Pav molecular marker closely linked with mei flower and branch character and its detection method and application

Abstract

The present application relates to the field of molecular biology and plant molecular breeding technology, and particularly relates to a PAV molecular marker closely linked with plum-blossom weeping trait, and a detection method and application thereof. The molecular marker is located at 1016141-1017922 base pairs of chromosome 7 of the genome of 'Fen-tai weeping' variety. The molecular marker of the present application is present in plum-blossom weeping trait individuals, and is absent in plum-blossom straight trait individuals. The molecular marker has good repeatability and high accuracy in plum-blossom weeping / straight trait identification of plum-blossom seedling cultivation populations, can realize molecular assisted selection of plum-blossom weeping / straight trait at the seedling stage, and is helpful to shorten the breeding period and improve the breeding efficiency. Based on the PAV molecular marker, the present application further provides a plum-blossom weeping trait regulatory gene Pmu_chr7_2839, which lays a solid foundation for the regulation and genetic breeding of plum-blossom weeping trait.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant molecular breeding technology, and in particular to PAV molecular markers closely linked to the drooping branch trait of plum blossoms, their detection methods and applications. Background Technology

[0002] Plum blossom (Prunus mume Sieb. et Zucc.) belongs to the genus Prunus in the family Rosaceae. It is one of China's ten most famous flowers, valued for both its ornamental and edible qualities. Because of its early spring bloom, the plum blossom is associated with a spirit of perseverance. Thousands of years of cultivation and breeding have greatly enriched the variety of ornamental plum blossoms; currently, over 300 varieties are known, each with different fragrances, flower shapes, leaf colors, and plant forms. Plum trees primarily exhibit two plant forms: upright and weeping. The weeping form, with its branches curving towards the ground, possesses unique ornamental value.

[0003] Among the Prunus genus, many ornamental trees exhibit weeping forms, including weeping peach, weeping cherry, and weeping plum. Significant progress has been made in the study of the tree form of Prunus species in terms of hormones and physiology (Nakamura et al., 1994; Werner and Chaparro, 2005; Yoshida et al., 1999). Yoshida proposed that GA (growth glycosides) inhibits downward growth in weeping cherry branches by forming stiff trunks (Yoshida et al., 1999). Furthermore, Sugano found that the expression level of the GA3 oxidase gene in the elongation zone of weeping cherry was higher than that in upright cherry, indirectly proving that the weeping trait in weeping cherry is related to GA (Sugano et al., 2004). Werner and Chaparro studied the interaction between columnar and weeping forms in peach trees, finding that the columnar trait exhibited a recessive epistatic relationship with the weeping trait, and a new arching trait appeared in the F2 generation of hybrids between columnar peach and weeping peach (Werner and Chaparro, 2005). Current research suggests that the drooping branches of weeping cherry and weeping peach in the genus *Prunus* are related to gravitational glycosides (GA), possibly because GA affects a certain stage of secondary growth in plants. Hollender, using BSA, discovered that a 1.8 kb deletion at the 5' end of the peach Ppa013325 gene resulted in the weeping trait in peach. Further RNAi analysis confirmed that Ppa013325 affects lateral branch growth and may be a potential gene for plants to sense or respond to gravity (Hollender et al., 2018).

[0004] Traditional methods of plum blossom variety improvement typically rely on propagation and selection, which are time-consuming, labor-intensive, and inefficient. With the development of molecular marker technology, PAV (pivot-assisted breeding) has become a highly efficient and precise approach. Using PAV molecular marker technology to study the drooping trait of plum blossoms not only helps to deepen our understanding of its genetic regulatory mechanisms but also provides technical support and methodological guidance for the efficient and precise breeding of new varieties with superior drooping traits. Summary of the Invention

[0005] To address the current lack of elucidation of the genetic regulatory mechanism of drooping branches in plum blossoms, the absence of molecular marker-assisted breeding technology, and the low breeding efficiency of drooping plum blossoms, this invention reveals the genetic mechanism of drooping branches through plum blossom pan-genome, PAV-GWAS, and transcriptome sequencing technologies. It mainly provides a PAV molecular marker closely related to the drooping branch trait of plum blossoms, primers for detecting the molecular marker, and a method for detecting the drooping / straight branch trait of plum blossoms.

[0006] Based on this, the following invention is proposed.

[0007] In a first aspect, the present invention provides a PAV molecular marker associated with the drooping trait of plum blossoms, which is located at base pairs 1016141 to 1017922 on chromosome 7 of the genome of the 'Fentai Drooping' variety.

[0008] The polymorphism of the molecular markers is manifested in the presence or absence of fragments.

