A method for identifying F1 hybrid of interspecific hybridization between betula nigra and betula fukienensis by SSR molecular marker

Abstract

The application provides a method for identifying F1 hybrids of interspecific cross of Betula nigra and Betula fukienensis by using SSR molecular markers, and relates to the technical field of biology.The application is a method for identifying F1 hybrids of interspecific cross of Betula nigra and Betula fukienensis by using SSR molecular markers, which uses the SSR marker technology to perform PCR amplification on the DNA of F1 hybrids of interspecific cross of Betula, uses capillary electrophoresis to detect the amplification products, and combines the bands and the plant morphology to determine the authenticity of the F1 hybrids.The SSR marker primer provided by the application has good repeatability and strong specificity;the method has the advantages of high efficiency, low cost, short cycle and early screening, can replace the traditional hybrid identification method, realizes accurate identification of F1 hybrids of interspecific cross of Betula nigra and Betula fukienensis, and has important practical application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for identifying F1 hybrids of interspecific hybrids of *Betula spp.* and *Betula fruticosa* using SSR molecular markers. Background Technology

[0002] Birch (Betula) is a genus within the family Betulaceae of the order Fagales, comprising approximately 100 species. It is primarily distributed in the cold temperate and temperate regions of the Northern Hemisphere, with a few species found in the Arctic Circle and subtropical mountainous regions. Birch wood has a wide range of uses, fine texture, and excellent processing properties, giving it high economic value. Interspecific hybridization is an important method for combining the superior traits of different species to cultivate superior plant varieties. River beech originates from North America and possesses strong stress resistance; Fujian beech is mainly distributed in Fujian province, is relatively fast-growing, has straight trunks, excellent wood quality, and a high yield. Interspecific hybridization between River beech and Fujian beech holds promise for obtaining superior birch varieties that combine stress resistance and high timber yield. Rapidly and accurately identifying true interspecific hybrid offspring of birch is fundamental to birch genetic breeding. SSR molecular markers are widely used for germplasm resource identification and kinship determination, but their application in identifying interspecific hybrids between River beech and Fujian beech has not yet been reported.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying F1 hybrids of interspecific hybrids between *Betula spp.* and *Betula fruticosa* using SSR molecular markers. This method is a rapid, simple, time-saving, and accurate method for hybrid identification, and can be effectively applied to the identification of the authenticity of F1 hybrids between *Betula spp.* and *Betula fruticosa*.

[0005] The objective of this invention is achieved by the following technical solution:

[0006] The method for identifying F1 hybrids from interspecific crosses between *Betula spp.* and *Betula fukiensis* using SSR molecular markers is as follows:

[0007] Step 1: DNA extraction from F1 hybrids and their parents from interspecific hybridization;

[0008] Step 2: Design specific SSR primers, establish the PCR reaction system and amplification reaction procedure;

[0009] Step 3: Use the Qsep100 fully automated nucleic acid and protein analysis system to perform capillary electrophoresis detection on the SSR amplification products;

[0010] Step 4, Identification of true hybrids: Analyze the results of capillary electrophoresis. If the interspecific hybrid F1 hybrid is found to have amplification bands of the parents or amplification bands specific to the father, it is judged to be a true hybrid.

[0011] Step 5: Observe the hybrid morphology to confirm the authenticity of the interspecific hybrid: Observe and count the leaf and lenticel morphological traits of the interspecific hybrid F1 hybrids. If the hybrid is found to have paternal-specific traits or new traits that neither parent has, then the authenticity of the hybrid is confirmed.

[0012] The SSR marker primers used for birch hybrid identification are as follows:

[0013] Primer number: FJH-29, upstream primer: 5'-CCATTCCAGTTAGCCACGAT-3' (SEQ ID NO.1), downstream primer: 5'-ATGGCAAGTAGATTCCTGCG-3' (SEQ ID NO.2);

[0014] Primer number: FJH-65, upstream primer: 5'-AAGATGCTAATGCCGATGGT-3' (SEQ ID NO.3), downstream primer: 5'-TGCCATCAACCCTATTCACC-3' (SEQ ID NO.4);

[0015] Primer number: FJH-67, upstream primer: 5'-GGCGCAAATCAAACGATAAT-3' (SEQ ID NO.5), downstream primer: 5'-GCTCCGTTGAATCCAGTCTC-3' (SEQ ID NO.6).

