A method for analyzing the source of the chromosomal genetic information component of an individual of the BC1 generation of olea europaea-elaeagnus oleaster

Abstract

The application provides a method for analyzing the chromosomal genetic information component source of an individual of the first generation of Olea europaea-Verhuellia acutifolia BC1, and belongs to the technical field of crop variety identification. The method disclosed by the application amplifies 46 fragments in the genome of the individual of the first generation of Olea europaea-Verhuellia acutifolia BC1 by using 46 pairs of primers shown in SEQ ID NO:1-SEQ ID NO:92, and sequencing, and the chromosomal genetic information component source is analyzed through the sequencing result.

Description

Technical Field

[0001] This invention belongs to the field of crop variety identification technology, specifically involving a method for analyzing the source of chromosomal genetic information components in individuals that are offspring of a cross between olive and osmanthus (backcross generation 1 individuals) (also referred to in this paper as olive-osmanthus BC1 individuals, which are individuals obtained by backcrossing olive as the female parent and olive as the male parent). Background Technology

[0002] The olive tree (Olea europaea L.) is an evergreen tree belonging to the genus Olea in the family Oleaceae. It is one of the world's renowned high-quality woody oilseed trees and fruit-bearing trees. Currently, olive trees exhibit incompatibility with climate and soil, and are also susceptible to fungal and bacterial diseases, such as leaf spot, anthracnose, and bacterial wilt. These diseases negatively impact olive yield and olive oil quality, leading to significant economic losses. Therefore, improving the resistance of olive varieties is an urgent problem to be solved for the olive industry.

[0003] The pointed-leaf osmanthus exhibits excellent resistance to disease. No major harmful diseases have been found in the artificial cultivation of this species. Its genetic components can be introduced into olive breeding populations to improve the adaptability and disease resistance of olives. The F1 generation produced by crossing pointed-leaf osmanthus with olive exhibits good resistance in various aspects, but the trees are tall with strong vegetative growth and weak reproductive growth, resulting in larger fruit pits and no increase in yield. Further genetic breeding is needed, for example, by backcrossing the F1 hybrid with olive as the male parent. Accurate identification of the genetic components of backcrossed offspring (e.g., backcross generation 1 (BC1)) is a major challenge in new variety development. Therefore, a reliable, rapid, and accurate identification system needs to be developed to identify the genetic origin of BC1 seedlings and assist in genomic selection breeding. Currently, there is no suitable method for analyzing the chromosomal genetic information source of olive-point-leaf osmanthus BC1 individuals. Summary of the Invention

[0004] To address one or more of the problems existing in the prior art, one aspect of the present invention provides a method for analyzing the source of chromosomal genetic information components in BC1 generation individuals of *Oliveum cuspidatum* and *Oliveum truncatum*, wherein the BC1 generation individuals of *Oliveum cuspidatum* are individuals obtained by backcrossing once between a hybrid offspring of *Oliveum cuspidatum* and *Oliveum* as the maternal parent and *Oliveum cuspidatum* as the paternal parent. The method includes amplifying 46 fragments from the genomic DNA of the BC1 generation individuals of *Oliveum cuspidatum* using 46 pairs of primers shown in SEQ ID NO:1-SEQ ID NO:92 and sequencing them. Each chromosome of the BC1 generation individuals of *Oliveum cuspidatum* corresponds to two fragments, and the genetic information of each chromosome is analyzed based on the sequencing results of the 46 fragments; wherein:

[0005] If the sequencing result of a certain amplified fragment at its specific location is a single peak, then the chromosomal genetic information of the olive-sharp-olive BC1 generation individual at that amplified location is entirely derived from olive.

[0006] If the sequencing result of a certain amplified fragment at its specific location is bimodal, then the chromosomal genetic information component at that amplified location in the olive-sharp-olive BC1 generation individual comes partly from olive and partly from olive.

[0007] In some embodiments, the hybrid offspring of the olive-sharp-oloursin olive is the olive variety Chemlar.

[0008] In another aspect, this invention provides an oligonucleotide combination, which is a combination of 46 primer pairs shown in SEQ ID NO:1-SEQ ID NO:92. The oligonucleotide combination provided by this invention can be used to analyze the source of chromosomal genetic information components in Olive-Oleander BC1 generation individuals. Therefore, the application of the oligonucleotide combination provided by this invention in analyzing the source of chromosomal genetic information components in Olive-Oleander BC1 generation individuals also falls within the scope of this invention.