[0009] If a segment from chromosome 7 (1016141 to 1017922 bp) is missing in the genome of the 'Fentai Chuizhi' variety, the plum blossoms will exhibit straight branches; if the segment is present, the plum blossoms will exhibit drooping branches.

[0010] Preferably, the molecular marker is obtained by amplification using the primers shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Secondly, the present invention provides an isolated or purified nucleic acid molecule containing a nucleic acid sequence as shown in SEQ ID NO.3.

[0012] The nucleic acid molecule is the plum blossom weeping branch trait regulatory gene Pmu_chr7_2839.

[0013] Thirdly, the present invention provides primers for amplifying the molecular marker or the nucleic acid molecule.

[0014] Preferably, the primers are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0015] Fourthly, the present invention provides a reagent or kit containing the nucleic acid molecule or the primers described herein.

[0016] Fifthly, the present invention provides the use of the said molecular marker, said nucleic acid molecule, said primer, or said reagent or kit in at least one of the following aspects:

[0017] (1) Detecting the characteristics of drooping branches in plum blossoms;

[0018] (2) Selecting or cultivating plum blossoms;

[0019] (3) Preparation of transgenic plum blossoms.

[0020] In a sixth aspect, the present invention provides a method for detecting the drooping branch trait of plum blossoms, comprising: using the genomic DNA of the plum blossom to be tested as a template, and performing PCR amplification using the primers described above.

[0021] Preferably, if the amplification product contains a nucleic acid sequence as shown in SEQ ID NO.3 or a gel electrophoresis band corresponding to the nucleic acid sequence, the morphology is drooping; if not, the morphology is straight.

[0022] Preferably, the PCR reaction program is as follows: 94℃~95℃, 3~5min; 94℃~95℃, 30~40s; 97℃~98℃, 30~40s; 72℃~74℃, 30~40s, for 35~40 cycles.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a PAV molecular marker associated with the drooping branch trait in plum blossoms, along with its detection method and application. The molecular marker of this invention is present in all individuals exhibiting the drooping branch trait in plum blossoms, but absent in those exhibiting the upright branch trait. This molecular marker demonstrates good repeatability and high accuracy in identifying drooping / upright branch traits in plum blossom seedling populations, enabling molecular-assisted selection of drooping / upright branch traits during the seedling stage, thus helping to shorten the breeding cycle and improve breeding efficiency. Based on this PAV molecular marker, this invention also provides the key regulatory gene Pmu_chr7_2839 for the drooping branch trait in plum blossoms, laying a solid foundation for the regulation and genetic breeding of this trait. Attached Figure Description

[0025] Figure 1 This is a line graph showing the significant difference in gene frequencies between the drooping and upright branch populations. The red line represents genes with higher frequencies in the drooping branch population, while the blue line represents genes with higher frequencies in the upright branch population.

[0026] Figure 2This is a Manhattan diagram of PAV-GWAS gene localization for the binary trait (Bin), branching angle during the growth period (A1), and branching angle during the dormant period (A2). Chr1-Chr8 represent chromosomes 1-8 of 'Pink Terrace Weeping Branch', respectively; Sca represents 'Pink Terrace Weeping Branch' scaffolds that failed to attach to chromosomes; Tor represents new sequences assembled from Prunus cerasifera in the pangenome; and Pangenome represents new sequences assembled de novo using resequencing data.

[0027] Figure 3 This is a Manhattan diagram of PAV-GWAS gene localization at branching angles T1 to T5 in the growth direction. Chr1-Chr8 represent chromosomes 1-8 of the 'Pink Terrace Weeping Branch', respectively; Sca represents the scaffold of the 'Pink Terrace Weeping Branch' that failed to attach to the chromosome; Tor represents a new sequence in the pangenome that represents a new sequence assembled from Prunus cerasifera; and Pangenome represents a new sequence assembled de novo using resequencing data.

[0028] Figure 4 This is a gel electrophoresis image of the PCR amplification products, where: M is the marker, 1-11 are 'single-pink weeping branch', 'pink platform weeping branch', 'eagle peak weeping branch', 'double green weeping branch', 'pink cinnabar', 'Jiangmei', 'golden sheng', 'six-petal pink', 'black feather jade', 'fragrant snow palace pink', and 'early green calyx', respectively. Nos. 1-4 show weeping branches, and nos. 5-11 show straight branches.

