Method for analyzing chromosome genetic information component sources of olea europaea-sweet clover olive BC1 generation individuals

Abstract

The invention provides a method for analyzing chromosome genetic information component sources of olea europaea-sweet clover olive BC1 generation individuals, and belongs to the technical field of crop variety identification. According to the method disclosed by the invention, 46 pairs of primers as shown in SEQ ID NO: 1-SEQ ID NO: 92 are used for amplification to obtain 46 fragments in a genome of an olive-sweet clover olive BC1 generation individual, the 46 fragments are sequenced, and a chromosome genetic information component source is analyzed according to a sequencing result.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop variety identification, and specifically relates to a method for analyzing the sources of chromosome genetic information components of backcross offspring individuals (first-generation backcross individuals) of hybridization of olive and sweet clover (also referred to as olive-sharp clover BC1 individuals herein, the BC1 individuals are individuals of the first generation of backcrossing of the hybridization offspring of olive and sweet clover as the female parent and olive as the male parent). Background Art

[0002] Olive (Olea europaea L.) is an evergreen tree belonging to the genus Olea in the family Oleaceae. It is one of the world's famous high-quality woody oil-bearing and fruit-bearing trees. At present, olives show incompatibility with climate and soil, and also have some fungal and bacterial disease problems, such as leaf spot, anthracnose, and bacterial wilt. The emergence of these diseases has some negative effects on the yield of olives and the quality of olive oil, and thus causes serious economic losses. Therefore, improving the resistance of olive varieties is an urgent problem to be solved in the olive industry.

[0003] Oleander has excellent resistance, so far. No major harmful diseases have been found in the artificial introduction and cultivation of this tree species, and its genetic components can be introduced into the olive breeding population to improve the adaptability and disease resistance of olive. The resistance performance of all aspects in the F1 generation produced after the hybridization of Oleander and olive is good, but the tree shape is tall, the vegetative growth is relatively strong, and the reproductive growth is weak, resulting in a larger fruit stone, and the yield is still not improved, and it is necessary to continue genetic breeding, such as using the hybrid F1 generation individual as the maternal parent and olive as the paternal parent for backcross breeding. In the new variety breeding process, the accurate identification of the genetic components of the backcross offspring (such as backcross 1 generation (BC1 generation)) after hybridization is a major current difficulty. Therefore, it is necessary to develop a reliable, fast, and accurate identification system for the identification of genetic component sources and auxiliary genome selection breeding for BC1 generation seedlings. At present, there is no method suitable for analyzing the chromosome genetic information source of olive-Oleander-Oleander BC1 generation individual. Summary of the invention

[0004] In view of one or more of the problems in the prior art, one aspect of the present invention provides a method for analyzing the sources of chromosome genetic information components of an individual of the BC1 generation of olive-Oleaceae, wherein the BC1 generation of olive-Oleaceae is an individual obtained by backcrossing a hybrid offspring of olive-Oleaceae as the female parent and olive as the male parent once, wherein the method comprises using 46 pairs of primers shown in SEQ ID NO:1-SEQ ID NO:92 to amplify 46 fragments from the genomic DNA of the BC1 generation of olive-Oleaceae and sequence them, wherein each chromosome of the BC1 generation of olive-Oleaceae corresponds to two fragments, and the genetic information of each chromosome is analyzed according to the sequencing results of the 46 fragments; wherein:

[0005] If the sequencing result of a certain amplified fragment at the locus of each specific position is a single peak, then the chromosome genetic information components of the olive-Oleaceae BC1 generation individual at the amplified position are all from olive;

[0006] If the sequencing result of a certain amplified fragment at the site of each specific position is a double peak, then the chromosomal genetic information component of the olive-Oleaceae BC1 generation individual at the amplified position is partly from olive and partly from Oleaceae.

[0007] In some embodiments, the olive-Olea europaea hybrid offspring is the olive variety - Chemlar.

