SSR (Simple Sequence Repeat) molecular marker primer for assisting orchid small plant type filial generation selection and application of SSR molecular marker primer

Abstract

The invention discloses an SSR (simple sequence repeat) molecular marker primer for assisting orchid small plant type filial generation selection and application of the SSR molecular marker primer. The SSR marker closely linked with the orchid small plant type is developed, the SSR marker primer is used for assisting in breeding of small plant type progeny plants, the breeding target of breeding of small plant type orchid progeny can be achieved more rapidly and accurately, the accuracy rate is larger than 80%, the breeding period is shortened, and the situation that breeding can only be conducted when progeny plants bloom in traditional breeding, and breeding time is shortened can be avoided. And early breeding cannot be realized. Meanwhile, a molecular marker-assisted small plant type orchid offspring selection technical system is established, amplification detection is performed through a molecular marker SSR32427 primer, orchid small plant type offspring selection is assisted, and selection can be advanced to the test tube seedling stage of hybrid offspring, so that the breeding period is shortened to about half of the original breeding period, the planting scale is reduced, and the breeding cost is reduced. The efficiency and benefit of orchid breeding are improved, and a technical foundation is laid for rapidly and directionally breeding new varieties of small-plant orchid.

Description

Technical Field

[0001] This invention relates to the field of molecular markers and plant breeding technology. More specifically, it relates to an SSR molecular marker primer for assisting in the selection of small-plant hybrid progeny of orchids and its application. Background Technology

[0002] Orchids are a general term for plants in the family Orchidaceae, but in my country, orchids specifically refer to plants in the genus Orchid (Orchidaceae). Cymbidium Orchids, including the terrestrial species known as Cymbidium goeringii (Liu Zhongjian et al., 2006), are one of the three most common types of orchids in the Chinese market. Cymbidium goeringii are elegant in form, beautiful in leaf shape, and have a delicate fragrance. They are a material carrier of traditional Chinese culture and have a cultivation history of over 2000 years in my country, with a broad market prospect. In the past, the breeding of Cymbidium goeringii varieties mainly relied on introduction and domestication, as well as selective breeding. However, with the industrialization of orchids and the development of biotechnology, sexual hybridization breeding has become the main method for breeding Cymbidium goeringii (He Ziyi et al., 2025).

[0003] Because orchid hybridization breeding has a long cycle, selecting a new variety typically takes more than 10 years. Plant form is an important ornamental trait and a primary breeding target trait for orchids. Small-sized orchids are lightweight and take up little space, making them widely used in home and office decoration. They are also easier to sell online, offering significant advantages in logistics and transportation costs. Therefore, small-sized orchids have become a market favorite in recent years, with broad market prospects. In traditional hybridization breeding, orchid plant form usually stabilizes after 3-5 years of cultivation until the plant matures and flowers, making it impossible to select plants with the target form earlier, requiring a long breeding cycle. At the same time, it is impossible to eliminate plants with non-target traits early in the breeding process, resulting in a large offspring population and high breeding costs. Therefore, researching techniques for selecting orchid plant form early in the breeding process is of great significance for shortening the breeding cycle, reducing planting scale, improving breeding efficiency, and quickly closing the gap with foreign breeding practices.

[0004] SSR markers, or simple sequence repeats (SSRs) and microsatellite markers, are numerous, co-dominant, exhibit Mendelian inheritance, and can distinguish between heterozygotes and homozygotes. They do not require high DNA quality; only a small amount of DNA tissue is needed for PCR analysis, making detection convenient. Polyacrylamide gel electrophoresis is commonly used to detect single-copy differences, and they contain a large amount of genetic information. By utilizing SSR markers closely linked to genes of the target breeding trait, genomic DNA can be extracted at different stages of plant growth for PCR and gel electrophoresis detection, enabling early selection of plants with the target trait. However, there are currently few SSR markers closely linked to small plant type developed for use in assisting the selection of hybrids with small plant types. To shorten the orchid breeding cycle and rapidly and directionally select new small-plant orchid varieties, this invention application is proposed. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the defects and shortcomings of existing orchid breeding, such as long cycle, inability to complete the selection of small plant type offspring in advance, and elimination of offspring with non-breeding target traits. This invention provides an SSR molecular marker primer to assist in the selection of small plant type hybrid offspring of orchids and its application.

