SSR molecular marker combination and application thereof in detection of carya illinoensis varieties

Abstract

The present application relates to the technical field of molecular marker, and more particularly to a kind of SSR molecular marker combination and its application in detecting pecan variety.The SSR molecular marker combination includes: SSR12-1 and SSR6-2;The SSR12-1 is located at Chr12:22685469bp-22685482bp, and repeat sequence is CT, and repeat 7 times;The SSR6-2 is located at Chr06:7785735bp-7785761bp, and repeat sequence is CTT, and repeat 9 times.The molecular marker provided by the present application can accurately and quickly identify pecan variety, and the result is stable and reliable, and it has important significance for the identification of pecan genetic resources.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to an SSR molecular marker combination and its application in detecting thin-shelled pecan varieties. Background Technology

[0002] Thin-shelled pecan (scientific name: *Carya illinoinensis* (Wangenh.) K. Koch), also known as American pecan, is a perennial, tall, deciduous tree belonging to the genus *Carya* in the family Juglandaceae. As a world-renowned high-grade nut, oilseed, and timber tree, it has high application value. The breeding of superior thin-shelled pecan varieties relies heavily on seed propagation. However, due to the long generation cycle of trees and the large coefficient of variation in offspring, introduced varieties are primarily used. Because of the long history of introduction, and the fact that early introductions were mostly based on cuttings, it is difficult to distinguish varieties morphologically, inevitably leading to confusion between varieties and resulting in different species with the same name or the same species having different names. This poses a significant challenge to the germplasm evaluation, genetic improvement, and widespread promotion of thin-shelled pecans.

[0003] The Caddo pecan variety is a protandrous cultivar. Caddo nuts have an average weight of 5.4g and are characterized by vigorous growth, high fruit set, high and stable yield, strong disease resistance, and adaptability. Pecan seedlings have a relatively long growth period, requiring 4-6 years, and are dioecious, with male and female flowers blooming at different times. Therefore, proper pairing of different varieties is often necessary to achieve sufficient pollination and stable yields. Thus, variety identification is particularly important in the early stages of orchard establishment. In production practice, the phenotypic traits of the same variety vary significantly at different developmental stages, under different geographical conditions, and under different cultivation conditions. Therefore, relying on empirical variety identification often leads to errors. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an SSR molecular marker combination and its application in detecting thin-shelled pecan varieties.

[0005] In a first aspect, the present invention provides an SSR molecular marker combination comprising: SSR12-1 and SSR6-2; wherein SSR12-1 is located at Chr12:22685469bp-22685482bp, with the repeat sequence being CT, repeated 7 times; and SSR6-2 is located at Chr06:7785735bp-7785761bp, with the repeat sequence being CTT, repeated 9 times.

[0006] Furthermore, the genomic version of the SSR molecular marker combination is Cillinoinensis_573_v1.1.

[0007] Secondly, the present invention provides a primer pair for detecting the SSR molecular marker combination, comprising: Chr12-SSR1 and Chr6-SSR2;

[0008] The Chr12-SSR1 includes:

[0009] Upstream primer: 5′-CCCCACTCCCCCAGATTTTC-3′

[0010] Downstream primer: 5′-AGGAGCTCAACATGAGTGCC-3′;

[0011] The Chr6-SSR2 includes:

[0012] Upstream primer: 5′-CAACAAGCAACACCCTCAGC-3′

[0013] Downstream primer: 5′-AAGGAAAGCGGCATCGAGAA-3′.

[0014] The present invention further provides a kit comprising the primer pair.

[0015] Thirdly, the present invention provides a method for detecting thin-shelled pecan varieties, comprising:

[0016] Genomic DNA was extracted from the thin-shelled pecans to be tested; the genomic DNA was amplified by PCR using the primer pair; and the variety of the thin-shelled pecans to be tested was determined based on the PCR amplification results.

[0017] Furthermore, the determination of the variety of the thin-shelled pecan to be tested based on the PCR amplification results includes:

[0018] If both primer pairs amplify two bands, then the thin-shelled pecan can be identified as the Caddo variety.

[0019] Furthermore, the statement that if both primer pairs amplify two bands includes:

[0020] Primer pair Chr12-SSR1 amplification yielded two bands of 237bp and 239bp, while primer pair Chr6-SSR2 amplification yielded two bands of 160bp and 163bp.

