Detection method for full-period screening of germplasm of mistletoe with autumn leaves turning red

Abstract

The invention relates to development of a Quercus aliena leaf color-changing molecular marker and application of the Quercus aliena leaf color-changing molecular marker in assisted selection. Two pairs of specific primers are developed by analyzing a natural variation sequence of a promoter of a key gene DFR1 of a mistletoe autumn colored leaf anthocyanin synthesis pathway, and are used for detecting molecular markers related to mistletoe autumn colored leaves. The molecular marker can quickly and accurately screen germplasm of the mistletoe with the autumn leaves turning red in different development stages of the leaves, and the selection efficiency and accuracy of breeding of the mistletoe are remarkably improved. The invention provides an important technical means for molecular assisted breeding of the mistletoe, and has a wide application prospect.

Description

1. Technical Field

[0001] The present invention relates to the technical field of plant genetic detection, in particular to a detection method for screening the red color change of the autumn leaves of Quercus aliena throughout the whole growth period. This method is applicable to the fields of plant genetics, forest tree breeding, horticulture, and landscape plant research, aiming to screen and identify the characteristics of the red color change of the autumn leaves of Quercus aliena through scientific means, providing technical support for the breeding of excellent varieties and genetic research. 2. Background Art

[0002] Quercus aliena, as one of the typical native oaks, is rich in resources and has economic, ecological, and ornamental values.

[0003] The leaves of Quercus aliena are beautiful in shape. Their leaves change color in autumn, presenting various colors such as red, yellow, and orange, with extremely high ornamental value. They are often used in the greening construction of urban landscape and ecological tourist areas. At present, through artificial breeding, Quercus aliena resources with great ornamental value can be screened out, and the color change mechanism has been preliminarily analyzed (Anthocyanin Biosynthesis Associated with Natural Variation in Autumn Leaf Coloration in Quercus aliena Accessions. Yang X, et al., 2022). However, there are many Quercus aliena resources, but the new varieties of colorful-leaved Quercus aliena are scarce, and the resource development and application are far from sufficient, seriously restricting the development of native colorful-leaved oaks. At the same time, with the increasing attention to the ecological environment and landscape diversity, the market prospect of native colorful-leaved tree species is becoming increasingly broad. Therefore, it is urgent to breed new varieties of native colorful-leaved Quercus aliena with high ornamental value to meet the growing market demand and ecological construction needs.

[0004] Therefore, the present invention proposes an efficient detection method for screening Quercus aliena var. acuteserrata germplasms with red autumn-colored leaves throughout the whole period, which can quickly screen colorful-leaved Quercus aliena var. acuteserrata. Currently, the molecular marker-assisted breeding technology (MAS), by detecting molecular markers closely related to target traits, can indirectly screen out individuals carrying target genes, thus significantly improving breeding efficiency and selection accuracy (Application and Prospect of Molecular Marker-Assisted Selection in Crop Breeding, Guo Ying, et al., 2023). This technology is not interfered by environmental factors and can be implemented at any stage of plant growth, especially suitable for the selection of recessive traits, sex-limited traits, traits difficult to measure or traits with high costs. Existing research has successfully developed two pairs of molecular markers specifically related to leaf color, which can efficiently distinguish the genotypes of purple-leaved and red-leaved mustard, providing strong support for the molecular breeding and germplasm resource identification of mustard (Brassica juncea) (Development and Application of Molecular Markers Related to Purple Leaves of Mustard, Heng Shuangping, et al., 2024). In addition, marker-assisted selection can also effectively distinguish the differences in inflorescence size and structure between cultivated and wild species of Brassica oleracea, quickly screen out individuals with excellent inflorescence traits, and thus shorten the breeding time (Molecular Markers for Detecting Inflorescence Size of Brassica oleracea L. Crops and B. oleracea Complex Species (n = 9) Useful for Breeding of Broccoli (B. oleracea var. italica) and Cauliflower (B. oleracea var. botrytis). Treccarichi S, et al., 2023). It can be seen that MAS has been very widely applied in plant breeding. Therefore, based on the previous research on the differences in anthocyanin synthesis in the autumn-colored leaves of Quercus aliena var. acuteserrata, the present invention uses MAS technology to develop specific detection markers, realize the rapid identification and screening of Quercus aliena var. acuteserrata germplasms with red autumn-colored leaves, and promote the creation of new varieties of excellent native oaks, which has important application value. III. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to quickly screen Quercus aliena var. acuteserrata germplasms with red autumn-colored leaves, develop and utilize high-quality Quercus aliena var. acuteserrata resources, breed new varieties of Quercus aliena var. acuteserrata with ornamental value, accelerate the breeding process of native oak trees, and provide new materials for landscaping. Therefore, the present invention provides an efficient detection method for screening Quercus aliena var. acuteserrata germplasms with red autumn-colored leaves throughout the whole period. Using the developed specific primers, it can accurately identify Quercus aliena var. acuteserrata at different developmental stages of its leaves.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] The present invention first provides specific primers for detecting the red color change of the autumn leaves of Quercus aliena var. acuteserrata, which are the following 2 pairs of primers: (1) primer pair HL-check-1 composed of primers with nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2; (2) primer pair HL-check-2 composed of primers with nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4.

