A method for identifying the authenticity of F1 hybrids between red ginger and white ginger using SSR markers

Abstract

The present invention discloses a method for identifying the authenticity of the interspecific hybrid F1 hybrid of red ginger flower and white ginger flower using SSR markers. The present invention extracts the genomic DNA of the parent red ginger flower, white ginger flower and the hybrid F1 generation thereof, performs SSR capillary electrophoresis amplification analysis, uses SSR marker primers HcgSSR115 and / or HcgSSR1951 for amplification, compares the characteristic peaks of the interspecific hybrid F1 generation with those of the parents, and identifies the authenticity of the hybrid. The SSR markers used in the present invention have strong specificity and good repeatability, and can screen out true hybrids at an early stage. They have the characteristics of short identification time, high accuracy, and high reliability, and can replace the traditional hybrid identification method using morphology, effectively improving the efficiency of identifying interspecific hybrids of ginger flower. The method provided by the present invention can quickly identify the authenticity of interspecific hybrids between red ginger flower and white ginger flower, provide an effective basis for ginger flower hybrid research, and be beneficial to the genetic improvement of ginger flower varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and more specifically, relates to a method for identifying the authenticity of interspecific hybrid F1 hybrids of red ginger flower and white ginger flower by using SSR markers. Background Art

[0002] Hedychium refers to a genus in the Zingiberaceae family, which is a perennial herb. There are about 50 species distributed worldwide, and about 30 species are distributed in my country. The plants in the genus Hedychium are diverse, colorful, and fragrant, and are mainly used for gardening and ornamental cut flowers. Among them, white ginger flower (H. coronarium) is currently the only main cultivated variety in China. It is a Lingnan specialty flower. It is loved by people because of its large, white flowers and strong fragrance. It is often produced and sold as cut flowers, but its color is single and it is difficult to meet people's requirements for rich colors. Red ginger flower (H. coccineum) is mainly a wild variety with bright petals but basically no fragrance. At present, cultivating flower varieties with unique colors and fragrance has become one of the goals of flower hybridization breeding, thereby enriching the germplasm resources of the genus Hedychium in my country. Through distant hybridization, hybrid vigor can be used to quickly cultivate new plant varieties with large trait variation, good comprehensive traits, and adaptability to industrial development.

[0003] At present, the key to hybrid breeding for excellent traits lies in the identification of the authenticity of the hybrid F1 generation. Traditional identification work is generally based on the morphological characteristics of the father and mother. However, due to the increasing convergence of the parents in high-yield breeding, morphological identification has problems such as long cycle, poor reliability, low accuracy and poor efficiency. In the prior art, there is a method of using molecular markers to assist in identification to identify the genetic traits of the hybrid F1 generation of hybrid breeding. There is no need to wait until the F1 generation grows into plants before making a judgment. The genetic traits of hybrid offspring can be accurately, conveniently and quickly identified. For example, the prior art discloses the use of SSR molecules for genetic analysis of flower color in the F1 generation of hybrids of white ginger flower and golden ginger flower (Zhou Yiwei, Xu Guoyu, Wang Qin, Yan Fulun, Yu Yunyi, Yu Rangcai, Fan Yanping. Genetic analysis of flower color in the F1 generation of hybrids of 'white ginger flower' × 'golden ginger flower' and development of related SSR molecular markers [J]. Acta Horticulturae Sinica, 2021, 48(10): 1921-1933.), but there are currently no reports on the use of SSR molecular marker technology to identify interspecific hybrids of ginger flower. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of traditional morphological trait identification methods, and provide a method for identifying the authenticity of F1 hybrids of interspecific hybridization between red ginger flower and white ginger flower using SSR markers. The method has strong specificity, good repeatability, and rapid identification. True hybrids can be screened out at an early stage, and can replace traditional hybrid identification methods using morphology, effectively improving the efficiency of interspecific hybrid identification of ginger flower.