[0016] Specifically, in step 1, leaves of interspecific hybrid F1 hybrids and parental plants are frozen and ground with liquid nitrogen, and DNA is extracted using the CTAB method. The DNA concentration and purity are detected using a spectrophotometer, and the integrity of the DNA is detected by 1% agarose gel electrophoresis.

[0017] Specifically, in step 2, the SSR reaction system is as follows: 1 μL template DNA (100 ng / μL), 5 μL 2×Premix Taq, 0.2 μL upstream primer (10 μmol), 0.2 μL downstream primer (10 μmol), and 3.6 μL ultrapure water.

[0018] Specifically, in step 2, the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and finally 72℃ extension for 10 min.

[0019] Specifically, in step 3, the capillary electrophoresis detection method is as follows: Take a 96-well plate containing PCR amplification products, add sterile water to each well to prevent it from drying out (for example, take a 96-well plate containing PCR amplification products, add 10 μL of sterile water to each well in rows A, B, and C, add 20 μL of sterile water to each well in rows D, E, and F, and add 30 μL of sterile water to each well in rows G and H), and then place the 96-well plate in the Qsep100 fully automated nucleic acid and protein analysis system. The electrophoresis parameters are set as follows: loading voltage 4 kV, loading time 10 s, sample separation voltage 6 kV, sample separation time 300 s, detection length range 15–1000 bp, resolution 2–4 bp, and Size Marker and Alignment Marker C109100-100A (Taiwan Guangding).

[0020] The Qsep100 fully automated nucleic acid and protein analysis system is from Taiwan Guangding.

[0021] Specifically, in step 4, the capillary electrophoresis results are analyzed as follows: (1) Using the alignment marker as a template, the peak values ​​of the size marker and sample peak table at 20bp and 1000bp are corrected; (2) The size marker peak table is opened, a reference scale is selected, fragment calculation is performed, and the size marker and sample peak values ​​are calibrated; (3) A new comparison file is created, the capillary electrophoresis results of the parent and offspring are selected, "Signal Alignment" and "Time Alignment" are integrated, the longitudinal offset of the signal between each sample is adjusted to 16, and the peak values ​​of the SSR sites are read for identification.

[0022] Specifically, if an interspecific hybrid F1 hybrid is detected to have amplification bands of the parents or a specific amplification band of the father, it is determined to be a true hybrid.

[0023] Specifically, in step 5, the authenticity of the hybrid is confirmed: the differences in leaves and lenticels between the interspecific F1 hybrids and the parents are observed and statistically analyzed. If the hybrid is found to have a paternal-specific trait or a new trait that neither parent has, it is determined to be a true hybrid.

[0024] The present invention has the following beneficial effects:

[0025] This invention discloses a method for identifying F1 hybrids from interspecific hybrids of *Betula spp.* and *Betula fruticosa* using SSR molecular markers. The method involves PCR amplification of the DNA of the F1 hybrids using SSR markers, detection of the amplified products using capillary electrophoresis, and confirmation of the F1 hybrid's authenticity by combining banding and plant morphology analysis. The SSR marker primers provided in this invention exhibit good repeatability and high specificity. The method itself offers advantages such as high efficiency, low cost, short cycle time, and early screening capabilities. It can replace traditional hybrid identification methods, enabling accurate identification of F1 hybrids from interspecific hybrids of *Betula spp.* and has significant practical application value. Attached Figure Description

[0026] The present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Figure 1 Image of DNA from F1 hybrids obtained through interspecific hybridization, detected by 1% agarose gel electrophoresis.

[0028] Figure 2 Amplification peak diagrams and electrophoresis images of interspecific hybrids F1 hybrids 17 and 24 and their parent FJH-29.