[0006] The first objective of this invention is to provide an SSR molecular marker primer that assists in the selection of hybrid offspring of small orchid plants.

[0007] A second objective of this invention is to provide applications of SSR molecular marker primers.

[0008] A third objective of this invention is to provide a reagent kit.

[0009] A fourth objective of this invention is to provide applications for the reagent kit.

[0010] The fifth objective of this invention is to provide a method for the assisted selection of small-sized orchid plants using SSR molecular markers.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides an SSR molecular marker primer to assist in the selection of hybrid offspring of small orchid plants, including an upstream primer: 5'-AATGTGCCTTCAGGGATGAG-3' and a downstream primer: 5'-TGAACGCTTTCAGTGGTCTG-3'.

[0012] This invention utilizes transcriptome sequencing of large and small-sized orchid hybrids to develop a SSR marker closely linked to the small-sized orchid. Using this SSR marker to assist in the selection of small-sized offspring allows for faster and more accurate breeding, achieving an accuracy rate greater than 80%, shortening the breeding cycle, and avoiding the limitations of traditional breeding methods that only allow selection at flowering time. Simultaneously, a molecular marker-assisted selection technology system for small-sized orchid offspring is established. Amplification and detection of the molecular marker SSR32427 assists in the selection of small-sized orchid offspring, enabling selection to begin as early as the in vitro seedling stage of hybrids. This shortens the breeding cycle to approximately half its original length, reduces planting scale, and improves the efficiency and effectiveness of orchid breeding, laying a technical foundation for the rapid and targeted selection of new small-sized orchid varieties.

[0013] Therefore, this invention provides the following applications of SSR molecular markers: Applications in selecting and breeding small orchid plants, or in distinguishing or identifying different orchid plant types.

[0014] Applications include preparing products that assist in the selection and breeding of small orchid plants, or in distinguishing or identifying different orchid plant types.

[0015] Application in the breeding of small orchid plants.

[0016] Preferably, the distinction or identification of different orchid plant types refers to the distinction or identification of large or small orchid plants; specifically, plants with a plant height of less than 30 cm and a compact plant type are defined as small plants, and plants with a height of 30 cm and above are defined as large plants.

[0017] This invention provides a kit containing the above-mentioned SSR molecular marker primers.

[0018] Preferably, the kit further contains reagents required for PCR amplification.

[0019] This invention provides a kit for the selection of small orchid plants or for distinguishing or identifying different orchid plant types.

[0020] This invention also provides a method for selecting small-sized orchid plants using SSR molecular markers, comprising the following steps: S1. Extract genomic DNA from the parent orchids to be selected and their hybrid offspring; S2. Using DNA as a template, PCR amplification was performed using SSR molecular marker primers; S3. Perform gel electrophoresis on the PCR amplification products and select offspring plants with the same banding pattern as the small-type parent. S4. Single plant identification: The selected offspring are continued to be cultivated, and when they flower, the target offspring are selected based on the plant type characteristics, which can then be used to breed small-sized plants.

[0021] Preferably, genomic DNA is extracted from orchid leaves in step S1.

[0022] Preferably, the PCR amplification system in step S2 is as follows: 1~2 μL DNA template, 5~10 μL 2×M5 PAGE TaqPCR Mix, 0.5~1 μL upstream primer, 0.5~1 μL downstream primer, and 3~6 μL ddH2O.

[0023] Preferably, the PCR amplification program in step S2 is as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, Tm annealing for 30 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 12℃.

[0024] The present invention has the following beneficial effects: This invention utilizes transcriptome sequencing of large and small-sized orchid hybrids to develop a SSR marker closely linked to the small-sized orchid. Using this SSR marker to assist in the selection of small-sized offspring allows for faster and more accurate breeding, achieving an accuracy rate greater than 80%, shortening the breeding cycle, and avoiding the limitations of traditional breeding methods that only allow selection at flowering time. Simultaneously, a molecular marker-assisted selection technology system for small-sized orchid offspring is established. Amplification and detection of the molecular marker SSR32427 assists in the selection of small-sized orchid offspring, enabling selection to begin as early as the in vitro seedling stage of hybrids. This shortens the breeding cycle to approximately half its original length, reduces planting scale, and improves the efficiency and effectiveness of orchid breeding, laying a technical foundation for the rapid and targeted selection of new small-sized orchid varieties. Attached Figure Description

[0025] Figure 1 This is a map showing the density distribution of SSR loci.

