An InDel molecular marker combination for identifying lemon nucellar embryo variation and application thereof

Abstract

This invention relates to the field of molecular marker technology, specifically to an InDel molecular marker combination for identifying nucellar embryo variations in lemons and its application. For two lemon varieties, Yunning 1 and Ferminalo, variety-specific InDel sites were identified based on whole-genome resequencing data: heterozygous deletions / insertions were found at Chr1:3756654 and Chr2:29145651 in the former, and heterozygous insertions were found at Chr1:949641 and Chr1:8872916 in the latter. Based on these, four pairs of specific primers (YN-InDel-1 / 2, FM-InDel-1 / 2) were designed. PCR amplification was performed using the sample's genomic DNA as a template, and the results were detected by agarose gel electrophoresis. The size and number of amplified bands distinguished the maternal parent from nucellar embryo variation materials. This invention offers high identification accuracy, low cost, and simple operation, effectively solving the technical challenge of distinguishing between zygotic and nucellar embryos in lemon hybridization breeding. It is suitable for large-scale early screening of seedlings.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to an InDel molecular marker combination for identifying variations in lemon nucellar embryos and its applications. Background Technology

[0002] Citrus fruits are among the world's most economically valuable fruit trees. After nearly two decades of rapid development, China's citrus industry has become the world's largest. With rising consumption levels, the market demands greater diversity in new citrus varieties. However, for the past thirty years, the breeding of new citrus varieties in my country has primarily relied on bud mutation and seedling selection, with hybridization accounting for a very low percentage. The fundamental reason for this phenomenon lies in the widespread apomixis, or polyembryony, in citrus. Polyembryony, also known as nucellar embryo, refers to an embryo that develops from the nucellus tissue rather than from the zygote. This asexual embryo results in offspring that are genetically identical to the maternal parent, severely hindering the process of citrus hybridization breeding.

[0003] Studies have shown that the nucellar embryo development rate varies significantly among different citrus varieties, with some varieties exhibiting a relatively high rate. Due to its polyembryonic nature, a single citrus seed can produce multiple asexual embryos developing from nucellar cells. These embryos do not undergo meiosis or fertilization; they develop directly from somatic cells into seeds, essentially representing asexual clones of the maternal parent, with a genetic background consistent with the mother. While this characteristic is beneficial for the stable inheritance of desirable traits, it also makes it difficult to distinguish between zygotic and nucellar embryos in hybrid offspring, significantly increasing the difficulty and workload of screening in hybridization breeding.

[0004] Currently, morphological marker methods are commonly used to identify citrus nucellar embryo variations. Preliminary screening relies on phenotypic characteristics such as leaf shape, flower shape, fruit shape, and tree form, combined with growth habits and physiological characteristics for a comprehensive assessment. However, the number of usable morphological markers is limited and easily affected by environmental conditions, making it difficult to guarantee the accuracy and stability of the identification results. This is especially true for materials with similar genetic backgrounds, where morphological markers often fail to provide accurate differentiation. Furthermore, while existing molecular marker technologies can assist identification to some extent, they generally suffer from poor universality and cannot accurately differentiate between specific varieties and their nucellar embryo variations.

[0005] Therefore, there is an urgent need in this field to develop a method for accurately, economically, and efficiently identifying variant materials in lemon nucellar embryos. Developing specific molecular markers for specific lemon varieties to achieve precise differentiation between the maternal parent and its nucellar embryo variants is of great significance for improving the efficiency of lemon hybridization breeding and accelerating the process of new variety selection. This invention is based on the resequencing data of two lemon varieties, Yunning 1 and Ferminalo, and their nucellar embryo variants. Through bioinformatics analysis, variety-specific InDel sites were obtained, and molecular markers capable of rapidly distinguishing between the maternal parent and nucellar embryo variants were developed, providing an effective technical means for the precise identification of lemon nucellar embryo variants. Summary of the Invention

[0006] The purpose of this invention is to provide an InDel molecular marker combination for identifying nucellar embryo variants in lemons and its application. For the first time, high-quality, high-specificity InDel marker primers designed based on resequencing data of Yunli 1 and Ferminalau lemons and their nucellar embryo variant materials are presented. A total of four primer pairs are designed to rapidly and accurately distinguish 'Yunli 1' and 'Ferminalau' from their nucellar embryo variant materials. The molecular markers and marker amplification primers of this invention can be easily and quickly applied to scientific research or production practices of related lemon varieties. Simultaneously, it provides an effective method and approach for identifying nucellar embryo variant materials of other multiembryonic citrus varieties and lays a solid foundation for this work.