[0005] The purpose of the present invention is to provide a method for identifying the authenticity of F1 hybrids of interspecific hybridization between red ginger flower and white ginger flower by using SSR markers.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention screened and analyzed 22 pairs of SSR marker primers (HcgSSR102-104, HcgSSR107, HcgSSR110-113, HcgSSR115-119, HcgSSR538, HcgSSR641, HcgSSR948, HcgSSR1166, HcgSSR1420, HcgSSR1501, HcgSSR1508, HcgSSR1751, and HcgSSR1951) with good amplification effects identified by the applicant's research team in the early stage in the genome of Zingiber officinale 'ZS'. Only markers HcgSSR115 and HcgSSR1951 can well distinguish between parents and hybrids, have obvious polymorphism, and have characteristic peaks of amplified fragments with good specificity. The use of markers HcgSSR115 or HcgSSR1951 can identify more than 60% of true hybrids, and the use of markers HcgSSR115 and HcgSSR1951 can further improve the accuracy, and can identify 84.21% of true hybrids. Compared with the traditional identification method of morphological trait observation, the method of the present invention has the advantages of convenience, short identification time, high accuracy, and strong reliability. It can effectively improve the efficiency of identifying interspecific hybrids of ginger flowers and reduce the error rate of the traditional identification method of morphological trait observation.

[0008] The present invention provides a method for identifying the authenticity of an interspecific hybrid F1 hybrid of red ginger flower and white ginger flower by using SSR markers, comprising the following steps:

[0009] S1. Extract genomic DNA from parental red ginger flower, white ginger flower and their interspecific hybrid F1 hybrids respectively;

[0010] S2. SSR capillary electrophoresis analysis: using the genomic DNA extracted in step S1 as a template, respectively, using SSR marker primers HcgSSR115 and / or HcgSSR1951 to perform SSR-PCR amplification detection, and collecting characteristic peaks of amplified products; the primer sequence of HcgSSR115 is shown in SEQ ID NO.1-2, and the primer sequence of HcgSSR1951 is shown in SEQ ID NO.3-4;

[0011] S3. Identification of hybrid authenticity: Compare the characteristic peaks of the interspecific hybrid F1 hybrid in step S2 with the characteristic peaks of the parents. If the hybrid has both characteristic peaks of the parents, it is a true hybrid.

[0012] Preferably, in step S1, young leaves of the parents and the interspecific hybrid F1 hybrid are extracted, and then the DNA purity is detected by 1% agarose gel electrophoresis.

[0013] Preferably, the 5' tail end of the labeled upstream primer is connected with a FAM fluorescent label.

[0014] Preferably, the system for SSR-PCR amplification in step S2 (25 μL): 50 ng·μL -1 Template DNA 1.0μL, 10μM dNTP (mix) 0.5μL, 10× Taq Buffer (with MgCl2) 2.5μL, 10mM upstream primer and downstream primer 1.0μL each, 5U / μL Taq enzyme 0.2μL, ultrapure water 19.8μL.

[0015] Preferably, the SSR-PCR reaction in step S2 is carried out in Veriti TM The reaction was carried out on a 96-well PCR instrument with the following procedure: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; and repair and extension at 72°C for 10 min.

[0016] The present invention provides the use of SSR marker primers HcgSSR115 and / or HcgSSR1951 shown in SEQ ID NOs. 1 to 4 in identifying the authenticity of F1 hybrids of interspecific hybridization between red ginger flower and white ginger flower.

[0017] The invention provides a kit for identifying the authenticity of an interspecific hybrid F1 hybrid of red ginger flower and white ginger flower, comprising SSR marker primers HcgSSR115 and / or HcgSSR1951, and the primer sequences are shown in SEQ ID NOs.1-4 in sequence.

[0018] Preferably, the kit further comprises reagents required for SSR-PCR amplification.

[0019] Preferably, the kit further comprises reagents required for extracting sample DNA.

[0020] The present invention has the following beneficial effects:

[0021] The present invention uses SSR molecular markers to effectively identify interspecific hybrids of white ginger flower and red ginger flower, and can quickly and accurately identify the true hybrid F1 generation of white ginger flower and red ginger flower. The present invention shows that the use of marker primers HcgSSR115 and / or HcgSSR1951 can well distinguish parents from hybrids, and has a characteristic peak of amplified fragments with good specificity, and the two markers have good complementarity. The use of marker primers HcgSSR115 or HcgSSR1951 can identify more than 60% of true hybrids, and the use of marker primers HcgSSR115 and HcgSSR1951 at the same time can further improve the accuracy rate, and can identify 84.21% of true hybrids. Compared with the traditional identification method based on morphological trait observation, the SSR markers used in the method of the present invention have strong specificity, good repeatability and rapid identification, and true hybrids can be screened out at an early stage. It has the advantages of short identification time, high accuracy and strong reliability, and can replace the traditional hybrid identification method based on morphology, effectively improving the efficiency of interspecific hybrid identification of ginger flower and reducing the error rate of the traditional identification method based on morphological trait observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison of the inflorescences of white ginger flowers, red ginger flowers, and some of their hybrid offspring;