[0026] Figure 2 This is a scatter plot of significantly enriched KEGG pathways (the vertical axis represents functional annotation information, and the horizontal axis represents the Rich factor corresponding to the function (the number of differentially expressed genes annotated to the function divided by the number of genes annotated to the function). The Q value is represented by the color of the dots, and the smaller the Q value, the closer the color is to red. The number of differentially expressed genes contained in each function is represented by the size of the dots.

[0027] Figure 3 The electrophoretic results of SSR32427 on 30 large-type F1 plants of the '13-52' combination (M: DL500 DNAMarker, P1: male parent 'Xin Zhongguo-1', P2: female parent 'Xiao Fenglan').

[0028] Figure 4 The results are electrophoretic analysis of SSR32427 against the '13-52' parental combination and 30 small F1 plants (M: DL500 DNA Marker, P1: male parent 'Xin Zhongguo-1', P2: female parent 'Xiao Fenglan').

[0029] Figure 5 The results are electrophoretic analysis of SSR32871 against the '13-52' parental combination and 30 large-type F1 plants (M: DL500 DNA Marker, P1: male parent 'Xin Zhongguo-1', P2: female parent 'Xiao Fenglan').

[0030] Figure 6 The results are electrophoretic analysis of SSR32871 against the '13-52' parental combination and 30 small F1 plants (M: DL500 DNA Marker, P1: male parent 'Xin Zhongguo-1', P2: female parent 'Xiao Fenglan').

[0031] Figure 7 The results are electrophoretic analysis of SSR32427 against the '17-49' combination parent and 57 F1 plants (M: DL500 DNA Marker, P1: male parent 'Da Fu Gui' Cymbidium goeringii, P2: female parent 'Xiao Feng Lan').

[0032] Figure 8 It is a combination of 5 large-type F1 offspring of 17-49.

[0033] Figure 9 It is the F1 offspring of 5 small plants from the 17-49 combination. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] The hybrid parents used in the examples, 'Xin Zhongguo-1' and 'Xiao Fenglan', are plant varieties cultivated by the inventor's research group, while 'Da Fugui' is a commercially available traditional Cymbidium goeringii variety.

[0037] Example 1: Transcriptome Sequencing and SSR Marker Screening 1. Transcriptome sequencing The plant height of the hybrid offspring of hybrid combination 13-52 (paternal parent 'Xin Zhongguo-1', maternal parent 'Xiao Fenglan') was measured. Plants with a compact plant type and a height of less than 30 cm were defined as small plants, and those with a height of 30 cm or more were defined as large plants. Both parents of combination 13-52 were large plants. Aseptic tissue culture seedlings approximately 4.5 cm tall from offspring of combinations 13-52-1907 (large plant type) and 13-52-1007 (small plant type) were used as materials. Total RNA was extracted using TRIzol reagent, with three biological replicates for each sample. The extracted RNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for quality control. Materials that passed quality control were sequenced using a high-throughput sequencing platform (Illumina NovaSeq 6000).

[0038] Sequencing results showed that the sequencing and assembly of the six samples yielded 108,589 unigenes and 278,926 transcripts, as shown in Table 1, with average lengths of 677.88 bp and 879.2 bp, respectively. Among the 108,589 unigenes, 20,962 contained SSR loci, of which 15,927 provided corresponding primers. The SSR locus density distribution per Mbp is shown in Table 1. Figure 1 As shown, SSR types are predominantly characterized by single-base repetition.

[0039] Table 1. Transcriptome sequencing data assembly statistics

[0040] 2. SSR tag filtering The sequencing results were compared with eight major databases (CDD, PFAM, KEGG, KOG, Swissprot, GO, NR, and NT) to comprehensively obtain functional information of genes and transcripts. Differential gene analysis was performed using DESeq26, with genes having |log2(FoldChange)|≥2 and FDR<0.05 defined as significantly differentially expressed genes. The results showed a total of 2527 significantly differentially expressed genes between 13-52-1007 and 13-52-1907, including 1209 upregulated genes and 1318 downregulated genes.