[0029] Figure 5 This is a graph showing the difference in gene expression levels between extreme drooping branches and extreme straight branches in seedlings and hybrid populations based on the quantitative fluorescence experiment Pmu_chr7_2839. In the graph, DFJ represents the stem of 'single pink drooping branch', DFJJ represents the stem tip of 'single pink drooping branch'; FTJ represents the stem of 'pink platform drooping branch'; FTJJ represents the stem tip of 'pink platform drooping branch'; SBJ represents the stem of 'double green drooping branch', SBJJ represents the stem tip of 'double green drooping branch'; JMJ represents the stem of 'Jiangmei', JMJJ ​​represents the stem tip of 'Jiangmei'; JSJ represents the stem of 'Jinsheng', JSJJ represents the stem tip of 'Jinsheng'; ZLJ represents the stem of 'early green calyx', ZLJJ represents the stem tip of 'early green calyx'; WJ represents the extreme drooping branch stem in the hybrid population, WJJ represents the stem tip of the extreme drooping branch in the hybrid population; UJ represents the extreme straight branch stem in the hybrid population, UJJ represents the stem tip of the extreme straight branch in the hybrid population. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0032] Example 1: PAV molecular marker mining

[0033] 1. Genome assembly and pangenome assembly of 'Pink Terrace Weeping Branch'

[0034] Genome sequencing materials were collected from the Wuhan Moshan Plum Garden. Leaves of 'Fentai Weeping Branch' were collected for genomic DNA extraction, while leaves, flowers, and stems were used for RNA extraction. Genomic DNA was sequenced using PacBio to obtain high-quality HiFireads. Preliminary assembly using Hifiasm (Cheng et al., 2021) yielded a contig-level genome draft. Fine assembly of the genome was performed using Hi-C technology and second-generation data-assisted error correction, ultimately obtaining the chromosome-level 'Fentai Weeping Branch' genome.

[0035] The plum resequencing data downloaded from NCBI had a sequencing depth of 19× and included 13 cultivar groups (weeping plum, apricot plum, Longyou plum, Gongfen plum, cinnabar plum, single-petal plum, jade butterfly plum, green calyx plum, jumping branch plum, wild plum, yellow fragrance plum, beauty plum, and fruit plum). De novo assembly of all individual individuals from 348 plum resequencing data downloaded from NCBI was performed using MaSuRCA (Zimin et al., 2013). Using the 'Fentai weeping plum' reference genome as a template, after alignment, filtering, and redundancy removal steps, non-redundant and uncontaminated non-reference sequences were finally obtained. The downloaded Longyou plum genome and wild plum genome were compared with the 'Fentai weeping plum' reference genome to obtain PVs sequences. Finally, the reference genome, the PVs sequences obtained from multi-version genome alignment, and the non-reference sequences obtained from second-generation sequencing data assembly were merged together to form the 'Fentai weeping plum' pan-genome.

[0036] 2. PAV-GWAS, transcriptome and gene frequency analysis

[0037] Using bwa (http: / / bio-bwa.sourceforge.net / ), the 348 resequencing data were re-aligned to the pangenome. Genes present in all individuals were defined as core genes, genes present in 99%–100% of individuals were defined as softcore core genes, genes present in 1%–99% of individuals were defined as shell genes, and genes present in 1% or less of individuals were defined as cloud genes (Gao et al., 2019).

[0038] Based on this, gene frequencies were calculated for other upright-branch plum and weeping-branch populations. Specifically, the frequencies of all genes in the 'Pink Terrace Weeping' pangenome were calculated in both populations, identifying genes in the weeping-branch population that were significantly higher or lower than those in other upright-branch plum populations. Six genes had high frequencies in the upright-branch population, while seven genes had high frequencies in the weeping-branch cultivar group. Figure 1 ).

[0039] Using the F1 generation of the hybrid 'six-petaled' and 'pink-platform weeping' plum blossoms as research material, 20 individuals with extreme weeping branches and 20 with straight branches were randomly selected. Tender stems and buds from each individual were collected, and transcriptome sequencing was performed on the materials. Differential expression analysis was conducted based on the 'pink-platform weeping' genome. Using 348 plum blossom seedling populations as research material, all populations were marked as binary traits (Bin) according to weeping and straight branches for GWAS mapping. Additionally, 214 plum blossom populations were selected, and the weeping trait was decomposed into 7 sub-traits: branching angle during the growth period (A1), branching angle during dormancy (A2), and the angle between the growth direction from the base to the tip and the direction of load-bearing force (T1-T5) divided into five equal parts. GWAS analysis of the weeping trait was performed based on PAV data.

[0040] Analysis results as follows Figure 2 and Figure 3 As shown, based on the combined results of PAV and GWAS, three candidate genes, Pmu_chr7_2434, Pmu_chr7_2456, and Pmu_chr7_2839, were screened. Quantitative real-time PCR verification revealed that only Pmu_chr7_2839 showed differential expression at the shoot tips of drooping and straight branches.