[0023] Figure 2 This is the agarose gel electrophoresis of the genomic DNA of white ginger flower, red ginger flower and some hybrid offspring;

[0024] Figure 3 Comparison of characteristic peaks of target products of parents and some hybrid offspring under marker HcgSSR115;

[0025] Figure 4 Comparison of characteristic peaks of the target product marked with HcgSSR1951 in parents and some hybrid offspring. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] Example 1 Identification method based on traditional morphological character observation

[0029] The parent ginger flowers used in the present invention are: red ginger flower and white ginger flower were introduced from Kunming Botanical Garden and Guangzhou Maoyuan Flower Farm in 2007, respectively, and are currently preserved as resources in the flower base of South China Agricultural University. Red ginger flower is a wild species with bright color and small flowers, but lacks fragrance; white ginger flower is a cultivated species with white color, large flowers and strong fragrance. The observation method of morphological traits refers to the "Guide to Plant Variety Specificity (Distinguishability), Consistency and Stability Testing-Ginger Flower Genus" issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China, and the main ornamental traits of the flower parts of the parents and their F1 hybrid offspring during the flowering period are observed. The main observation indicators mainly include lip color, lip length, lip width, lateral degenerate stamen color, lateral degenerate stamen length, lateral degenerate stamen width, filament color, filament length, anther color, anther length, etc. The parent white ginger flower and red ginger flower used in the present invention and the F1 hybrids obtained by positive and negative crosses are all planted in the planting base of the Flower Research Center of South China Agricultural University.

[0030] During the cultivation process, the hybrid F1 of white ginger flower♀×red ginger flower♂ was numbered as BH, and the hybrid F1 of red ginger flower♀×white ginger flower♂ was numbered as HB, and the above-mentioned main observation indicators of the parents and hybrid F1 generation were counted. When the plants grew to the flowering stage, the main morphological traits of the F1 generation plants showed that the main morphological traits of the parents were different.

[0031] Taking some hybrid F1 samples BH04, BH10, HB17 and HB47 as examples for analysis, the inflorescences of white ginger flowers, red ginger flowers and some hybrid offspring of positive and negative crosses are shown in Figure 2. Figure 1The results of the comparison of their morphological traits are shown in Table 1. Among them, the hybrid F1 generations BH04 and BH10 obtained by hybridizing white ginger flower as the female parent and red ginger flower as the male parent, their lip length, width, lateral degenerate stamen length, width and anther length are between the parents, and the main color of the lip has changed significantly; the hybrid F1 generations HB17 and HB47 obtained by hybridizing red ginger flower as the female parent and white ginger flower as the male parent, their lip length, width and lateral degenerate stamen width are between the parents, and the main color of the lip and the main color of the anther have changed significantly. It can be preliminarily determined that BH04, BH10, HB17 and HB47 are all true hybrids. At the same time, the same morphological trait observation method was used to conduct traditional morphological trait observation and identification on the 86 F1 hybrid offspring obtained, and 38 were preliminarily determined to be true hybrids, including BH01, BH02, BH04, BH06, BH07, BH08, BH10, BH11, HB01, HB02, HB04, HB05, HB07, HB10, HB11, HB12, HB16, HB17, HB18, HB19, HB20, HB30, HB46, HB47, HB52, HB54, HB56, HB57, HB58, HB61, HB63, HB64, HB65, HB66, HB68, HB69, HB70 and HB71.

[0032] Table 1 Comparison of morphological traits of F1 hybrids from reciprocal crosses between white ginger flower and red ginger flower

[0033]

[0034] Example 2 Screening of SSR markers

[0035] The SSR molecular markers used in the present invention are from 22 pairs of SSR primers with good amplification effects (HcgSSR102-104, HcgSSR107, HcgSSR110-113, HcgSSR115-119, HcgSSR538, HcgSSR641, HcgSSR948, HcgSSR1166, HcgSSR1420, HcgSSR1501, HcgSSR1508, HcgSSR1751, HcgSSR1951) selected from 266,825 SSR markers identified in the genome of Zingiber officinale 'ZS' by the research team of the applicant in the early stage. For details of the specific SSR markers, please refer to: Zhou YW, Wei X, Abbas F, et al. Genome-wide identification of simple sequence repeats and assessment of genetic diversity in Hedychium[J]. Journal of Applied Research on Medicinal and Aromatic Plants,2021,24:100312.