[0007] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution: An InDel molecular marker ensemble for identifying variations in lemon nucellar embryos, comprising markers developed based on resequencing data from *Lemon 1* and *Lemon Ferminalo* and their nucellar embryo variation materials: The first InDel molecular marker used to distinguish Yunli No. 1 lemon from Yunli No. 1 nucellar embryo variants; and The second InDel molecular marker used to distinguish Ferminalo lemon from Ferminalo nucellar embryo variants.

[0008] Furthermore, the first InDel molecular marker includes the following two InDel molecular markers: lemon reference genome Chr1: 3756654 site and lemon reference genome Chr2: 29145651 site; The second InDel molecular marker includes the following two InDel molecular markers: lemon reference genome Chr1: 949641 site and lemon reference genome Chr1: 8872916 site.

[0009] The principle behind the InDel molecular marker combination described in this invention for distinguishing between the maternal parent and nucellar embryo variant materials is as follows: Through whole-genome resequencing and alignment analysis, InDel sites with heterozygous deletions or insertions in the maternal parent (Yunning 1 or Ferminalo) relative to its nucellar embryo variant materials are screened. In the maternal parent, because this site is heterozygous, PCR amplification produces two characteristic bands of different lengths (double bands); while the nucellar embryo variant materials, developed from the nucellar cells of the maternal parent through somatic cell asexual reproduction, become homozygous at this site due to loss of heterozygosity (LOH), producing only one characteristic band after PCR amplification (single band). Therefore, through simple PCR amplification and agarose gel electrophoresis detection, the maternal parent and its nucellar embryo variant materials can be rapidly and accurately identified based on the number and size of the amplified bands.

[0010] On the other hand, the present invention proposes a set of primer pairs for amplifying the above-mentioned InDel molecular markers, the primer pair combinations comprising: At least one pair of primers for amplifying the first InDel molecular marker; and At least one pair of primers used to amplify the second InDel molecular marker.

[0011] Furthermore, the primer pair used to amplify the first InDel molecular marker is selected from at least one of the following primer pairs: Primer pair YN-InDel-1: The forward primer sequence is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; Primer pair YN-InDel-2: The forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The primer pair used to amplify the second InDel molecular marker is selected from at least one of the following primer pairs: Primer pair FM-InDel-1: The forward primer sequence is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; Primer pair FM-InDel-2: The forward primer sequence is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8.

[0012] On the other hand, the present invention proposes a kit for identifying variations in lemon nucellar embryos, the kit comprising the aforementioned primer pair combination.

[0013] On the other hand, the present invention proposes a method for identifying variations in lemon nucellar embryos, comprising the following steps: S1: Extract genomic DNA from the lemon sample to be tested; S2: Using the genomic DNA extracted in step S1 as a template, perform PCR amplification using the primer pair combination described above; S3: Detect the amplification products from step S2, and determine whether the lemon sample to be tested is the parent plant or a nucellar embryo variant material based on the fragment size of the amplification products.

[0014] Furthermore, the PCR amplification using the above-mentioned primer pair combination includes: amplification using primer pairs for amplifying the first InDel molecular marker, and determining whether the sample to be tested is Yunli No. 1 lemon or Yunli No. 1 nucellar embryo variant material based on whether the first characteristic band appears in the amplification product.

[0015] Furthermore, the first feature band is 521bp or 394bp, wherein the 521bp band corresponds to the Yun Ning No. 1 pearl embryo variant material, and the 394bp band corresponds to the Yun Ning No. 1 lemon.

[0016] Furthermore, the PCR amplification using the above-mentioned primer pair combination includes: amplification using primer pairs for amplifying the second InDel molecular marker, and determining whether the sample to be tested is Ferminerau lemon or Ferminerau nucellar embryo variant material based on whether the second characteristic band appears in the amplification product.

