A method for parentage identification of prunus plant

Abstract

The application discloses a parent-offspring identification primer group and method of Prunus. The parent-offspring identification primer group of Prunus disclosed by the application is composed of 30 single-stranded DNAs shown in sequence 1 to sequence 30 in a sequence list. The primer group and method of the application can identify the genetic relationship of Prunus armeniaca, Prunus dulcis and their offspring, establish a parent-offspring identification system, and are simple to operate. The primer group and method of the application can quickly obtain genetic information of Prunus parents and offspring and determine the source of the offspring, can early determine and retain hybrid offspring between Prunus armeniaca and Prunus dulcis at the seedling stage, which is beneficial to quickly determine the true outcrossing seedling at the seedling stage, thereby removing hybrid offspring caused by natural pollination and eliminating the hybrid offspring at the seedling stage, so as to save land resources, reduce the identification cost in the later period, speed up the breeding process, and also provide a reference for parent-offspring identification of other fruit plants.

Description

Technical Field

[0001] This invention relates to the field of Prunus genus breeding technology, specifically a method for parentage identification in Prunus genus plants. Background Technology

[0002] Both *Prunus armeniaca* and *Prunus spp.* belong to the *Prunus* genus, but they are distantly related, exhibiting significant hybrid vigor. Therefore, distant hybridization between *Prunus armeniaca* and *Prunus spp.* is currently an important method for obtaining superior *Prunus armeniaca* germplasm. However, due to their distant kinship, obtaining hybrid offspring through distant hybridization is extremely difficult. To improve hybridization efficiency, the so-called "emasculation" method is used for pollination. However, because this method does not involve bagging for isolation, it is necessary to perform paternity testing on the assumed paternal parent to eliminate the influence of natural pollination and determine whether the obtained hybrid seedlings are truly distant hybrids between *Prunus armeniaca* and *Prunus spp.*. With the development of molecular technology, the main methods currently used for paternity testing include microsatellite markers (SSR), SNP markers, and mitochondrial markers. Among these, SSR marker technology is considered the preferred method for paternity testing. SSR markers exhibit high polymorphism, co-dominant inheritance, and conform to Mendelian segregation laws. The technique is simple to operate and highly reliable. It has been widely used for paternity testing in humans and animals with good results. However, research on its application in plant paternity testing is relatively limited. Microsatellite markers have been widely used in resource identification and genetic breeding research of apricot and almond, as well as in genetic diversity research, variety identification, kinship analysis and genetic linkage map construction. However, their application in parentage testing has not yet been reported. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to identify the parents of hybrid offspring of apricot and almond.

[0004] To address the aforementioned technical problems, this invention first provides a primer set, which consists of the following primers named UDP98-406-P, UDP98-409-P, UDP98-411-P, UDP98-412-P, P04-P, BPPCT029-P, BPPCT030-P, BPPCT039-P, Pchcms4-P, Pchgms4-P, PaCITA15-P, PaCITA7-P, UDAp422-P, UDAp404-P, and AMPA095-P.

[0005] The UDP98-406-P consists of two single-stranded DNA molecules named as Sequence 1 and Sequence 2 in the sequence listing, respectively.

[0006] The UDP98-409-P consists of two single-stranded DNA molecules named as shown in sequence 3 and sequence 4 of the sequence listing, respectively.

[0007] The UDP98-411-P consists of two single-stranded DNA molecules named as shown in sequence 5 and sequence 6 of the sequence listing, respectively.

[0008] The UDP98-412-P consists of two single-stranded DNA molecules named as shown in sequence 7 and sequence 8 of the sequence listing, respectively.

[0009] The P04-P consists of two single-stranded DNAs named as shown in sequence 9 and sequence 10 of the sequence listing, respectively.

[0010] The BPPCT029-P consists of two single-stranded DNAs named as shown in sequence 11 and sequence 12 in the sequence listing, respectively.