[0036] The genomic DNA of the parents and some of the interspecific hybrid F1 generations in Example 1 were extracted and analyzed by Veriti TM SSR analysis was performed on a 96-well PCR instrument. The reaction system (20 μL) contained 50 ng·μL -1 Template DNA 1.0μL, 2×Taq PCRStarMix 10.0μL, 10mM upstream primer and downstream primer 1.0μL each, ultrapure water 7.0μL. Reaction procedure: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 30s, 35 cycles; 72℃ repair extension for 7min. SSR-PCR products were analyzed by 8% polyacrylamide gel electrophoresis.

[0037] After screening and analysis, after amplification with the above 22 pairs of SSR marker primers, only the markers HcgSSR115 and HcgSSR1951 had obvious polymorphism between the parents, and could identify the authenticity of the hybrids of some interspecific hybrid F1 generations used. SSR capillary electrophoresis was further performed to analyze all interspecific hybrid F1 individuals. The 5' tail of the upstream primers of the above two SSR markers was connected to the FAM fluorescent marker, which was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Most of the hybrid offspring had amplified fragment characteristic peaks with good specificity. The characteristic peak statistics of the parent white ginger flower and red ginger flower are shown in Table 2 below, where in the marker HcgSSR115, the white ginger flower has characteristic peaks at 308.0bp and 313.5bp, while the red ginger flower has characteristic peaks at 302.6bp and 306.6bp; in the marker HcgSSR1951, the white ginger flower has characteristic peaks at 174.8bp and 183.1bp, while the red ginger flower has characteristic peaks at 151.0bp, 169.9bp and 196.7bp. Therefore, the characteristic peaks amplified from the two markers of the parents are significantly different and have good polymorphism, so the present invention selects markers HcgSSR115 and HcgSSR1951 as primers for identifying the authenticity of the interspecific hybrid F1 hybrid of red ginger flower and white ginger flower, and their specific sequences are shown in Table 3.

[0038] Table 2 Statistics of characteristic peaks of white ginger flower and red ginger flower

[0039] mark White ginger flower characteristic peak Red ginger flower characteristic peak HcgSSR115 308.0bp、313.5bp 302.6bp、306.6bp HcgSSR1951 174.8bp、183.1bp 151.0bp, 169.9bp, 196.7bp

[0040] Table 3 Primer sequences of SSR markers HcgSSR115 and HcgSSR1951 of the present invention

[0041]

[0042] Example 3 Identification of the authenticity of F1 hybrids between red ginger flower and white ginger flower based on SSR markers

[0043] 1. Extraction of genomic DNA:

[0044] Fresh and tender leaves of the parents used in Example 1 and 38 hybrid F1 plants confirmed to be true hybrids were cut, and genomic DNA was extracted using the quick plant genomic DNA extraction kit (DP321) of Tiangen Biochemical Technology (Beijing) Co., Ltd., with the specific steps strictly referring to the extraction instructions; the extracted genomic DNA was then detected by 1.0% agarose gel electrophoresis.

[0045] The results are as follows Figure 2Shown are agarose gel electrophoresis images of genomic DNA of red ginger flower, white ginger flower and some hybrid offspring. The electrophoresis bands are relatively clear without any diffusion or tailing. It can be considered that the DNA meets the requirements of subsequent SSR-PCR reactions.

[0046] 2. SSR capillary electrophoresis analysis:

[0047] The 5' tails of the primers for the SSR markers HcgSSR115 and HcgSSR1951 screened in Example 2 were connected to a FAM fluorescent label and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. SSR-PCR amplification was performed using the same amplification conditions and procedures as in Example 2; the amplified products were placed on a 3730XL genetic analyzer for detection and analysis.

[0048] 3. Characteristic peak comparison: Compare the characteristic peaks of the F1 hybrid SSR markers detected in the above steps with the characteristic peaks of the SSR markers of the parents to determine the authenticity of the interspecific hybrid.