[0008] The above-mentioned HL-check specific primers provided by the present invention can be applied to the screening of Quercus aliena var. acuteserrata germplasms with red autumn leaves throughout the whole period, including: (1) extracting genomic DNA of the Quercus aliena var. acuteserrata sample to be tested; (2) performing PCR amplification using the above-mentioned HL-check specific primers; (3) separating the amplification products in step (2) by electrophoresis and counting the results.

[0009] Furthermore, the present invention provides a screening kit for Quercus aliena var. acuteserrata germplasms with red autumn leaves throughout the whole period, which includes a buffer solution, dNTP, Taq DNA polymerase and primers, wherein the primers are the above-mentioned Quercus aliena var. acuteserrata HL-check specific primers.

[0010] Detailed description of the overall technical solution of the present invention

[0011] By using the gene expression data and sequence alignment results of Quercus aliena var. acuteserrata germplasms with color change differences, the present invention designed and developed specific HL-check markers and carried out relevant verification.

[0012] The present invention verified three populations of Quercus aliena var. acuteserrata using two pairs of specific primers (HL-check-1 and HL-check-2). The results showed that through the PCR amplification results of HL-check-1, it is possible to judge the red color change characteristics of the autumn leaves of Quercus aliena var. acuteserrata germplasms by whether an amplification band appears. If an amplification band of 296bp appears in the PCR product, it indicates that the germplasm is a Quercus aliena var. acuteserrata germplasm with red autumn leaves, and its anthocyanin content in autumn leaves is relatively high, and the red color change effect is significant; if there is no 296bp band, it means that the germplasm does not turn red in autumn or has a poor red color change effect, and its anthocyanin content in autumn leaves is relatively low. Using the PCR amplification results of HL-check-2, it is possible to judge the red color change characteristics of the autumn leaves of Quercus aliena var. acuteserrata germplasms by whether two amplification bands appear. If two amplification bands of 176bp and 472bp appear simultaneously in the PCR product, it indicates that the germplasm is a germplasm with red autumn leaves and has a higher anthocyanin accumulation ability, and the red color change effect is more significant. If there is no 472bp band and only a 176bp band, it means that the germplasm does not turn red in autumn or has a poor red color change effect, and its anthocyanin content in autumn leaves is relatively low. IV. Description of the Drawings

[0013] Figure 1DFR1 promoter sequence alignment of two new Quercus aliena var. acuteserrata varieties

[0014] Figure 2 Specific primer site design

[0015] Figure 3 Detection of specific loci and anthocyanin content in three Quercus aliena var. acuteserrata populations

[0016] Figure 4 Analysis of anthocyanin content in Quercus aliena var. acuteserrata populations V. Specific implementation manners

[0017] The present invention will be further described below in conjunction with specific embodiments.

[0018] Example 1 Development of specific primers for Quercus aliena var. acuteserrata HL-check and their application in population resource screening

[0019] 1 Experimental materials

[0020] The experimental materials of the present invention were collected from Dadongliu Nursery, Jufeng Forest Park and Jufeng Forest Farm in Beijing respectively. Mature leaves at the color-changing stage were selected as subsequent experimental samples. The specific material source information is shown in Table 1.1.

[0021] Table 2.1 Experimental materials and their sources Table 2.1 The germplasm materials and their sources

[0022]

[0023]

[0024]

[0025] 2 Experimental methods

[0026] 2.1 Extraction of genomic DNA

[0027] According to the physiological characteristics of the materials, Quercus aliena var. acuteserrata leaves at the peak color-changing stage were collected in the field. After recording the numbers, they were immediately put into liquid nitrogen and taken back, stored at -80 °C for use. About 100 mg of fresh Quercus aliena var. acuteserrata leaves were taken, and after adding liquid nitrogen and grinding thoroughly, the modified CTAB extraction method was used to extract DNA from Quercus aliena var. acuteserrata samples.