[0029] Figure 3 Amplification peak diagrams and electrophoresis images of interspecific hybrids F1 hybrids 17 and 24 and their parent FJH-65.

[0030] Figure 4 Amplification peak diagrams and electrophoresis images of interspecific hybrids F1 hybrids 17 and 24 and their parent FJH-67.

[0031] Figure 5 Comparison of leaves of F1 hybrids from interspecific hybridization and their parents. Detailed Implementation

[0032] Example

[0033] Step 1: DNA extraction from F1 hybrids and their parents from interspecific hybridization;

[0034] Specifically, leaves from the F1 hybrids and parental lines were cryogenically ground in liquid nitrogen, and DNA was extracted using the CTAB method. DNA concentration and purity were detected using a spectrophotometer, and DNA integrity was assessed by 1% agarose gel electrophoresis. Results are as follows: Figure 1 As shown.

[0035] Step 2: Design specific SSR primers, establish the PCR reaction system and amplification reaction procedure;

[0036] Specifically, the primers are:

[0037] Primer number: FJH-29, upstream primer: 5'-CCATTCCAGTTAGCCACGAT-3' (SEQ ID NO.1), downstream primer: 5'-ATGGCAAGTAGATTCCTGCG-3' (SEQ ID NO.2);

[0038] Primer number: FJH-65, upstream primer: 5'-AAGATGCTAATGCCGATGGT-3' (SEQ ID NO.3), downstream primer: 5'-TGCCATCAACCCTATTCACC-3' (SEQ ID NO.4);

[0039] Primer number: FJH-67, upstream primer: 5'-GGCGCAAATCAAACGATAAT-3' (SEQ ID NO.5), downstream primer: 5'-GCTCCGTTGAATCCAGTCTC-3' (SEQ ID NO.6).

[0040] Specifically, the SSR reaction system consisted of: 1 μL template DNA (100 ng / μL), 5 μL 2×Premix Taq, 0.2 μL upstream primer (10 μmol), 0.2 μL downstream primer (10 μmol), and 3.6 μL ultrapure water.

[0041] Specifically, the PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and finally 72℃ extension for 10 min.

[0042] Step 3: Use the Qsep100 fully automated nucleic acid and protein analysis system to perform capillary electrophoresis detection on the SSR amplification products;

[0043] Specifically, the capillary electrophoresis detection method is as follows: Take a 96-well plate containing PCR amplification products, add 10 μL of sterile water to each well in rows A, B, and C, 20 μL of sterile water to each well in rows D, E, and F, and 30 μL of sterile water to each well in rows G and H. Then place the 96-well plate on the Qsep100 fully automated nucleic acid and protein analysis system (Taiwan Guangding). The electrophoresis parameters are set as follows: loading voltage 4 kV, loading time 10 s, sample separation voltage 6 kV, sample separation time 300 s, detection length range 15–1000 bp, resolution 2–4 bp, and Size Marker and Alignment Marker C109100-100A (Taiwan Guangding).

[0044] Step 4, Identification of true hybrids: Analyze the results of capillary electrophoresis. If the interspecific hybrid F1 hybrid is found to have amplification bands of the parents or amplification bands specific to the father, it is determined to be a true hybrid.

[0045] Specifically, the capillary electrophoresis results were analyzed as follows: (1) Using the alignment marker as a template, the peak values ​​of the size marker and sample peak table at 20bp and 1000bp were corrected; (2) The size marker peak table was opened, a reference scale was selected, fragment calculations were performed, and the size marker and sample peak values ​​were calibrated; (3) A new comparison file was created, the capillary electrophoresis results of the parent and offspring were selected, and “Signal Alignment” and “Time Alignment” were integrated. The longitudinal offset of the signal between each sample was adjusted to 16, and the peak values ​​of the SSR sites were read for identification.

[0046] Step 5: Observe the hybrid morphology to further confirm the authenticity of the interspecific hybrid; observe and count the morphological traits such as leaves and lenticels of the interspecific hybrid F1 hybrids. If the hybrids are found to have paternal-specific traits or new traits not present in either parent, the authenticity of the hybrids will be further confirmed.

