Development and application of InDel marker for identifying east-west pears and hybrids of east-west pears

Abstract

The invention develops an InDel marker (namely DX15) for identifying east and west pears and hybrids thereof, and further provides a method for identifying the east and west pears and the hybrids thereof by using the InDel marker. The InDel marker (namely DX15) has polymorphism between the east pear and the west pear, and can quickly identify whether any pear variety contains the genetic background of the western pear or not.

Description

Technical Field

[0001] The invention belongs to the field of sand pear molecular breeding and relates to an InDel marker for identifying eastern and western pears and hybrids thereof and application thereof. Background Art

[0002] Pear is one of the main fruits in the world and the third most popular fruit in my country after apples and citrus. In the past decade, my country's pear production has maintained more than two-thirds of the world's total. Pears originated in the mountainous areas of western or southwestern my country in the Tertiary period and spread eastward and westward respectively. The eastward spread formed the Oriental pear population, including sand pear (P.pyrifolia Nakai), autumn pear (P.ussuriensis Max.), white pear (P.×bretschneideri Rehd.), and Xinjiang pear (P.×sinkiangensis Yu), which are the main cultivated pear species in East Asia; while the western pear (P.communis L.) gradually formed by the westward spread is the main cultivated pear species in the main pear producing countries outside East Asia.

[0003] Eastern and Western pears have completely different fruit characteristics. Oriental pears are crisp-fleshed fruits (except for the Autumn Pear), which can be eaten right after harvest and have almost no fragrance; Western pears are soft-fleshed fruits, which need to be fully ripened before being eaten, and have a strong fragrance. Improving varietal traits through hybridization of Eastern and Western pears has always been a hot topic in pear breeding research. There is currently a lack of effective molecular identification technology systems to quickly determine whether a variety has Western pear ancestry and clarify its genetic background. Summary of the invention

[0004] To solve the above problems, the present invention provides an InDel marker (named as DX15) for identifying eastern and western pears and their hybrids. The InDel marker is -850 bp upstream of Ppy15g2510.1, with a 285 bp insertion fragment. The forward primer of the InDel marker is DX15-F, SEQ ID NO.1, 5'-GTGGGTCCCTTAGGCATTTT-3', and the reverse primer of the InDel marker is DX15-R, SEQ ID NO.2, 5'-TAGGGCGTTCACCATTACCT-3'.

[0005] The present invention also provides a method for identifying the eastern and western pears and their hybrids, comprising the following steps:

[0006] Step 1, extracting the genomic DNA of the pear to be tested;

[0007] Step 2: Using the pear genomic DNA to be tested as a template, perform PCR amplification using the above primer pair to obtain a PCR amplification product.

[0008] Step 3: Use 2% agarose gel electrophoresis to detect the size of the PCR amplification product: 468 bp homozygous is Western pear (containing a 285 bp insert), 183 bp homozygous is Oriental pear, and 183 / 468 heterozygous is a hybrid of Eastern and Western pears.

[0009] Furthermore, the total volume of the PCR amplification system in step 2 is 20 μl, including 2 μl of 10 ng genomic template DNA, 10 μl of 2×Es Taq MasterMix, 2 μl each of the 10 mM forward and reverse primers mentioned above, and 4 μl of ddH2O.

[0010] Furthermore, the PCR amplification program in step 2 is pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing temperature at 60°C for 30 s, extension at 72°C for 150 s, 35 cycles; and finally extension at 72°C for 10 min.