[0017] Furthermore, the second characteristic band is 381bp or 465bp, wherein the 381bp band corresponds to Ferminerau pearl embryo variant material, and the 465bp band corresponds to Ferminerau lemon.

[0018] The beneficial effects of this invention are: This invention does not employ conventional SSR or random amplification markers. Instead, it performs whole-genome resequencing on Yunning 1, Ferminalo, and their respective nucellar embryo variants. Through FastQC quality control, BWA alignment, GATK 4.2.0 variant detection, and IGV 2.10.3 visualization verification, it identifies truly existing InDel differential loci between the maternal parent and the variant materials. Specifically, Yunning 1 exhibits a 127bp heterozygous deletion at Chr1:3756654 and a 59bp heterozygous insertion at Chr2:29145651; Ferminalo exhibits an 84bp heterozygous insertion at Chr1:949641 and a 160bp heterozygous insertion at Chr1:8872916. The key characteristic of these loci is that the maternal parent is heterozygous, while the nucellar embryo variants are homozygous due to loss of heterozygosity (LOH) during somatic cell asexual reproduction. Based on this, the four primer pairs (YN-InDel-1 / 2, FM-InDel-1 / 2) produced distinctly different banding patterns after PCR amplification: the maternal parent showed double bands due to heterozygosity at the locus, while the nucellar embryo variant material showed only a single band due to homozygosity. In the comparative examples, published SSR markers (cAGG9, Ma2-1556, Mest88) and the citron-specific marker XY-InDel-3 could not distinguish between the maternal parent and the nucellar embryo variant material, directly demonstrating the irreplaceable accuracy of the variety-specific InDel markers of this invention in terms of identification.

[0019] Traditional morphological identification relies on the phenotypic characteristics of mature seedlings, such as tree structure, leaf shape, flower shape, and fruit shape (e.g., Figure 4 (As shown), this process takes several years and is subject to environmental interference. This invention operates entirely at the DNA level: after extracting genomic DNA from the sample, PCR amplification is performed using 2×Taq plus master mix, 35 cycles, annealing at 55°C. The amplified products are then genotyped by 2.5% agarose gel electrophoresis. This system requires no fluorescent labeling, capillary electrophoresis, or sequencing, and has low hardware requirements. As described in the examples, each milliliter of 2×Taq plus master mix costs 15 yuan and can amplify 100 samples, with a single sample reagent cost of only 0.15 yuan. This allows breeding units to conduct early screening of thousands of hybrid offspring seedlings without significantly increasing their budgets, precisely investing limited resources in a very small number of hybrid individuals developing zygotic embryos, significantly improving the scale efficiency and resource utilization of lemon hybrid breeding.

[0020] Morphological markers are easily influenced by cultivation environment, tree age, nutritional status, and observer experience. Especially when the genetic background of the maternal parent and the variant material is highly similar, reliable identification based solely on phenotypic differences in leaves and fruits is difficult. This invention directly uses agarose gel electrophoresis bands as the interpretation criterion, with clear and unique standards: For Yunling 1, a single 521bp band amplified by YN-InDel-1 primers indicates a nucellar embryo variant material, while a double band of 521bp+394bp indicates the maternal parent; a single 303bp band amplified by YN-InDel-2 primers indicates a variant material, while a double band of 362bp+303bp indicates the maternal parent. For Ferminalo, a single 381bp band amplified by FM-InDel-1 primers indicates a variant material, while a double band of 465bp+381bp indicates the maternal parent; a single 327bp band amplified by FM-InDel-2 primers indicates a variant material, while a double band of 487bp+327bp indicates the maternal parent. The interpretation model is based on the stable genomic characteristic of heterozygosity inherent in the maternal InDel locus, which is unaffected by environmental factors or plant growth status. Therefore, completely consistent identification results can be obtained between different operators and laboratories. For institutions that need to conduct lemon hybridization breeding across regions and years, this ensures the uniformity of identification standards and the comparability of data, providing a standardized technical foundation for the identification of nucellar embryo variant materials.