[0041] KEGG enrichment analysis of differentially expressed genes yielded 162 differentially expressed metabolic pathways. The functional enrichment scatter plot is shown below. Figure 2As shown, differential metabolic pathways related to plant height include plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, circadian rhythm-plant, photosynthesis, photosynthesis-antenna proteins, zeatin biosynthesis, N-glycosylation biosynthesis, and the MAPK signaling pathway-plant.

[0042] Four SSR primers were identified based on the differentially expressed gene IDs in the aforementioned metabolic pathways. An additional 68 SSR primers were obtained from other Unigene genes and transcripts related to plant height in the transcriptome. The selected SSR marker primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their specific sequence information is shown in Table 2.

[0043] Table 2 SSR molecular marker primer sequences

[0044] Example 2: Screening of SSR molecular markers linked to plant height 1. Sample DNA extraction Leaves from 60 F1 progeny (30 large-type and 30 small-type) of combinations 13-52 and their parent plants were collected, and DNA was extracted from each sample using the CTAB method.

[0045] 2. PCR amplification and gel electrophoresis detection The extracted leaf DNA was amplified by PCR using the primers synthesized in Table 2. The PCR amplification system was 10 μL: 1 μL DNA template (50-100 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer (10 uM / μL), 0.5 μL downstream primer (10 uM / μL), and 3 μL ddH2O or 5 μL (each component volume halved). The amplification program is shown in Table 3. The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis at 300 V for 120-150 min. After electrophoresis, silver staining and color development were performed, and finally, the samples were placed on a white plastic plate for photographing and reading.

[0046] Table 3 PCR reaction procedure

[0047] 3. Screening of SSR molecular markers linked to small plant types The parental samples were amplified using the 72 primer pairs listed in Table 2. 63 clear bands were amplified, resulting in an effective amplification rate of 87.5%. Further electrophoresis of PCR products from 60 F1 strains (combinations 13-52) and their parents was performed using these 63 primer pairs. Molecular markers closely linked to the small-type strain were screened based on criteria including clear main bands, a significant difference in the proportion of small-type and large-type bands, and a high proportion of small-type bands.

[0048] Some measurement results are as follows Figures 3-6 As shown, the banding patterns of the SSR32427 marker hybrid progeny are divided into those with specific bands and those without specific bands. The specific bands are approximately 200 bp in size. Figure 3 and Figure 4 Offspring with specific bands are large-sized plants, while those without specific bands are small-sized plants, excluding... Figure 4 The 14th band was unclear, and there were 20 non-specific band patterns, 17 of which were for small plants. The accuracy of selecting small plants based on the non-specific band was 85.0%. However, using other markers, such as SSR32871, showed that both small and large plants predominated with two bands, making it difficult to efficiently distinguish between them. Figure 5 and Figure 6 Therefore, the SSR32427 marker can accurately distinguish between small and large orchid plants and can be used for auxiliary selection of small-sized offspring of hybrid orchids. The SSR32427 marker is a 2-base repeat with a start position of 248 to 279, a sequence size of 32 bp, a repeat unit of TC, and a primer Tm value of 60.1.

[0049] Example 3: Assisted selection of small-plant-type orchid progeny using the molecular marker SSR32427 1. Selection of parent lines and obtaining hybrid fruits With the breeding goal of selecting small-plant varieties, 'Xiaofenglan' (large-plant type) was selected as the female parent and 'Dafugui' (small-plant type) as the male parent. A hybrid combination was prepared and the fruits were harvested. The combination code is 17-49.

[0050] 2. Hybrid offspring seedling production After cutting off the fruit and rinsing off surface dust with detergent, soak it in 75% alcohol for 8 minutes in a clean bench, rinse it three times with sterile water, cut the pod open with a scalpel, and sow the seeds into a culture medium (medium preparation: MS + 6-BA 0.5 mg / L + NAA 0.2 mg / L + AC 0.3 g / L + sucrose 30 g / L + carrageenan 7.5 g / L). Culture in the dark at 25℃ to obtain rhizomes, and then culture the single-seed rhizomes under light to form offspring seedlings. When the seedlings grow to 4-5 leaves, the leaves can be harvested for DNA extraction.