[0041] Example 2: PAV molecular marker PCR and quantitative real-time PCR verification

[0042] 1. Experimental materials

[0043] Young leaves from seedling populations were collected using the following varieties: 'Single Pink Weeping Branch', 'Pink Terrace Weeping Branch', 'Eagle Peak Weeping Branch', 'Double Green Weeping Branch', 'Pink Cinnabar', 'Jiangmei', 'Golden Sheng', 'Six-Petal Pink', 'Black Lobe Jade', 'Fragrant Snow Palace Pink', and 'Early Green Calyx'.

[0044] 2. Extraction of genomic DNA and total RNA

[0045] Genomic DNA was extracted according to the instructions of the DNAsecure Novel Plant Genomic DNA Extraction Kit (DP320-2) from Tiangen Biotech Co., Ltd. Total RNA was extracted using the EASYspin Plus Polysaccharide Polyphenol / Complex Plant RNA (RN5301) Rapid Extraction Kit from Beijing Adley Biotechnology Co., Ltd. Nucleic acid samples that passed electrophoresis and NanoDrop 2000 testing were used for PCR amplification and quantitative real-time PCR experiments.

[0046] 3. Validation using Pmu_chr7_2839 labeling and quantitative real-time PCR.

[0047] PCR amplification was performed using primers SEQ ID NO.1 and SEQ ID NO.2, and the amplification products were detected by 1% agarose gel electrophoresis.

[0048] The PCR reaction system is shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] The PCR reaction program was as follows: 95℃, 3 min; 95℃, 30 sec; 98℃, 30 sec; 72℃, 30 sec, for 40 cycles.

[0053] The results are as follows Figure 4 As shown, the results indicate that the Pmu_chr7_2839 gene exists in the weeping varieties 'Danfen Chuizhi', 'Fentai Chuizhi', 'Jiufeng Chuizhi', and 'Shuangbi Chuizhi' (corresponding to 1-4 respectively), but not in the straight-branch varieties 'Fenhong Zhusha', 'Jiangmei', 'Jinsheng', 'Liubanfen', 'Wuyuyu', 'Xiangxuegongfen', and 'Zaolve' (corresponding to 5-11 respectively).

[0054] Using the F1 progeny of 'Six-Petal' × 'Pink Terrace Weeping Branch', including extreme straight and extreme weeping branches, as well as 'Early Green Calyx', 'Eagle Peak Weeping Branch', 'Single Pink Weeping Branch', and 'Double Green Weeping Branch' as ​​materials, the expression levels of three candidate genes, Pmu_chr7_2434, Pmu_chr7_2456, and Pmu_chr7_2839, were verified by real-time quantitative PCR. The results showed that Pmu_chr7_2434 and Pmu_chr7_2456 were not expressed in the tissues, while only Pmu_chr7_2839 showed a difference in expression levels between the straight and weeping branch populations.

[0055] Based on the results of the fluorescence quantitative experiment, as follows Figure 5 As shown, white represents the drooping branch population and black represents the straight branch population. The expression level of the Pmu_chr7_2839 gene in the hybrid population is more than 7 times higher in the extreme drooping branches than in the extreme straight branches, and the expression level of the drooping branch population in the seedlings is 2 to 3 times higher than that in the straight branch population.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PAV molecular marker associated with the weeping branch trait of plum blossoms, characterized in that, It is located at base pairs 1016141 to 1017922 on chromosome 7 of the 'Fentai Chuizhi' variety genome; the molecular marker was obtained by amplification using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. An isolated or purified nucleic acid molecule, characterized in that, It is the nucleic acid sequence shown in SEQ ID NO.

3.

3. A reagent or kit, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. The use of the molecular marker of claim 1, the nucleic acid molecule of claim 2, or the reagent or kit of claim 3 in at least one of the following aspects: (1) Detect the drooping branch characteristics of plum blossoms; (2) Screening or cultivating plum blossoms with drooping branches.

5. A method for detecting the drooping branch trait of plum blossoms, characterized in that, include: Using the genomic DNA of the plum blossom to be tested as a template, PCR amplification was performed using the primers shown in SEQ ID NO.1 and SEQ ID NO.2; If the amplified product contains a gel electrophoresis band the size of the amplified product obtained by the primers shown in SEQ ID NO.1 and SEQ ID NO.2, the morphology is drooping; if it does not exist, the morphology is straight.

6. The detection method according to claim 5, characterized in that, The PCR reaction program was as follows: 94℃~95℃, 3~5 min; 94℃~95℃, 30~40 s; 97℃~98℃, 30~40 s; 72℃~74℃, 30~40 s, for 35~40 cycles.

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