[0021] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A schematic diagram illustrating the visualization of site variation information using IGV 2.10.3 software; Figure 2 A schematic diagram of genome sequence alignment at a specific location; Figure 3 A schematic diagram showing the identification results of primers YN-InDel-1, YN-InDel-2, FM-InDel-1, and FM-InDel-2; Figure 4 Schematic diagram of leaf and fruit phenotypes of lemon and its nucellar embryo variants; Figure 5 This is a schematic diagram of the identification results for XY-InDel-3 labeled primers; Figure 6 This is a schematic diagram of the identification results for SSR marker primers. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0025] The InDel molecular marker combinatorial used to identify variations in lemon nucellar embryos described in this embodiment includes markers developed based on resequencing data from Yunli No. 1 and Ferminalo lemons and their nucellar embryo variation materials, comprising: (1) In this invention, the Yunli No. 1 and Ferminalo lemon and their nucellar embryo variants were resequencing. Two pairs of InDel-labeled primers were designed for Yunli No. 1 and Ferminalo lemon respectively, for a total of 4 pairs. The primer information was sent to Youkang Biotechnology Co., Ltd. for primer synthesis.

[0026] (2) Using the two primer pairs YN-InDel-1 and YN-InDel-2 provided for Yun Ning No. 1 in (1) above, PCR amplification was performed on Yun Ning No. 1 and its nucleus embryo variant material. Both InDel primer pairs were able to distinguish Yun Ning No. 1 from the nucleus embryo variant material. The PCR amplification was performed in a 20 μL system. Due to the high quality and high specificity of the primer design, the relatively inexpensive 2 x Taq plus master mix could be used for amplification (each mix tube contains 1 ml, costs 15 yuan, and can identify 100 samples).

[0027] (3) Further use of the two primer pairs FM-InDel-1 and FM-InDel-2 provided by (1) to perform PCR amplification on Ferminalo and its nucellar embryo variants. The amplification products were detected by 2.5% agarose gel electrophoresis. Based on the amplification results, the unique InDel markers of Ferminalo nucellar embryo variants were screened, and it was determined that these two primer pairs could significantly distinguish Ferminalo and its nucellar embryo variants. A total of 4 effective primer pairs were obtained that can be used to identify lemon nucellar embryo variants. Example 2

[0028] Primer design and validation for YN-InDel-1, YN-InDel-2, FM-InDel-1, and FM-InDel-2 Material selection The test materials were Yun Ning No. 1 Ferminalo and the seed nucellar embryo variants of both from the Ruili Station Citrus Germplasm Resource Garden of the Institute of Tropical and Subtropical Economic Crops, Yunnan Academy of Agricultural Sciences (Table 1). Figure 3 , Figure 5 , Figure 6 The numbers 1 to 6 in the table correspond one-to-one with the citrus varieties represented by the numbers in Table 1. Genomic DNA of the citrus varieties was extracted using the CTAB method. The extracted DNA from the lemon material was then purified to meet the quality requirements for resequencing.

[0029] Table 1 Summary of information on citrus and nucellus embryo variants YN-InDel-1, YN-InDel-2, FM-InDel-1, and FM-InDel-2 labeling design and PCR amplification validation Using the published Taiwan lemon genome (Yu et al. Horticulture Research, 2024) as a reference genome, bioinformatics methods were employed to analyze the resequencing data of Yunli No. 1 Ferminal and nucellar embryo variants. FastQC software was used for quality control and filtering of the raw resequencing data, removing adapter sequences and low-quality reads. BWA software was then used to align the quality-controlled reads to the Taiwan lemon genome. GATK 4.2.0 software was used for whole-genome InDel variant detection and gvcf file merging and filtering. The vcf files containing InDel information were sorted according to InDel fragment size to determine variant site information, and IGV 2.10.3 software was used for further visualization to verify the variant information. Figure 1 ).

[0030] For Yunning No. 1 and its nucleus embryo variants, variant sites 1 and 2 were screened, and the site information was retrieved for sequence alignment. Figure 2The results showed that, compared to YN-MT, YN-WT had a 127 bp heterozygous deletion at mutation site 1 (Chr1: 3756654), i.e., the 127 bp YN-InDel-1 gene sequence at Chr1: 3756654 was GCCAATTGGAGTTTGGGGCAAATTACTGATATTTTCTGTCAATAAATCCCCACCTTGAACTTGATAACTGGCTACAGAAATGTCCTTCTCTTCTTCCAAATTATCCTGTTCAGCAGAACTTCGAGCC; YN-WT had a 59 bp heterozygous insertion at mutation site 2 (Chr2: 29145651), i.e., the 59 bp YN-InDel-2 gene sequence at Chr2: 29145651 was ATTAGGACAAGTAATGATTAATGAATTATTTGATAATGAGTTAATTTGTGTTTAATAAC. Two specific, high-quality primer pairs were designed upstream and downstream of YN-InDel-1 and YN-InDel-2 (Table 2).