[0009] The method provided above for analyzing the source of chromosomal genetic information components in the BC1 generation of olive-scented sweet olive (Olive-Sweet Olive) using 46 pairs of primers can achieve the following objectives:

[0010] 1. Determine whether the genetic information of each chromosome in the BC1 generation of olive-scented osmanthus comes from olive or osmanthus.

[0011] 2. To determine whether recombination occurs between the beginning and end of chromosomes during gamete formation in olive-olive-olive BC1 generation individuals. If no recombination occurs between the beginning and end of chromosomes, then the variant sites at the beginning and end of the same chromosome will both be heterozygous or homozygous; otherwise, recombination occurred during gamete formation in olive-olive-olive BC1 generation individuals.

[0012] 3. By counting the number of loci containing the genetic information of Osmanthus fragrans and dividing by 46, the proportion of Osmanthus fragrans genetic information in the BC1 generation of olive-Osmanthus fragrans individuals can be obtained.

[0013] These results will significantly improve the efficiency of early genome-assisted selection in olive seedlings, which is of great significance to their genetic breeding work. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0015] Figure 1 The sequencing results are those of amplified genome fragments from 25 Oleaceae samples and 66 Olive samples, obtained using Chr01-1 primers. Detailed Implementation

[0016] When the inventors studied the genomic information of 25 *Oleander* genome sequences and 66 *Oleander* genome sequences (the genomic sequence information of *Oleander* and *Oleander* obtained by the applicant's laboratory) using bioinformatics methods, they discovered that each chromosome (a total of 23 chromosomes) of both *Oleander* and *Oleander* contains at least one sequence that can reliably distinguish between the two species, their hybrid offspring (as the female parent), and the backcross offspring (BC1 generation) of *Oleander* (as the male parent). That is, these sequences contain variation sites. Therefore, this invention designed primers for this type of variation site upstream and downstream of the 23 chromosomes, obtaining a total of 46 primer pairs (2 pairs for each chromosome) (as shown in Table 1 below). Using these 46 primer pairs, 46 fragments can be amplified from the 23 chromosomes of each sample. Each fragment contains variant sites that can be used to distinguish between olive and sweet olive (Oleander), as well as the BC1 generation of olive-sweet olive. Specifically, the variant sites for olive in these 46 fragments are, in order: C, T, A, A, C, T, T, G, T, A, C, G, G, G, A, A, A, A, C, T, A, A, C, T, A, A, C, T, A, G, A, C, A, A, A, A, G, C, C, G The variation sites in these 46 fragments of *Oleander arborescens* are, in order: G, C, G, C, A, C, A, A, A, G, G, T, T, C, C, G, G, G, G, T, C, G, T, T, G, A, T, A, G, T, C, G, G, A, G, T, T, C, G, G, T, C, G, G, G (see Table 1 below for details). Figure 1 As shown, it exemplifies the sequencing results of the amplified genome fragments of primer Chr01-1 from these 46 primer pairs for 25 Osmanthus fragrans samples and 66 olive samples, respectively. Since the F1 generation of hybrids of olive and sweet olive (Oleander) all showed bimodal sequencing results at these loci, the backcross generation 1 individuals (i.e., olive-sweet olive BC1 individuals) obtained after backcrossing the F1 generation (using olive as the maternal parent) with olive as the paternal parent for one generation may show single-peak sequencing results and / or bimodal sequencing results at these 46 fragments. A single-peak sequencing result indicates that the genetic information at this locus comes from olive, while a bimodal sequencing result indicates that the genetic information at this locus comes partly from olive and partly from sweet olive. Therefore, the sweet olive genetic component at these 46 loci can be counted for each BC1 individual, thereby calculating the proportion of sweet olive genetic component in the individual, and also clarifying whether each locus of the BC1 individual retains sweet olive genetic component.

[0017] Table 1: Amplification Primer Information

[0018]

[0019]

[0020]

[0021] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the following description is to be considered exemplary in nature and not restrictive.

[0022] The present invention will be described in detail below with reference to the embodiments.