[0049] The comparison results of the characteristic peaks of the target products amplified by marker HcgSSR115 in the parent white ginger flower and red ginger flower and some hybrid F1 generations are as follows Figure 3 As shown, the characteristic peaks of the target product amplified by labeled HcgSSR1951 are as follows Figure 4 As shown in the figure, it can be seen that the hybrids with both parental characteristic peaks are true hybrids. The identification of the authenticity of white ginger flowers, red ginger flowers and 38 F1 hybrids is shown in Table 4. In the marker HcgSSR115, 23 (60.53%) F1 hybrids have both parental characteristic peaks; in the marker HcgSSR1951, 24 (63.16%) F1 hybrids have both parental characteristic peaks. When the two markers are used for identification together, 32 (84.21%) of the 38 F1 hybrids can be identified as true hybrids, and the identification efficiency is greatly improved compared to using a single marker.

[0050] Table 4 Identification of authenticity of 38 F1 hybrids using two SSR markers

[0051]

[0052]

[0053] In summary, compared with the traditional morphological trait identification method, the method of identifying the authenticity of F1 hybrids using SSR molecular markers in the present invention has the following advantages:

[0054] The morphological trait identification method has the disadvantages of long cycle, susceptibility to environmental conditions, and requiring a large planting space. The SSR molecular markers of the present invention can save a lot of time and space while identifying the authenticity of hybrids, have high accuracy, and can be used to identify the authenticity of hybrids in the hybrid seedling stage, which is conducive to accelerating the process of genetic improvement of ginger flower varieties.

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Sequence Listing <110> South China Agricultural University <120> A method for identifying the authenticity of F1 hybrids between red ginger and white ginger using SSR markers <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> HcgSSR115-F(SIPOSequenceListing 1.0) <400> 1 attccacgtg caaatcattg 20 <210> 2 <211> 20 <212> DNA <213> HcgSSR115-R(SIPOSequenceListing 1.0) <400> 2 ccagacttcc atcacctcct 20 <210> 3 <211> 20 <212> DNA <213> HcgSSR1951-F(SIPOSequenceListing 1.0) <400> 3 taccaaacca gcccaaaaag 20 <210> 4 <211> 20 <212> DNA <213> HcgSSR1951-R(SIPOSequenceListing 1.0) <400> 4 ggatagagcc caacgacgta 20

Claims

1. A method for identifying the authenticity of F1 hybrids of interspecific hybridization between red ginger flower and white ginger flower using SSR markers, characterized in that: The following steps are involved: S1. Extract genomic DNA from parental red ginger flower, white ginger flower and their interspecific hybrid F1 hybrids respectively; S2. SSR capillary electrophoresis analysis: using the genomic DNA extracted in step S1 as a template, respectively, and using SSR marker primers HcgSSR115 and / or HcgSSR1951 to perform SSR-PCR amplification detection, and collecting characteristic peaks of amplified products; the upstream primer sequence of HcgSSR115 is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2; the upstream primer sequence of HcgSSR1951 is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4; S3. Identification of hybrid authenticity: Compare the characteristic peaks of the interspecific hybrid F1 hybrid in step S2 with the characteristic peaks of the parents. If the hybrid has both characteristic peaks of the parents, it is a true hybrid.

2. The method according to claim 1, characterized in that: In step S1, young leaves of the parent and the interspecific hybrid F1 hybrid are extracted.

3. The method according to claim 1, characterized in that: In step S1, the extracted genomic DNA was subjected to 1% agarose gel electrophoresis to detect the DNA purity.

4. The method according to claim 1, characterized in that: The 5' tail end of the upstream primer of the labeled primer is connected with a FAM fluorescent label.

5. The method according to claim 1, characterized in that: System for SSR-PCR amplification in step S2: 25 μL: 50 ng·μL -1 Template DNA 1.0 μL, 10 μM dNTP mix 0.5 μL, 10× Taq Buffer containing MgCl2 2.5 μL, 10 mM upstream primer and downstream primer 1 μL each, 5 U·μL -1 0.2 μL of Taq enzyme and 19.8 μL of ultrapure water.

6. The method according to claim 1, characterized in that: The SSR-PCR amplification reaction program in step S2 was as follows: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, for 35 cycles; and repair and extension at 72 °C for 10 min.

7. Application of SSR marker primers HcgSSR115 and / or HcgSSR1951 in identifying the authenticity of F1 hybrids of interspecific hybridization between red ginger and white ginger, characterized in that: The primer sequences of HcgSSR115 are shown in SEQ ID NOs. 1 to 2, and the primer sequences of HcgSSR1951 are shown in SEQ ID NOs. 3 to 4.

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