[0011] The BPPCT030-P consists of two single-stranded DNAs named as shown in sequence 13 and sequence 14 in the sequence listing, respectively.

[0012] The BPPCT039-P consists of two single-stranded DNAs named as shown in sequence 15 and sequence 16 in the sequence listing, respectively.

[0013] The Pchcms4-P consists of two single-stranded DNAs named as shown in sequence 17 and sequence 18 in the sequence listing, respectively.

[0014] The Pchgms4-P consists of two single-stranded DNAs named as shown in sequence 19 and sequence 20 in the sequence listing, respectively.

[0015] The PaCITA15-P consists of two single-stranded DNAs named as shown in sequence 21 and sequence 22 in the sequence listing, respectively.

[0016] The PaCITA7-P consists of two single-stranded DNAs named as shown in sequence 23 and sequence 24 in the sequence listing, respectively.

[0017] The UDAp422-P consists of two single-stranded DNAs named as shown in sequence 25 and sequence 26 in the sequence listing, respectively.

[0018] The UDAp404-P consists of two single-stranded DNAs named as shown in sequence 27 and sequence 28 in the sequence listing, respectively.

[0019] The AMPA095-P consists of two single-stranded DNA molecules named as shown in sequence 29 and sequence 30 of the sequence listing, respectively.

[0020] In the primer sets mentioned above, the even-numbered sequences of UDP98-406-P, UDP98-409-P, BPPCT039-P, and PaCITA7-P, and the odd-numbered sequences of other primer pairs indicate that the 5′ ends of the primers are labeled with fluorescent substances such as HEX, FAM, HEX, or TAMRA.

[0021] The number of moles of each primer in the primer set is equal.

[0022] The following applications of the primer set also fall within the scope of protection of this invention:

[0023] X1) Identify or assist in identifying parentage relationships in Prunus species;

[0024] X2) Prepare products for identifying or assisting in the identification of parentage relationships in Prunus species;

[0025] X3) Identify or assist in identifying the parents of hybrid offspring of apricot kernel and almond;

[0026] X4) Prepare, identify or assist in the identification of parent products of hybrid offspring of apricot kernel and almond.

[0027] The present invention also provides a kit containing the primer set.

[0028] The following applications of the reagent kit also fall within the scope of protection of this invention:

[0029] X1) Identify or assist in identifying parentage relationships in Prunus species;

[0030] X2) Prepare products for identifying or assisting in the identification of parentage relationships in Prunus species;

[0031] X3) Identify or assist in identifying the parents of hybrid offspring of apricot kernel and almond;

[0032] X4) Prepare, identify or assist in the identification of parent products of hybrid offspring of apricot kernel and almond.

[0033] This invention also provides a method for identifying the parent plants of a *Prunus* species to be tested. The method includes: using the genomic DNA of the hybrid seedling to be tested and the assumed almond parent as templates, performing PCR amplification using the primer pair, and detecting the PCR products of the hybrid seedling and the assumed almond parent to determine whether the 15 SSR loci of the hybrid seedling and the assumed almond parent conform to Mendelian inheritance laws. If all 15 SSR loci conform to Mendelian inheritance laws, the hybrid seedling and the assumed almond parent have or are candidate to have a parent-child relationship; if none of the 15 SSR loci conform to Mendelian inheritance laws, the hybrid seedling and the assumed almond parent do not have or are candidate to have a parent-child relationship; if some of the 15 SSR loci do not conform to Mendelian inheritance laws, the hybrid seedling and the assumed almond parent are determined to have a parent-child relationship based on the cumulative RCP value: cumulative RCP value = 1 - (1 - RCP1)(1 - RCP2)...(1 - RCP15).

[0034] The cumulative RCP value of the tested Prunus species is greater than or equal to 99.73%, and the presumed parent has or is a candidate parent-child relationship; the cumulative RCP value of the tested Prunus species is less than 99.73%, and the presumed parent does not have or is a candidate parent-child relationship.