[0031] For Ferminalo and its nucellar embryo variants, variant sites 1 and 2 were screened, and the site information was retrieved for sequence alignment. Figure 2 The results showed that, compared to FM-MT, FM-WT had an 84 bp heterozygous insertion at mutation site 1 (Chr1: 949641), specifically, the 84 bp FM-InDel-1 gene sequence at Chr1: 949641 was TGAAAAACCAAGCATCTAACATCTCATTCCACCATTCCTTGATTTCCAATCATGCATAGATTCAAATCTTTTCAAGTCATTTGT; FM-WT also had a 160 bp heterozygous insertion at mutation site 2 (Chr1: 8872916), specifically, the 160 bp heterozygous insertion at Chr1: 8872916... The FM-InDel-2 gene sequence of bp is TCATATATATATAGAAAATTTTCAATCTAGAATTTTAAAATGCAGGAACGCTTTAAAACCCTGCCCGTTTTAATGCTTTAATTCCATAATCTTTAAAACCATGTAATTTTAAAGTTTTTCTGGATTTTTCCGCATATATATATATATTATATACATATAA. Two pairs of specific, high-quality primers were designed upstream and downstream of FM-InDel-1 and FM-InDel-2 (Table 2).

[0032] The effectiveness of InDel-labeled primer pairs was screened. PCR amplification was performed on YN-WT, YN-MT and FM-WT, FM-MT materials, and the amplified bands were detected by electrophoresis. High-quality, specific differentially expressed InDel markers, such as Yunli No. 1 and Ferminalo, were selected as candidate primers for identifying effective nucellar embryo variant materials. The specific operational steps are as follows: The PCR reaction system consisted of 20 µL of components: 10.0 µL 2X Taq Mix, 0.5 µL F (forward primer 10 mmol / L), 0.5 µL R (reverse primer 10 mmol / L), 1.0 µL DNA (template 300 ng / L), and 8.0 µL ddH2O. The PCR program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 10 min. The product was then stored at 4℃. After PCR amplification, 8 μL of the amplified product was electrophoresed on a 2.5% agarose gel (0.5×TBE buffer), developed using Goldview (Guangzhou Jianyang Biotechnology Co., Ltd.), and observed under a gel imaging system. Figure 3 As shown, YN-InDel-1 and YN-InDel-2 can both significantly distinguish YN-WT from YN-MT; FM-InDel-1 and FM-InDel-2 can both significantly distinguish FM-WT from FM-MT.

[0033] PCR amplification results using YN-InDel-1 primers showed that when a 521 bp line appeared on the agarose gel (CATAAGCACCTTGGACTTGAGTGCTGCCTGAAACATTTCTTACATATCGATCTGGGCTATAAAACTTTGGCAAATTACTTGGCGTTGGAGAAGAAACTTTGGTACTTTCTTCTGATAAATCAGTGCCATTCAATTGATTGCATGTAACAAAAATATCTCTTACATCTTGAGGTGGATGAGAAAACCTCGGCAGCCAATTGGAGTTTGGGGCAAATTACTGATATTTTCTGTCAATAAATCCCCACCTTGAACTTGATAACTGGCTACA), When the bands of GAAATGTCCTTCTCTTCTTCCAAATTATCCTGTTCAGCAGAACTTCGAGCCCCAACTGGGTTTGCAGTAAGGCGATTGATGTTTTCTTCACACTCATCAGCACCTCTAACCTTTTCACTAACTACAGATTCGTCTGTGGCATCATTTCCGCTCTTTCTGACCACCACTTGGAAACCCAATTGTGCTTTTAGAAGTGAAAATTAAAAGATTCTTTCCACTTTCAATAATTGGTTCTCTCTTCTCATGGAC) are found, it is determined to be a variant material of Yunli No. 1 pearl embryo.When a 521 bp and a 394 bp band (CATAAGCACCTTGGACTTGAGTGCTGCCTGAAACATTTCTTACATATCGATCTGGGCTATAAAACTTTGGCAAATTACTTGGCGTTGGAGAAGAAACTTTGGTACTTTCTTCTGATAAATCAGTGCCATTCAATTGATTGCATGTAACAAAAATATCTCTTACATCTTGAGGTGGATGAGAAAACCTCGGCACCAACTGGGTTTGCAGTAAGGCGATTGATGTTTTCTTCACACTCATCAGCACCTCTAACCTTTTCACTAACTACAGATTCGTCTGTGGCATCATTTCCTCGCTCTTTCTGACCACCACTTGGAAACCCAATTGTGCTTTTAGAAGTGAAAATTAAAAGATTCTTTCCACTTTCAATAATTGGTTCTCTCTTCTCATGGAC) are displayed on the agarose gel, it is identified as Yunli No. 1.