[0047] like Figure 2 The maternal parent, *Betula spp.*, showed a characteristic peak and band at 229 bp, while the paternal parent, *Betula fruticosa*, showed a characteristic peak and band at 239 bp. The interspecific hybrids F1, hybrids 17 and 24, showed characteristic peaks and bands at 229 bp and 239 bp, respectively, indicating that the interspecific hybrids F1, hybrids 17 and 24, simultaneously possess the characteristic peaks and bands of both parents, especially the characteristic peak and band of the paternal parent, *Betula fruticosa*, at 239 bp (indicated by the arrow). Based on this, they are determined to be true hybrids.

[0048] like Figure 3 The maternal parent, *Betula spp.*, showed a characteristic peak and band at 217 bp, while the paternal parent, *Betula fruticosa*, showed a characteristic peak and band at 188 bp. The interspecific hybrids F1, hybrids 17 and 24, showed characteristic peaks and bands at 217 bp and 188 bp, respectively, indicating that the interspecific hybrids F1, hybrids 17 and 24, simultaneously possess the characteristic peaks and bands of both parents, especially the characteristic peak and band of the paternal parent, *Betula fruticosa*, at 188 bp (indicated by the arrow). Based on this, they are determined to be true hybrids.

[0049] like Figure 4 The maternal parent, *Betula spp.*, showed a characteristic peak and band at 225 bp, while the paternal parent, *Betula fruticosa*, showed a characteristic peak and band at 244 bp. The interspecific hybrids F1, hybrids 17 and 24, showed characteristic peaks and bands at 225 bp and 244 bp, respectively, indicating that the interspecific hybrids F1, hybrids 17 and 24, simultaneously possess the characteristic peaks and bands of both parents, especially the characteristic peak and band of the paternal parent, *Betula fruticosa*, at 244 bp (indicated by the arrow). Based on this, they are determined to be true hybrids.

[0050] like Figure 5 Among them, the maternal parent, *Betula spp.*, has fewer leaf margin serrations, which are sparsely distributed and relatively blunt; the paternal parent, *Betula fukiensis*, has more leaf margin serrations, which are densely distributed and relatively sharp; the interspecific hybrid F1 hybrid has leaf margin serrations, distribution, and sharpness that are all between the parents, representing a new trait different from the parents.

[0051] Table 1. Lenticulum density of F1 hybrids from interspecific hybridization and stems of the maternal parent *Betula spp.* and paternal parent *Betula fruticosa*.

[0052]

[0053] As shown in Table 1, the lenticel density of the mother plant, *Betula spp.*, is 114.24 lenticels / cm³. 2 The lenticel density of the paternal parent, *Birchia fruticosa*, is 7.49 lenticels / cm². 2 The lenticel density of the interspecific hybrid F1 was 43.10 lenticels / cm². 2 Between the maternal and paternal parents, an analysis of variance was performed on the lenticel density of three birch varieties. The results showed that the significance value (p value) was 4.3846E-10, indicating that there was a significant difference in lenticel density among the three species: Fujian birch, river birch, and interspecific hybrids. In other words, lenticel density is a new trait that is different from the parent species.

[0054] In summary, based on the comparison results of SSR molecular markers and morphological traits, it is proven that interspecific hybrids F1 hybrids 17 and 24 are true hybrids of *Betula spp.* and *Betula fruticosa*.