[0011] The beneficial effects of the present invention are:

[0012] The present invention develops an InDel marker (i.e., DX15) for identifying Eastern and Western pears and their hybrids, and also provides a method for identifying Eastern and Western pears and their hybrids using the InDel marker. The InDel marker (i.e., DX15) of the present invention is polymorphic between Eastern and Western pears, and can quickly identify whether any pear variety contains the genetic background of Western pears. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Amplification profiles of different eastern and western pear varieties and their hybrids based on DX15 marker. DETAILED DESCRIPTION

[0014] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0015] 1. Obtaining InDel markers

[0016] In the study of the ripening mechanism of pear fruit, it was found that there was an insertion fragment of 285 bp in length at -850 bp upstream of Ppy15g2510.1 only in the European pear varieties. Therefore, primers were designed at both ends of this site to develop an InDel marker (named DX15): the forward primer of the InDel marker is 5'-DX15-F, SEQ ID NO.1, GTGGGTCCCTTAGGCATTTT-3', and the reverse primer of the InDel marker is DX15-R, SEQ ID NO.2, 5'-TAGGGCGTTCACCATTACCT-3'.

[0017] 2. Application of InDel markers in identification of hybrids of eastern and western pears

[0018] Twenty representative varieties of pears and their hybrids and two varieties of unknown parents were used as test materials (Table 1). Genomic DNA was extracted and amplified by PCR and detected by agarose gel electrophoresis using the developed InDel marker DX15 ( Figure 1 ). The total volume of the PCR amplification system was 20 μl, including 2 μl of 10 ng genomic template DNA, 10 μl of 2×Es Taq MasterMix, 2 μl of each of the above 10 mM forward and reverse primers, and 4 μl of ddH2O; the PCR amplification program was 94°C pre-denaturation for 5 min; 94°C denaturation for 10 s, annealing temperature of 60°C for 30 s, extension at 72°C for 150 s, 35 cycles; and finally extension at 72°C for 10 min.

[0019] Table 1 Application of InDel markers in identification of hybrids between eastern and western pears

[0020]

[0021]

[0022] The identification results are as follows Figure 1 As shown in Table 1, the DX15 genotypes of the five western pear varieties from different origins were all homozygous for 468, indicating that the insertion fragment was highly conserved in western pears, while the DX15 genotypes of the nine varieties of oriental pears belonging to different species such as white pear, sand pear, autumn pear, and Xinjiang pear were all homozygous for 183. In addition, the DX15 genotypes of the six varieties with clear genetic backgrounds and hybridization of eastern and western pears were all heterozygous for 183 / 468, and the genotypes of the two varieties with unknown parents, 'July Early Red Pear' and 'Wai Hulu Pear', were all heterozygous for 183 / 468, suggesting that they contain western pear ancestry and are hybrids of eastern and western pears.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An InDel marker for identifying Eastern and Western pears and their hybrids, characterized in that: The InDel marker is -850 bp upstream of Ppy15g2510.1, a 285 bp insertion fragment.

2. A primer pair for detecting the InDel marker of claim 1, characterized in that: The forward primer of the primer pair is DX15-F, SEQ ID NO. 1, 5'-GTGGGTCCCTTAGGCATTTT-3', and the reverse primer of the primer pair is DX15-R, SEQ ID NO. 2, 5'-TAGGGCGTTCACCATTACCT-3'.

3. A method for identifying Eastern and Western pears and their hybrids, characterized in that: The following steps are involved: Step 1, extracting the genomic DNA of the pear to be tested; Step 2: using the pear genomic DNA to be detected as a template, performing PCR amplification using the primer pair described in claim 2 to obtain a PCR amplification product; Step 3: Use 2% agarose gel electrophoresis to detect the size of the PCR amplification product: 468bp homozygous is Western pear, 183bp homozygous is Oriental pear, and 183 / 468 heterozygous is a hybrid of Eastern and Western pears.

4. A method for identifying Eastern and Western pears and their hybrids according to claim 3, characterized in that: The total volume of the PCR amplification system in step 2 is 20 μl, including 2 μl of 10 ng genomic template DNA, 10 μl of 2×Es TaqMasterMix, 2 μl of each of the 10 mM forward and reverse primers mentioned above, and 4 μl of ddH2O.

5. A method for identifying Eastern and Western pears and their hybrids according to claim 3, characterized in that: The PCR amplification program in step 2 is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 150 s, 35 cycles; and finally extension at 72°C for 10 min.

Images