[0035] RCP1, RCP2, RCP3, RCP4, RCP5, RCP6, RCP7, RCP8, RCP9, RCP10, RCP11, RCP12, RCP13, RCP14, and RCP15 are the RCP values ​​of UDP98-406, UDP98-409, UDP98-411, UDP98-412, P04, BPPCT029, BPPCT030, BPPCT039, Pchcms4, Pchgms4, PaCITA15, PaCITA7, UDAp422, UDAp404, and AMPA095, respectively; RCP value = PI / (PI+1);

[0036] The 15 SSR sites are UDP98-406, UDP98-409, UDP98-411, UDP98-412, P04, BPPCT029, BPPCT030, BPPCT039, Pchcms4, Pchgms4, PaCITA15, PaCITA7, UDAp422, UDAp404, and AMPA095.

[0037] In the above method, the maternal parent of the hybrid seedling to be tested is known, while the paternal parent is unknown.

[0038] In the above method, the plum species to be tested can be apricot kernel, almond, or a hybrid offspring of apricot kernel and almond.

[0039] The method for identifying the parent plants of the genus *Prunus* described herein, when applied in the hybridization breeding of *Prunus chinensis* and *Prunus spp.*, also falls within the scope of protection of this invention.

[0040] The primer set and method of this invention can identify the kinship between apricot kernel and almond and their offspring, establish a parentage identification system, and are simple to operate. The primer set and method of this invention can quickly obtain the genetic information of the parents and offspring of plum plants and determine the source of the offspring. It can identify and retain the true hybrid offspring between apricot kernel and almond early in the seedling stage. This is beneficial for quickly identifying the true distant hybrid seedlings in the seedling stage, thereby removing mixed offspring caused by natural pollination and removing them in the seedling stage, saving land resources, reducing the cost of later identification, accelerating the breeding process, and can also provide a reference for the parentage identification of other fruit trees.

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0042] Figure 1 A dendrogram of SSR cluster analysis for 6 materials. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0044] In the following examples, the assumed paternal parent 1 (AF1) is the almond variety 'Monterey', provided by the Shandong Provincial Fruit Tree Research Institute, as described in the article "A Brief Introduction to Several Introduced American Almond Varieties, Chen Xiuhui et al., Southwest Horticulture, Vol. 30, No. 4, 2002". The public can obtain this biological material from the applicant. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0045] In the following examples, the assumed paternal parent 2 (AF2) is the almond variety 'Thompson', provided by the Shandong Provincial Fruit Tree Research Institute, as described in the article "A Brief Introduction to Several Introduced American Almond Varieties, Chen Xiuhui et al., Southwest Horticulture, Vol. 30, No. 4, 2002". The public can obtain this biological material from the applicant. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0046] Example 1: Preparation of primer sets for parentage testing of Prunus species

[0047] This embodiment provides a primer set for parentage identification of Prunus species. Information on each primer in the primer set is shown in Table 1. The number of moles in each primer in the primer set is equal. The reverse primers (R) of UDP98-406-P, UDP98-409-P, BPPCT039-P and PaCITA7-P, and the forward primers (F) of other primer pairs are labeled with fluorescent material at the 5′ end.

[0048] Table 1. SSR primer sequence information

[0049]

[0050]

[0051] Application of primer sets from Example 2 and Example 1 in parentage testing of Prunus species

[0052] The samples to be tested included: leaves from two hypothetical male almonds, 'Monterey' (AF1) and 'Thompson' (AF2); leaves from the female almond 'Longwangmao' (M); and leaves from three distant hybrid seedlings (A1, B1, and B2).

[0053] 1. Genomic DNA extraction

[0054] Genomic DNA was extracted from the leaves of each sample using the CTAB method. DNA quality was determined by 1% agarose gel electrophoresis, and concentration was determined by a K5800 micro spectrophotometer.