[0034] YN-InDel-2 primer PCR amplification results showed that when a 303 bp band (CTCACTTGCTAGCAAATGGCGTTGACTGATTAATTTAAGTCATTGTATTGGAAGGTCTATATAGTCAAAGACGGTGGGCGAGTGATAAATTTGATAATGAGTTAATTTGTGTTTAATAACGTCATTAATTATTTTCCATGATATTAATGAATTATTGCAGAGACGCTTATAAGGTCTACACCACATATTGAGTATCGACTAACATTATTGCAGAGACGCTTGTTTTGTTAATTACTAGATCGATTTTTCCTTAATCATGAAGTTAATCATAAAAATTTGAATATTTTTGCGGGGAAATTTAAG) was displayed on the agarose gel, it was identified as a variant material of Yunli No. 1 nucleus embryo; when a 362 bp band was displayed on the agarose gel, it was identified as a variant material of Yunli No. 1 nucleus embryo. BP GTAATGATTAATGAATTATTTGATAATGAGTTAATTTGTGTTTAATAACGTCTACACCACATATTGAGTATCGACTAACATTATTGCAGAGACGCTTGTTTTGTTAATTACTAGATCGATTTTTCCTTAATCATGAAGTTAATCATAAAAATTTGAATATTTTTGCGGGGAAATTTAAG) and a 303 bp band, it was determined to be Yun Ning No. 1.

[0035] FM-InDel-1 primer PCR amplification results showed that when a 381 bp band (CATCATGGCCTTTTCCACTCATTATAGAATGATACAAACAAAATGCTGCGTGTTTATATAAATGCATGTATTCACCAGTCATCACTTACAAAATGGGCAATGAACTTCACTATTACAATTCGCACGGGACAGTAATGTACATATGAATTGCAATTTACTCAAATTGCCTGCTGAAACGGACATTCTCCGTTGAAGCATATTAGTGTTCAGAATCATATGATACTGATTTAGGAAGAAATGGCAGCCCGTTTTCAACTGATCCAACCAAAATCATCTTTTGGAGAGGAAAGATGGTATGCTTCAAGAAACACAGTTTTGATAATAAGCAAAACGTATTTACGAGAGTCAGATACACTCCAAGCATGTAGCTAAGACTGTTCTG) was displayed on the agarose gel, it was identified as Ferminalo nucellar embryo variant material; when a 465 bp band was displayed on the agarose gel, it was identified as Ferminalo nucellar embryo variant material. BP CCAATCATGCATAGATTCAAATCTTTTCAAGTCATTTGTTGAAGCATATTAGTGTTCAGAATCATATGATACTGATTTAGGAAGAAATGGCAGCCCGTTTTCAACTGATCCAACCAAAATCATCTTTTGGAGAGGAAAGATGGTATGCTTCAAGAAACACAGTTTTGATAATAAGCAAAACGTATTTACGAGAGTCAGATACACTCCAAGCATGTAGCTAAGACTGTTCTG) and a 381 bp band, it was determined to be Fermi-resistant.