[0008] On the other hand, the present invention provides an oligonucleotide combination, which is a combination of 46 pairs of primers shown in SEQ ID NO: 1-SEQ ID NO: 92. The oligonucleotide combination provided by the present invention can be used to analyze the source of the chromosome genetic information components of the BC1 generation of individuals of Olea europaea-Oleracea acuminata. Therefore, the use of the oligonucleotide combination provided by the present invention in analyzing the source of the chromosome genetic information components of the BC1 generation of individuals of Olea europaea-Oleracea acuminata also belongs to the content of the present invention.

[0009] The method for analyzing the sources of chromosome genetic information components of individuals of the BC1 generation of olive-Olea europaea using 46 pairs of primers provided by the above technical solution can achieve the following purposes:

[0010] 1. Clarify whether the genetic information components of each chromosome of the BC1 generation of individuals of the olive-Oleaceae family come from the olive or the Oleaceae family.

[0011] 2. Identify whether recombination occurs between the head and tail of chromosomes when the BC1 generation of olive-Oleaceae individuals form gametes. If recombination does not occur between the head and tail of chromosomes, then the variant sites before and after the same chromosome will all be heterozygous or homozygous, otherwise recombination occurs during the gamete formation process of the BC1 generation of olive-Oleaceae individuals.

[0012] 3. By counting the number of loci containing genetic information components of Olea europaea and dividing it by 46, we can obtain the proportion of genetic components of Olea europaea in the BC1 generation of individuals of the olive-Olea europaea.

[0013] These results will significantly improve the efficiency of early genome-assisted selection of olive seedlings and are of great significance to its genetic breeding work. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0015] Figure 1 The results are shown in Figure 2, which are the sequencing results of the amplified fragments from the genomes of 25 Olea europaea samples and 66 Olea europaea samples using the Chr01-1 primer. DETAILED DESCRIPTION

[0016] When the inventors studied the genomic information of 25 Olea europaea genome sequences and 66 Olea europaea genome sequences (the genomic sequence information of the above Olea europaea and Olea europaea obtained by the applicant's laboratory) by bioinformatics methods, they found that there was at least one sequence on each chromosome of Olea europaea and Olea europaea (a total of 23 chromosomes) that could reliably distinguish the two and their hybrid offspring (as the maternal parent) from the backcross offspring (BC1 generation) of Olea europaea (as the paternal parent), that is, there were variant sites in these sequences. Therefore, the present invention designed primers for this type of variant site upstream and downstream of the 23 chromosomes, and obtained a total of 46 pairs of primers (2 pairs of primers for each chromosome) (as shown in Table 1 below). Using these 46 pairs of primers, 46 fragments can be amplified for the 23 chromosomes of each sample, and There are mutation sites on each fragment that can be used to identify and distinguish between olive and Olea europaea and the olive-Olea europaea BC1 generation. Specifically, the mutation sites of olive on these 46 fragments are C, T, A, A, C, T, T, G, T, A, C, G, G, G, A, A, A, A, C, T, A, A, C, T, T, A, G, A, C, A, A, A, G, C, C, G , T, A, C, C, G, C, A, A, A, A; the mutation sites of Oleaceae on these 46 fragments are G, C, G, C, A, C, 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, T, G, T, C, T, C, G, G, G (as shown in Table 1 below). Figure 1 As shown, it exemplarily shows the sequencing results of the amplified fragments of the genomes of 25 Oleaceae samples and 66 Olea europaea samples respectively using primer Chr01-1 among the 46 primer pairs. Since the hybrid offspring (F1 generation) of olive and Olea europaea all have bimodal sequencing results at these sites, the first-generation backcross individuals obtained by backcrossing the hybrid F1 generation as the mother with olive as the father for one generation (i.e., olive-Olea europaea BC1 generation individuals) may show single-peak sequencing results and / or double-peak sequencing results at the variation sites on these 46 fragments, wherein the single-peak sequencing result represents that the genetic information component of the site comes from olive, and the double-peak sequencing result represents that part of the genetic information component of the site comes from olive and the other part from Olea europaea; thereby, the Olea europaea genetic components at these 46 sites of each BC1 generation individual can be counted to calculate the proportion of Olea europaea genetic components in the individual, and it can also be clarified whether the Olea europaea genetic components are retained at each site of the BC1 generation individual.