[0055] 2. PCR amplification and product detection

[0056] The reaction system consisted of 10 μl of the following components: 1 μl of 10×Buffer I, 0.8 μl of 2.5 mM dNTPs, 0.8 μl of the primer set from Example 1, 0.1 μl of HS Taq (5 U / μl), 1.0 μl of genomic DNA from the sample to be tested, and ddH2O to a final volume of 10 μl. In this system, the concentration of each primer in the primer set from Example 1 was 0.4 μM. 10×Buffer I was a product of Beijing Yuewei Gene Technology Co., Ltd., catalog number: 202105; HS Taq (5 U / μl) was a product of TAKAR Corporation, catalog number: AL52290A.

[0057] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles, and finally 60℃ extension for 30 min.

[0058] Capillary electrophoresis was used for detection, as follows: 9 μl of a mixture of molecular weight internal standard and formamide (0.5:8.5) and 1.0 μl of PCR product were added to each well of a 96-well plate; the plate was denatured at 95℃ for 3 min, and then detected by capillary electrophoresis using an ABI 3730 gene analyzer.

[0059] 3. Data Analysis and Processing

[0060] The raw data files obtained from the detection were imported into the analysis software Genemapper ID3.2 for statistical analysis of experimental results. Individual genotyping analysis was performed on the electrophoresis results, and Cervus3.0 software was used to analyze the number of alleles, allele frequencies, and polymorphism information content (PIC).

[0061] Sequencing analysis of six samples revealed 78 alleles detected at 15 SSR loci, averaging 5.2 alleles per locus, exceeding the requirement of at least 4 alleles for microsatellite loci used in paternity testing. The loci with the highest number of alleles detected were UDP98-411 and BPPCT039, each with 7 alleles. Even the locus with the fewest alleles detected had 4 alleles (Tables 2 and 3). These 15 loci exhibited high polymorphism, with a mean PIC value of 0.679. Except for Pchgms4 (0.5 > PIC = 0.393 > 0.25), which was considered moderately polymorphic, the other 14 loci had PIC values ​​greater than 0.5, indicating highly polymorphic loci (Table 3).

[0062] Table 2. Distribution of allele frequencies at 15 SSR loci in 6 samples.

[0063]

[0064] Table 3. Polymorphic Information Content

[0065] SSR sites Number of alleles Polymorphic information content SSR sites Number of alleles Polymorphic information content UDP98-406 4 0.599 Pchcms4 4 0.599 UDP98-409 5 0.726 Pchgms4 4 0.393 UDP98-411 7 0.782 PaCITA15 6 0.719 UDP98-412 6 0.777 PaCITA7 6 0.777 P04 5 0.726 UDAp422 5 0.643 BPPCT029 6 0.719 UDAp404 4 0.622 BPPCT030 4 0.622 AMPA095 5 0.692 BPPCT039 7 0.782

[0066] 4. Determination of parentage

[0067] Parentage is determined by genotyping and calculating cumulative RCP values. When the maternal parent is known but the paternal parent is unknown, the standard triplet method (hypothetical father-son-mother) is generally used to genotype the assumed parents and offspring, and the applicable formula is determined. This involves grouping the amplification results of all SSR loci according to the allele types of the two parents and offspring loci, and using the corresponding paternity index (PI) calculation formula for each group. The PI calculation formula is based on the simplified PI calculation method given by Yang Qing'en (1998) and Lu Huiling et al. (2001), as shown in Table 4.

[0068] The probability of a patriarchal relationship is RCP = PI / (PI+1).

[0069] Table 4. Simplified Calculation Formula for PI Corresponding to Standard Triad SSR Genomes

[0070]

[0071] Note: P, Q, R, and S represent the different alleles at each SSR locus; p and q are the true paternal gene frequencies. The same applies below.

[0072] Verification of the reliability of the parentage identification method: UPGMA clustering was performed on the parental and offspring materials based on SSR locus typing data.