[0036] FM-InDel-2 primer PCR amplification results showed that when a 327 bp band (GCCAAAATGTTTAGGGAAGCAATTGCAGGTCAATTAAATTTCTTTTTTTTTTTTAATTGATTTTTTTTTCAGTTTTTTCTAAATTTATGACCTTGGTGTGAATGAGAATTTCTAAATTTCTCCACACAATTGTTTATAAGAAGGATCCCTATTTTTAAAACTAATCAATATTTGATATATAATGTCATTTTAACTTCCTTCAGTTTAATTATCTCAAGGTACTCAACCACCGTGGCAAGTTATTTTTGTCATATGTATCAACTTCTCAAGTTTTTAAACACTTGAAAGTAGAGATGAAGTTTTGTGAGTTTAGATACTTAGGTATTG) was displayed on the agarose gel, it was identified as Ferminalo nucellar embryo variant material; when a 487 bp band was displayed on the agarose gel, it was identified as Ferminalo nucellar embryo variant material. BP CGTTTTAATGCTTTAATTCCATAATCTTTAAAACCATGTAATTTTAAAGTTTTTCTGGATTTTTCCGCATATATATATATATTATATACATATAAAATGTCATTTTAACTTCCTTCAGTTTAAT TATCTCAAGGTACTCAACCACCGTGGCAAGTTATTTTTGTCATATGTATCAACTTCTCAAGTTTTTAAACACTTGAAAGTAGAGATGAAGTTTTGTGAGTTTAGATACTTAGGTATTG) and a 327 When the bp band is detected, it is identified as Fermineron.

[0037] Table 2. Information on InDel primers used for identifying variations in lemon nucleus embryos. Comparative Example 1 Existing SSR markers and citron InDel markers cannot distinguish between nucleus embryo variants. Initially, a hybridization pollination combination using Yun Ning No. 1 Ferminalo as the female parent and citron as the male parent was conducted at the Ruili Station of the Yunnan Academy of Agricultural Sciences. Later, two hybrid populations were obtained through seed propagation. The hybrid populations were observed and recorded according to the "Specifications and Data Standards for Citrus Germplasm Resources Description," and two superior lines, YN-MT and FM-MT, were ultimately selected for propagation. Because lemons exhibit zygotic embryo / nucleate embryo phenomena, YN-MT and FM-MT may be nucleate embryo seedlings (…). Figure 4 ).

[0038] Based on the XY-InDel-3 marker primer pair previously developed by the team (the development and application of an InDel molecular marker for identifying citron, for which no patent has been published yet), PCR amplification and verification were then performed using the XY-InDel-3 marker primer pair for XY, YN-WT, FM-WT, YN-MT, FM-MT, and GDX. Figure 5 The amplified products were electrophoresed in a 2.5% agarose gel (0.5×TBE buffer) and observed under a gel imaging system after being developed with Goldview. The bands of YN-MT, FM-MT, and the maternal parents YN-WT, FM-WT, and GDX were of the same size and clearly distinguishable from the paternal parent XY. This indicates that YN-MT and FM-MT share the same genetic background as their maternal parents and are non-hybrids.

[0039] To further confirm that YN-MT and FM-MT are nucellar embryo variants rather than zygotic embryos, PCR amplification was performed on YN-WT, FM-WT, YN-MT, and FM-MT using published SSR markers cAGG9, Ma2-1556, and Mest88 for verification. Figure 6 The band sizes of YN-MT and FM-MT are the same as those of the maternal parents YN-WT and FM-WT. YN-MT and FM-MT both have the same genetic background as their maternal parents and are therefore non-hybrids. The specific operational steps are as follows: According to the above embodiments, genomic DNA from 6 citrus materials was obtained. The DNA was qualified by agarose gel electrophoresis, and the concentration was detected by spectrophotometer. The concentration was then diluted to 300 ng / ul for later use.

[0040] The PCR reaction system consisted of 20 µL of components: 10.0 µL 2X Taq Mix, 0.5 µL F (forward primer 10 mmol / L), 0.5 µL R (reverse primer 10 mmol / L), 1.0 µL DNA (template 300 ng / L), and 8.0 µL ddH2O. The PCR program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 10 min. The mixture was then stored at 4℃.

[0041] From the above embodiments, it can be concluded that the Indel molecular marker for identifying lemon nucellar embryo variants described in this invention can be used rapidly and accurately for identifying Yunli No. 1 Ferminalo nucellar embryo variant materials, especially for identifying variant materials with obvious fruit papillae and increased leaf shape index. Figure 4 ).

