWB CMS cytoplasmic male sterile gene, and the expression thereof was fertile, indicating that they also contained NWB CMS restorer gene, which provides the basis for breeding.

(III) creation of sterile source of NWB CMS and breeding of sterile line

Hybridizing black radish materials 3, 4 and 5 serving as female parents and material 2 serving as male parents to obtain F1 generation, and then using 2m for single plant of F1 generation material³Covering with a gauze of 80 meshes, selfing and pollinating 40 osmia bees to obtain a large number of F2-generation populations, selecting sterile plants from the F2-generation populations as NWB CMS sterile sources, carrying out continuous backcross for 5 generations by taking the sterile sources as female parents and various radish materials (6-11) as backcross male parents, and selfing the male parents to breed various corresponding NWB CMS sterile lines. The results are shown in FIG. 3.

Creation and breeding of NWB CMS restoring line

Taking a long-rooted black radish 4 as a female parent, taking a long-rooted radish material 8 (a hybrid variety progeny from YR Baiyuchun containing Ogura CMS) as a male parent, obtaining an F1 generation material (figure 4-a), then taking F1 as a female parent, taking the material 8 as a recurrent male parent, selecting a fertile backcross progeny as the female parent for each generation, carrying out continuous backcross for 3 generations, carrying out selfing after the backcross progeny has basically the same properties with the recurrent parent, selecting a fertile material of the selfed progeny, and carrying out continuous selfing for 5 generations to obtain the mosaic white skin white meat long cylindrical radish NWBCMS restoring line (figure 4-b). Similarly, the round-root radish material 7 is used as a male parent, and the round-white radish NWB CMS restorer can be bred by the transformation method (fig. 4-c).

(V) NWB CMS sterile hybrid variety utilization

Taking radish material 1(NWB CMS sterile variety Shinong 301) as a female parent, carrying out hybridization on the white long radish NWB CMS restoring line obtained by transformation in the step four to obtain a male parent, carrying out hybridization to obtain an F1 generation material, then carrying out F1 net covering and bee breeding for selfing to obtain a large number of F2 generation populations, separating 2 leaf type materials from flower leaves and leaf type F2 generation populations, selecting each type of excellent fertile material for selfing, and carrying out continuous selfing for 5 generations to obtain the leaf, flower leaf white skin white meat long root type selfing line, thereby achieving the purpose of utilizing the excellent genes of the NWB CMS sterile variety.

While the embodiments of the invention have been described in detail in connection with the drawings and examples, the invention should not be construed as limited to the scope of the claims. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims (1)

1. A breeding utilization method of black radish germplasm resources is characterized by comprising the following steps:

(1) creating sterile source and breeding sterile line

S1: specific gene in NWB CMS mitochondrial genomeorf463Designing an amplification primer, wherein the sequence of a forward primer of the amplification primer is shown as SEQ ID NO.1, and the sequence of a reverse primer is shown as SEQ ID NO. 2;

s2: can be amplified outorf463The fertile black radish with specific gene is used as female parent and can not be amplifiedorf463Hybridizing fertile radish materials with specific genes as male parents to obtain F1 generation populations; then selfing the F1 generation population to obtain an F2 generation population; separating sterile plants from F2 generation population to obtain sterile source for NWB CMS sterile line breeding;

s3: using the sterile source as female parent and using the sterile source as male parentorf463The method comprises the following steps of taking a specific gene fertile radish material as a male parent, carrying out continuous backcross for 6 generations, selfing the male parent, simultaneously carrying out character evaluation and screening on a backcross population and a selfed population in each generation, and breeding into an NWB CMS sterile line and a maintainer line;

(2) creation and breeding recovery line

S1: hybridizing by taking black radishes as female parents and radish materials to be transferred as backcross male parents to obtain F1 generation populations; backcrossing with backcross male parent as female parent in F1 generation material, and continuous backcrossing for 3 generations with each generation of bred fertile backcross generation as female parent; backcross population is continuously selfed for 5 generations to obtain the productorf463NWB CMS restoring line of specific gene;

s2: hybridizing black radish serving as a male parent and a fertile radish material to be transferred serving as a female parent to obtain an F1 generation group; backcrossing F1 generation material as female parent and radish to be transferred as backcross male parent, test crossing the backcross progeny with NWB CMS sterile line, screening backcross progeny capable of restoring the fertility of NWB CMS sterile line as female parent, backcrossing for 3 generations continuously, test crossing fertility identification, continuously selfing for 5 generations, test crossing the selfed progeny and NWB CMS sterile line to identify fertility, and breeding to obtain the product without backcross progenyorf463Specific gene NWB CMS restorer line;

(3) NWB CMS sterile variety good character fixation

Taking NWB CMS sterile varieties containing excellent properties in the market as female parents, hybridizing the NWB CMS recovery line created in the step (2) as male parents, selfing the F1 generation, selecting fertile materials in the F2 generation for selfing, simultaneously screening and evaluating the properties, selecting materials containing more than one excellent properties of the NWB CMS sterile varieties for selfing, selfing continuously for 5 generations, and breeding a series of selfing lines containing the excellent properties of the NWB CMS sterile varieties for variety breeding.

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