[0028] 2.2 PCR amplification

[0029] 1) Primer design

[0030] Based on the DFR1 promoter sequence of natural variation of Quercus aliena var. acuteserrata obtained by our research group previously ( Figure 1 ), two pairs of primers were designed at the specific insertion fragment. The schematic diagram of the relative positions of the primers is shown in the figure (Figure 2 ) The specific information of the primers is shown in Table 2.

[0031] Table 2 Specific detection primers used for analysis of Q. aliena

[0032]

[0033] 2) PCR reaction system and procedure

[0034] A suitable PCR reaction system is essential for obtaining good experimental results. Based on the existing experimental foundation in the laboratory, the PCR reaction system adopted in this experiment is a 10 μl reaction system, and the specific reaction system is shown in Table 3.

[0035] Table 3 PCR reaction system

[0036]

[0037] The PCR reaction procedure is set in three parts. The first part is pre-denaturation at 95 °C for 3 minutes; the second part is pre-denaturation at 95 °C for 15 seconds, annealing at 56 °C for 15 seconds, and extension at 72 °C for 10 seconds, with 34 cycles set; the third part is final extension at 72 °C for 10 minutes. After the reaction is completed, it is detected by agarose gel electrophoresis and the band size is confirmed.

[0038] 3 Test results

[0039] 3.1 Detection of specific loci and anthocyanins in Q. aliena

[0040] When screening three Q. aliena population samples, it was found that among the Q. aliena population in Dadongliu Nursery, 17 samples showed different characteristics: Using the HL-check-1 specific primer for detection, it was found that 5 of the samples had an insertion of a 296 bp fragment, and their anthocyanin content was higher than 100 mg / g; while the other 12 samples did not detect the insertion of this fragment; the detection results of the HL-check-1 specific primer also showed that the above 5 samples simultaneously had two fragments of 472 bp and 176 bp, while the other 12 samples only detected the 176 bp band. In the Q. aliena population in Jiufeng Forest Park, using the HL-check-2 specific primer for detection, it was found that all 9 samples only detected the 176 bp band, and their anthocyanin content was relatively low. Among the 16 Q. aliena samples in Jiufeng Forest Farm, the detection results of the HL-check-2 specific primer showed that 5 samples simultaneously had two fragments of 472 bp and 176 bp, with insertion and anthocyanin higher than 100 mg / g, and the remaining 11 samples only detected the 176 bp band (Figure 3 )。

[0041] 3.2 Analysis of Anthocyanin Content in Quercus aliena var. acuteserrata Populations

[0042] Based on the above detection data, we divided the Quercus aliena var. acuteserrata populations into two groups and conducted a correlation analysis of anthocyanin content and the presence of fragment insertions. The t-test results showed that the anthocyanin content of individuals with fragment insertions was significantly higher than that of individuals without fragment insertions (P < 0.001), indicating a significant correlation between fragment insertions and anthocyanin content( Figure 4 )。

Claims

1. A method for detecting a specific site of Quercus aliena, comprising the following steps: Extract DNA samples from oak leaves; Perform PCR amplification on DNA samples using specific primers; The presence of the target fragment in the PCR product was detected by gel electrophoresis; If the target fragment is detected, it is determined that the Quercus sample has the specific site.

2. The method according to claim 1, wherein the specific primers are a primer pair HL-check-1 consisting of primers whose nucleotide sequences are shown in SEQ ID No.1 and SEQ ID No.2, and a primer pair HL-check-2 consisting of primers whose nucleotide sequences are shown in SEQ ID No.3 and SEQ ID No.

4.

3. A method for quantitatively detecting anthocyanin content in Quercus serrata leaves, comprising the following steps: Collect leaf samples of Quercus truncatula at the peak of color change and remove the veins; The leaf samples were added to 1% (V / V) hydrochloric acid-methanol solution for extraction; Incubate at 4°C in the dark for 24 hours; The supernatant was separated by centrifugation, and the absorbance was measured at 530 nm and 657 nm using a UV-visible spectrophotometer; The anthocyanin content was calculated according to the formula.

4. The method according to claim 3, wherein the anthocyanin content is calculated as follows: Anthocyanin content = (A530-A657×calibration factor) / fresh weight.

Images