[0042] In summary, this invention proposes an InDel molecular marker combination for identifying nucellar embryo variants in lemons and its application. For two lemon varieties, Yunning 1 and Ferminalo, variety-specific InDel sites were identified based on whole-genome resequencing data: heterozygous deletions / insertions were found at Chr1:3756654 and Chr2:29145651 in the former, and heterozygous insertions were found at Chr1:949641 and Chr1:8872916 in the latter. Based on these, four pairs of specific primers (YN-InDel-1 / 2, FM-InDel-1 / 2) were designed. PCR amplification was performed using the sample's genomic DNA as a template, and the maternal parent and nucellar embryo variant materials could be distinguished by the size and number of amplified bands, as detected by agarose gel electrophoresis. This invention offers high accuracy, low cost, and simple operation, effectively solving the technical challenge of distinguishing between zygotic and nucellar embryos in lemon hybridization breeding, and is suitable for large-scale early screening of seedlings.

[0043] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A set of InDel molecular markers for identifying variations in lemon nucellar embryos, characterized in that, The molecular marker combination includes: The first InDel molecular marker used to distinguish Yunli No. 1 lemon from Yunli No. 1 nucellar embryo variants; and The second InDel molecular marker used to distinguish Ferminalo lemon from Ferminalo nucellar embryo variants.

2. The InDel molecular marker combination as described in claim 1, characterized in that, The first InDel molecular marker includes the following two InDel molecular markers: lemon reference genome Chr1: 3756654 site and lemon reference genome Chr2: 29145651 site; The second InDel molecular marker includes the following two InDel molecular markers: lemon reference genome Chr1: 949641 site and lemon reference genome Chr1: 8872916 site.

3. A set of primer pairs for amplifying the InDel molecular marker as described in claim 1 or 2, characterized in that, The primer pair combinations include: At least one pair of primers for amplifying the first InDel molecular marker; and At least one pair of primers used to amplify the second InDel molecular marker.

4. The primer pair combination as described in claim 3, characterized in that, The primer pair used to amplify the first InDel molecular marker is selected from at least one of the following primer pairs: Primer pair YN-InDel-1: The forward primer sequence is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; Primer pair YN-InDel-2: The forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The primer pair used to amplify the second InDel molecular marker is selected from at least one of the following primer pairs: Primer pair FM-InDel-1: The forward primer sequence is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6; Primer pair FM-InDel-2: The forward primer sequence is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.

8.

5. A kit for identifying variations in lemon nucellar embryos, characterized in that, The kit contains the primer pair combination as described in claim 3 or 4.

6. A method for identifying variations in lemon nucellar embryos, characterized in that, Includes the following steps: S1: Extract genomic DNA from the lemon sample to be tested; S2: Using the genomic DNA extracted in step S1 as a template, perform PCR amplification using the primer pair combination as described in claim 3 or 4; S3: Detect the amplification products from step S2, and determine whether the lemon sample to be tested is the parent plant or a nucellar embryo variant material based on the fragment size of the amplification products.

7. The method as described in claim 6, characterized in that, The PCR amplification using the primer pair combination as described in claim 3 or 4 includes: amplification using the primer pair for amplifying the first InDel molecular marker, and determining whether the sample to be tested is Yunli No. 1 lemon or Yunli No. 1 nucellar embryo variant material based on whether the first characteristic band appears in the amplification product.

8. The method as described in claim 7, characterized in that, The first feature band is 521bp or 394bp, where the 521bp band corresponds to the Yun Ning No. 1 pearl embryo variant material, and the 394bp band corresponds to the Yun Ning No. 1 lemon.

9. The method as described in claim 6, characterized in that, The PCR amplification using the primer pair combination as described in claim 3 or 4 includes: amplification using primer pairs for amplifying the second InDel molecular marker, and determining whether the sample to be tested is Ferminerau lemon or Ferminerau nucellar embryo variant material based on whether a second characteristic band appears in the amplification product.

10. The method as described in claim 9, characterized in that, The second characteristic band is 381bp or 465bp, where the 381bp band corresponds to Ferminerau pearl embryo variant material and the 465bp band corresponds to Ferminerau lemon.

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