[0073] The results of amplification, detection, and sequencing analysis of the six experimental materials using 15 pairs of primers are shown in Table 5. Analysis of the SSR genotyping results showed that offspring A1 and its known maternal parent (M) 'Dragon King Hat' and presumed paternal parent (AF1) 'Monterey' conformed to Mendelian inheritance, indicating a kinship between the presumed paternal parent (AF1) 'Monterey' and A1. However, the genetic inheritance of offspring B2 and its known maternal parent (M) 'Dragon King Hat' and presumed paternal parent (AF2) 'Thompson' violated Mendelian inheritance; no alleles of the presumed paternal parent (AF2) 'Thompson' were found in offspring B2 at any of the 15 loci, thus ruling out a kinship between them. Offspring B1 showed segregation at 12 loci consistent with Mendelian inheritance from its known maternal parent 'Dragon King Hat' and hypothetical paternal parent (AF2) 'Thompson', while 3 loci violated Mendelian inheritance. Whether the hypothetical paternal parent (AF2) 'Thompson' is related to B1 needs to be confirmed through further paternity index analysis.

[0074] Table 5. Typing of 15 SSR loci in distant hybrids of apricot and almond.

[0075]

[0076] Further paternity index analysis was performed on two pairs of samples that might have a father-son relationship, conforming to Mendelian inheritance laws. Based on the comparison of DNA genotype maps of mother-son or father-son pairs, the essential genes passed from the mother to the child and the genes from the biological father could be determined. The amplification results of 15 SSR loci were divided into 9 groups (Table 5) according to the allele types of the A1 loci in the parents and offspring: UDP98-406 and BPPCT039 in one group, UDP98-409, BPPCT030, and UDAp422 in another, UDP98-411, UDP98-412, PaCITA15, and PaCITA7 in yet another, and the other 6 loci each in their own group. The corresponding PI value calculation formula was used for each group. The paternity identification types and PI value calculations for apricot-almond distant hybrids are shown in Tables 2 and 5. When using multiple loci for paternity identification, the cumulative RCP value is generally used for determination. As shown in Table 6, the cumulative RCP of offspring A1 across 15 SSR loci is 1 - (1 - RCP1)(1 - RCP2)...(1 - RCP15) = 99.999998%, indicating that the distant hybrid offspring A1 originated from the paternal parent 'Monterey'. According to domestic and international paternity testing practices, an RCP value greater than 99.73% is generally considered to indicate a parent-child relationship between the paternal parent and the hybrid.

[0077] Table 6. Genotyping results of 15 SSR loci in distant hybrids of apricot and almond.

[0078]

[0079] Based on SSR data, UPGMA cluster analysis was performed on the six materials, and the results are as follows: Figure 1 As shown, the six samples were divided into two groups (I and II) at a genetic similarity coefficient of 0.525. B2, being more distantly related to the other samples, formed a separate group (I), while the other varieties formed a separate group (II). At a genetic similarity coefficient of 0.79, AF1 ('Monterey') in group II clustered with A1, indicating the closest relationship. AF2 ('Thompson'), although relatively distantly related to B1 and M ('Dragon King Hat'), also clustered with these two samples at a similarity coefficient of 0.525. This clustering result indicates that AF1 ('Monterey') is more closely related to its offspring A1, AF2 ('Thompson') is relatively closely related to its offspring B1, and most distantly related to its offspring B2, consistent with the previous paternity identification results.