[0017] Table 1: Amplification primer information

[0018]

[0019]

[0020]

[0021] Hereinafter, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the following description is considered to be exemplary and non-restrictive in nature.

[0022] The present invention is described in detail below with reference to embodiments.

[0023] Example 1: Analysis of the source of genetic information components of seedlings (Olea europaea-Oleaceae BC1 individuals)

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

[0025] (1) Determine whether each chromosome of the sown seedlings contains chromosomes with genetic information components from Oleaceae.

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

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

[0028] (4) Identify which chromosome of the seedlings contains the genetic components of Olea europaea.

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

[0030] 1. Obtaining DNA from leaf samples of seedlings: Collect two leaves from a single seedling to be identified, dry them with silica gel, and then cut leaf samples (1 cm×1 cm in size) for DNA extraction; wherein the seeds used to produce the single seedlings are collected from Chemlar (olive variety), the Chemlar plant is a hybrid F1 individual of olive (Fouao) and Oleaceae, and the seeds on the Chemlar plant are obtained by backcrossing Chemlar as the female parent and olive (Fouao) as the male parent;

[0031] 2. PCR amplification: Use a conventional PCR instrument and method to amplify the PCR product, wherein the primers listed in Table 1 are used, and the specific amplification procedure is shown in Table 2 below;

[0032] Table 2: PCR amplification program

[0033]

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

[0035] 4. Sequence comparison and identification: Referring to the sequence information listed in Table 1, the genetic information of each site of the seedlings was analyzed according to the sequence information of each variable site. The standards and results are shown in Table 3.

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

[0037] By counting the number of loci containing Olea europaea components and dividing it by 46, we can obtain the proportion of genetic components containing Olea europaea in backcross individuals.

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

[0039] Table 3: Genetic information test results of 10 olive-Olea europaea BC1 individuals

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing the source of chromosome genetic information components of an individual of the BC1 generation of olive-Oleaceae, wherein the BC1 generation of olive-Oleaceae is an individual obtained by backcrossing a hybrid offspring of olive-Oleaceae as the female parent and olive as the male parent once, characterized in that: The method comprises amplifying 46 fragments from the genomic DNA of the olive-Oleaceae BC1 generation individual using 46 pairs of primers shown in SEQ ID NO: 1-SEQ ID NO: 92 and sequencing the fragments, wherein each chromosome of the olive-Oleaceae BC1 generation individual corresponds to two fragments, and analyzing the genetic information of each chromosome according to the sequencing results of the 46 fragments; wherein: If the sequencing result of a certain amplified fragment at the locus of each specific position is a single peak, then the chromosome genetic information components of the olive-Oleaceae BC1 generation individual at the amplified position are all from olive; If the sequencing result of a certain amplified fragment at the site of each specific position is a double peak, then the chromosomal genetic information component of the olive-Oleaceae BC1 generation individual at the amplified position is partly from olive and partly from Oleaceae.

2. The method according to claim 1, wherein the olive-Oleaceae hybrid offspring is an olive variety-Qimlar.

3. An oligonucleotide combination, which is a combination of 46 pairs of primers shown in SEQ ID NO: 1 to SEQ ID NO:

92.

4. Use of the oligonucleotide combination described in claim 3 in analyzing the source of chromosomal genetic information components of olive-Oleaceae BC1 generation individuals, wherein the olive-Oleaceae BC1 generation individuals are individuals obtained by backcrossing once with olive-Oleaceae hybrid offspring as the female parent and olive as the male parent.

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