[0051] 3. Molecular marker-assisted selection of small orchid progeny (1) Amplification and detection: DNA was extracted from leaves of the parental lines and 57 progeny lines from combination 17-49 using the CTAB method. PCR amplification was performed using SSR32427-labeled primers (upstream sequence: 5'-AATGTGCCTTCAGGGATGAG-3', downstream sequence: 5'-TGAACGCTTTCAGTGGTCTG-3'). The PCR amplification system was 10 μL: 1 μL DNA template (50-100 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer (10 uM / μL), 0.5 μL downstream primer (10 uM / μL), 3 μL ddH2O or 5 μL (each component volume halved). The amplification procedure was the same as in Table 3. The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis at 300 V for 120-150 min. After electrophoresis, silver staining and color development were performed, and finally, the samples were placed on a white plastic plate for photographing and reading.

[0052] The measurement results are as follows Figure 7 As shown, the maternal parent 'Xiaofenglan' has distinctive bands, while the paternal parent 'Dafugui' Cymbidium goeringii does not. Among the 57 offspring, 21 have no distinctive bands and are identified as small-sized offspring; 36 have distinctive bands and are identified as large-sized offspring.

[0053] (2) Verification of plant type in offspring: The hybrid offspring were cultivated, and the plant height of the flowering offspring was measured after flowering. The results showed that most of the 36 offspring with specific bands were large-sized, and some offspring had plant types such as... Figure 8As shown. Of the 21 offspring without specific bands after testing, 17 had a plant height of less than 30 cm, resulting in an accuracy rate of 81.0% for auxiliary selection. Combined with other traits, 5 superior small-plant-type offspring were selected, such as... Figure 9 As shown.

[0054] In summary, this invention developed an SSR molecular marker primer linked to plant height from transcriptome sequencing results of orchid hybrids of different plant types. This primer can be used to assist in the selection of small-plant hybrids in orchids, with an accuracy rate greater than 80%. It allows for the selection of small-plant hybrids as early as the in vitro seedling stage of the hybrids. Combined with tissue culture rapid propagation technology, the breeding cycle can be shortened by half. Combined with observation of other breeding target traits, it can be used to select superior small-plant hybrids. Simultaneously, offspring that do not meet the breeding objectives can be directly eliminated, reducing the planting scale and improving the efficiency and benefits of small-plant hybrid orchid breeding, thereby achieving targeted, precise, economical, and efficient breeding of small-plant hybrids in orchids.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An SSR molecular marker primer for assisting in the selection of hybrid offspring of small orchid plants, characterized in that, Including upstream primer: 5'-AATGTGCCTTCAGGGATGAG-3', downstream primer: 5'-TGAACGCTTTCAGTGGTCTG-3'.

2. The application of the primers described in claim 1 in the selection of small-sized orchid plants, or in distinguishing or identifying different orchid plant types.

3. The application of the primers described in claim 1 in the preparation of products that assist in the selection and breeding of small orchid plants, or in distinguishing or identifying different orchid plant types.

4. The application of the primers described in claim 1 in the breeding of small-plant orchids.

5. A reagent kit, characterized in that, Contains the primers described in claim 1.

6. The application of the kit according to claim 5 in the selection of small-sized orchid plants, or in distinguishing or identifying different orchid plant types.

7. A method for selecting small-sized orchid plants using SSR molecular markers, characterized in that, Includes the following steps: S1. Extract genomic DNA from the parent orchids to be selected and their hybrid offspring; S2. Using DNA as a template, perform PCR amplification using the primers described in claim 1; S3. Perform gel electrophoresis on the PCR amplification products and select offspring plants with the same banding pattern as the small-type parent. S4. Single plant identification: The selected offspring are continued to be cultivated, and when they flower, the target offspring are selected based on the plant type characteristics, so that small-sized plants can be bred.

8. The method according to claim 7, characterized in that, Genomic DNA was extracted from orchid leaves in step S1.

9. The method according to claim 8, characterized in that, The PCR amplification system in step S2 is as follows: 1~2μL DNA template, 5~10μL 2×M5 PAGE Taq PCR Mix, 0.5~1μL upstream primer, 0.5~1μL downstream primer, and 3~6μL ddH2O.

10. The method according to claim 8, characterized in that, The PCR amplification program in step S2 is as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, Tm annealing for 30 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 12℃.

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