[0080] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for identifying the parent plants of a species in the genus *Prunus*, comprising: Using genomic DNA from the hybrid seedlings to be tested and the presumed almond parents as templates, PCR amplification was performed using primer sets. The PCR products of the hybrid seedlings and the presumed almond parents were detected to determine whether the 15 SSR loci of the hybrid seedlings to be tested and the presumed almond parents conformed to Mendelian inheritance laws. The hybrid seedlings to be tested and the presumed almond parents that conformed to Mendelian inheritance laws had or were candidate for parentage. The hybrid seedlings to be tested and the presumed almond parents that did not conform to Mendelian inheritance laws had or were candidate for parentage. The hybrid seedlings to be tested and the presumed almond parents that did not conform to Mendelian inheritance laws at some of the 15 SSR loci were determined to be parentage based on the cumulative RCP value: cumulative RCP value = 1 - (1 - RCP1)(1 - RCP2) ... (1 - RCP15). The cumulative RCP value of the tested Prunus species is greater than or equal to 99.73%, and the presumed parent has or is a candidate for parent-child relationship; the cumulative RCP value of the tested Prunus species is less than 99.73%, and the presumed parent does not have or is a candidate for parent-child relationship. RCP1, RCP2, RCP3, RCP4, RCP5, RCP6, RCP7, RCP8, RCP9, RCP10, RCP11, RCP12, RCP13, RCP14, and RCP15 are the RCP values ​​of UDP98-406, UDP98-409, UDP98-411, UDP98-412, P04, BPPCT029, BPPCT030, BPPCT039, Pchcms4, Pchgms4, PaCITA15, PaCITA7, UDAp422, UDAp404, and AMPA095, respectively; RCP value = PI / (PI+1); The 15 SSR sites are UDP98-406, UDP98-409, UDP98-411, UDP98-412, P04, BPPCT029, BPPCT030, BPPCT039, Pchcms4, Pchgms4, PaCITA15, PaCITA7, UDAp422, UDAp404, and AMPA095; The primer set consists of UDP98-406-P, UDP98-409-P, UDP98-411-P, UDP98-412-P, P04-P, BPPCT029-P, BPPCT030-P, BPPCT039-P, Pchcms4-P, Pchgms4-P, PaCITA15-P, PaCITA7-P, UDAp422-P, UDAp404-P, and AMPA095-P; The UDP98-406-P consists of two single-stranded DNA sequences shown in Sequence 1 and Sequence 2 of the sequence listing. The UDP98-409-P consists of two single-stranded DNA sequences shown in sequences 3 and 4 of the sequence listing; The UDP98-411-P consists of two single-stranded DNA sequences shown in sequences 5 and 6 of the sequence listing. The UDP98-412-P consists of two single-stranded DNA sequences shown in sequences 7 and 8 of the sequence listing; The P04-P consists of two single-stranded DNA sequences shown in sequences 9 and 10 of the sequence listing; The BPPCT029-P consists of two single-stranded DNA sequences shown in sequence 11 and sequence 12 in the sequence listing; The BPPCT030-P consists of two single-stranded DNA sequences shown in sequences 13 and 14 of the sequence listing; The BPPCT039-P consists of two single-stranded DNA sequences shown in sequences 15 and 16 of the sequence listing. The Pchcms4-P consists of two single-stranded DNA sequences shown in sequences 17 and 18 of the sequence listing. The Pchgms4-P consists of two single-stranded DNA sequences shown in sequence 19 and sequence 20 of the sequence listing; The PaCITA15-P consists of two single-stranded DNA sequences shown in sequences 21 and 22 in the sequence listing; The PaCITA7-P consists of two single-stranded DNA sequences shown in sequences 23 and 24 of the sequence listing; The UDAp422-P consists of two single-stranded DNA sequences shown in sequences 25 and 26 of the sequence listing; The UDAp404-P consists of two single-stranded DNA sequences shown in sequences 27 and 28 of the sequence listing; The AMPA095-P consists of two single-stranded DNA sequences shown in sequence 29 and sequence 30 of the sequence listing; The plum species to be tested were apricot kernels, almonds, or hybrids of apricot kernels and almonds.

2. The method according to claim 1, characterized in that: Even-numbered sequences of UDP98-406-P, UDP98-409-P, BPPCT039-P, and PaCITA7-P, and odd-numbered sequences of other primer pairs, are used to label the 5′ ends of the primers with fluorescent material.

3. The application of the method according to claim 1 or 2 in the crossbreeding of apricot kernel and almond.

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