Claims

1. A method for identifying F1 hybrids of interspecific hybrids between *Betula spp.* and *Betula fruticosa* using SSR molecular markers, characterized in that... The steps are as follows: Step 1: DNA extraction from F1 hybrids and their parents from interspecific hybridization; Step 2: Design specific SSR primers, establish the PCR reaction system and amplification reaction procedure; Step 3: Use the Qsep100 fully automated nucleic acid and protein analysis system to perform capillary electrophoresis detection on the SSR amplification products; Step 4, Identification of true hybrids: Analyze the results of capillary electrophoresis. If the interspecific hybrid F1 hybrid is found to have amplification bands of the parents or amplification bands specific to the father, it is judged to be a true hybrid. Step 5: Observe the hybrid morphology to confirm the authenticity of the interspecific hybrid: Observe and count the leaf and lenticel morphological traits of the interspecific hybrid F1 hybrids. If the hybrid is found to have paternal-specific traits or new traits that neither parent has, then the authenticity of the hybrid is confirmed. In step 2, the primers are designed as follows: Primer number: FJH-29, upstream primer: 5'-CCATTCCAGTTAGCCACGAT-3', downstream primer: 5'-ATGGCAAGTAGATTCCTGCG-3'; Primer number: FJH-65, upstream primer: 5'-AAGATGCTAATGCCGATGGT-3', downstream primer: 5'-TGCCATCAACCCTATTCACC-3'; Primer number: FJH-67, upstream primer: 5'-GGCGCAAATCAAACGATAAT-3', downstream primer: 5'-GCTCCGTTGAATCCAGTCTC-3'.

2. The method for identifying the F1 hybrid of the interspecific hybrid between Betula nigra and Betula fargesiana by SSR molecular markers according to claim 1, wherein, In step 1, leaves of interspecific hybrid F1 hybrids and parental plants were frozen and ground in liquid nitrogen, and DNA was extracted using the CTAB method. The DNA concentration and purity were detected using a spectrophotometer, and the integrity of the DNA was detected by 1% agarose gel electrophoresis.

3. The method for identifying the F1 hybrid of the interspecific hybrid between Betula nigra and Betula fargesiana by SSR molecular markers according to claim 1, characterized in that, In step 2, the SSR reaction system consisted of: 1 μL of template DNA solution with a concentration of 100 ng / μL, 5 μL of 2 × PremixTaq, 0.2 μL of upstream primer solution with a concentration of 10 μmol, 0.2 μL of downstream primer solution with a concentration of 10 μmol, and 3.6 μL of ultrapure water.

4. The method for identifying the F1 hybrid of the interspecific hybrid between Betula nigra and Betula fargesiana by SSR molecular markers according to claim 1, wherein, In step 2, the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and finally 72℃ extension for 10 min.

5. The method for identifying F1 hybrids of interspecific hybrids of *Betula spp.* and *Betula fruticosa* using SSR molecular markers as described in claim 1, characterized in that... In step 3, the capillary electrophoresis detection method is as follows: Take a 96-well plate containing PCR amplification products, add sterile water to each well to prevent it from drying out, and then place the 96-well plate in the Qsep100 fully automated nucleic acid and protein analysis system. The electrophoresis parameters are set as follows: loading voltage 4kV, loading time 10s, sample separation voltage 6kV, sample separation time 300s, detection length range 15~1000bp, resolution 2~4bp, and size marker and alignment marker C109100-100A. The Qsep100 fully automated nucleic acid and protein analysis system is from Taiwan Guangding.

6. The method for identifying F1 hybrids of interspecific hybrids of *Betula spp.* and *Betula fruticosa* using SSR molecular markers as described in claim 1, characterized in that... Step 4, analysis of capillary electrophoresis results: (1) Using the alignment marker as a template, correct the peak values ​​of the size marker and sample peak table at 20bp and 1000bp; (2) Open the size marker peak table, select the reference scale, perform fragment calculation, and complete the calibration of the size marker and sample peak; (3) Create a new comparison file, integrate "SignalAlignment" and "Time Alignment", adjust the longitudinal offset of the signal between each sample to 16, and read the peak value of the SSR site for identification.

7. The method for identifying F1 hybrids of interspecific hybrids of *Betula spp.* and *Betula fruticosa* using SSR molecular markers as described in claim 1, characterized in that... In step 5, the authenticity of the hybrid is confirmed: the differences in leaves and lenticels between the interspecific F1 hybrids and the parents are observed and statistically analyzed. If the hybrid is found to have a paternal-specific trait or a new trait that neither parent has, it is determined to be a true hybrid.

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