[0023] Example 1: Analysis of the source of genetic information components in seedlings (Olive-Oleander BC1 generation individuals)

[0024] This embodiment aims to analyze the source of genetic information components in seedlings (Olive-Sweet Olive BC1 generation individuals), specifically:

[0025] (1) Determine whether each chromosome of the seedling has a chromosome containing genetic information from Osmanthus fragrans.

[0026] (2) To identify whether recombination occurred between the head and tail of each chromosome during gamete formation in the BC1 generation of olive-sharp-sweet olive.

[0027] (3) The proportion of genetic components of Osmanthus fragrans in seedlings.

[0028] (4) Determine which chromosome of the seedling contains the genetic material of Osmanthus fragrans.

[0029] The method provided in this embodiment specifically includes the following steps:

[0030] 1. Obtaining DNA from seedling leaf samples: Two leaves were collected from each seedling to be identified, dried with silica gel, and then leaf samples (1cm×1cm in size) were cut for DNA extraction. The seeds used to produce the seedlings were collected from Chamlar (an olive variety). The Chamlar plant is a hybrid F1 individual of olive (Foao) and osmanthus fragrans. The seeds on the Chamlar plant were obtained by backcrossing Chamlar as the female parent and olive (Foao) as the male parent.

[0031] 2. PCR amplification: PCR products were amplified using a conventional PCR instrument and method. The primers listed in Table 1 were used, and the specific amplification procedure is shown in Table 2 below.

[0032] Table 2: PCR amplification program

[0033]

[0034] 3. Sequencing: The PCR products obtained in step 2 are sent to a sequencing company for sequencing using the Sanger method to obtain the sequencing results;

[0035] 4. Sequence alignment and identification: Referring to the sequence information listed in Table 1, the genetic information of each site of the seedling was analyzed based on the sequence information of each variant site. The standards and results are shown in Table 3.

[0036] If no recombination occurs at the beginning or end of the chromosome, then the variant sites at the beginning and end of the same chromosome will all be heterozygous or homozygous. Otherwise, recombination occurs during gamete formation in the BC1 generation of olive-sharp-olive clover.

[0037] By counting the number of loci containing the Osmanthus fragrans component and dividing by 46, the proportion of the Osmanthus fragrans genetic component in backcross individuals can be obtained.

[0038] The genetic information of 10 olive-sharp-olive BC1 individuals (samples 1-10) and their parents collected from the field were tested using steps 1-4 of the above method. The results are shown in Table 3 below.

[0039] Table 3: Genetic information detection results of 10 Olive-Olive Tree BC1 individuals

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for analyzing the origin of chromosomal genetic information components in BC1 generation individuals of *Olive* and *Osmanthus fragrans*, wherein the BC1 generation individuals of *Olive* and *Osmanthus fragrans* are obtained by backcrossing once between a hybrid offspring of *Olive* and *Olive* as the maternal parent and *Olive* as the paternal parent, characterized in that... The method includes amplifying 46 fragments from the genomic DNA of the *Olive-Sweet Olive* BC1 generation individuals using 46 primer pairs shown in SEQ ID NO:1-SEQ ID NO:92, and sequencing them. Each chromosome of the *Olive-Sweet Olive* BC1 generation individual corresponds to two fragments. The genetic information of each chromosome is analyzed based on the sequencing results of the 46 fragments. If the sequencing result of a certain amplified fragment at its specific location is a single peak, then the chromosomal genetic information of the olive-sharp-olive BC1 generation individual at that amplified location is entirely derived from olive. If the sequencing result of a certain amplified fragment at its specific location is bimodal, then the chromosomal genetic information component at that amplified location in the olive-sharp-olive BC1 generation individual comes partly from olive and partly from olive BC1. The hybrid offspring of the olive-sharp-sweet olive variety is the olive variety Chemlar; The information of the 46 primer pairs and the specific positions and site information of the 46 fragments are shown in the table below: 。 2. An oligonucleotide combination comprising 46 primer pairs shown in SEQ ID NO:1-SEQ ID NO:

92.

3. The application of the oligonucleotide combination of claim 2 in analyzing the source of chromosomal genetic information components of olive-scented osmanthus BC1 generation individuals, wherein the olive-scented osmanthus BC1 generation individuals are individuals obtained by backcrossing once between the offspring of olive-scented osmanthus hybrids as the maternal parent and olive as the paternal parent. The hybrid offspring of the olive-sharp-sharp-olive-olive hybrid is the olive variety—Chamlar.

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