[0021] Furthermore, the reaction conditions for the PCR amplification include:

[0022] 95-97℃, 5-10 min; 94-98℃ denaturation 30-45 s, 54-58℃ annealing 45-60 s, 72-75℃ extension 1-2 min, 29-32 cycles; 72-75℃ extension 7-10 min.

[0023] The present invention further provides the application of the SSR molecular marker combination, the primer pair, or the kit in the detection of thin-shelled pecan varieties.

[0024] Furthermore, the thin-shelled pecan variety is the Caddo thin-shelled pecan variety.

[0025] The present invention has the following beneficial effects:

[0026] This invention identifies two SSR loci through bioinformatics screening and designs corresponding primer pairs for these loci. PCR detection using these primer pairs targeting these two loci allows for the identification of thin-shelled pecan varieties; specifically, when both primer pairs amplify two bands, the detected pecan variety is identified as Caddo. This invention provides SSR molecular markers that can accurately distinguish the Caddo pecan variety (other varieties cannot amplify two bands with both primer pairs), which is of great significance for the identification of existing thin-shelled pecan varieties. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products of 18 varieties using the two primer pairs Chr12-SSR1 and Chr6-SSR2 provided in Example 1 of this invention.

[0029] Figure 2 This is a capillary electrophoresis image of the PCR amplification product of the thin-shelled pecan variety Caddo, obtained by amplification with the primer pair Chr12-SSR1 provided in Example 1 of this invention.

[0030] Figure 3 This is a capillary electrophoresis image of the PCR amplification product of the thin-shelled pecan variety Caddo, obtained by amplification with the primer pair Chr6-SSR2 provided in Example 1 of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] The 18 thin-shelled pecan varieties used in the following examples were all obtained from the germplasm resource bank of the Subtropical Forestry Research Institute of the Chinese Academy of Forestry. The variety names are as follows: Mcmillan, Greenriver, Creek, Golby, Mohawk, Peruque, Forkert, Kanza, Syrup Mill, Barton, Caddo, Hopi, Sruart, Choctaw, Gloriu Grande, Rosey, Candy, and Mouidan.

[0033] Example 1: Development of SSR Molecular Markers

[0034] 1. In this embodiment, more than 410,000 SSR sites were discovered from the whole genome sequence of pecan, and more than 340,000 pairs of specific primers were designed in batches. These primers were further screened, and the main screening criteria were as follows: (1) the type of SSR site is 2 to 6 base repeats; (2) the annealing temperature difference between the upstream and downstream primers does not exceed 1℃; (3) the target product is 150bp to 350bp. 32 pairs of primers were initially screened and synthesized.

[0035] 2. Extracting plant genomic DNA using the CTAB method

[0036] (1) Grind 0.5g of tender leaves of thin-shelled pecan thoroughly with liquid nitrogen and put them into a 2ml centrifuge tube.

[0037] (2) Add 600 μl of preheated CTAB extraction buffer to the centrifuge tube, incubate at 65°C for 30 min, and invert and mix several times every 10 min.

[0038] (3) Add 600 μl of phenol: chloroform: isopropanol (25:24:1), mix by inverting, centrifuge at 12000 rpm for 10 min, and transfer the upper aqueous phase to a new centrifuge tube.

[0039] (4) Add an equal volume of chloroform:isopropanol (24:1), mix by inverting, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0040] (5) Add 0.4 times the volume of isopropanol and place at -20℃ for more than 30 minutes.

[0041] (6) Centrifuge at 12000 rpm for 30 min, discard the supernatant, and wash twice with 75% ethanol;

[0042] (7) After opening the centrifuge tube and letting it dry, add 100 μl ddH2O to dissolve the DNA. Use Nanodrop2000 to detect the DNA concentration and purity, and use 0.8% agarose gel electrophoresis to detect its integrity.

[0043] 3. PCR detection

[0044] The SSR primers used for initial screening were synthesized by Zhejiang Shangya Biotechnology Co., Ltd., while the primers with adapters and universal fluorescent primers were synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd.

[0045] The PCR amplification system and reaction conditions are as follows:

[0046] Table 1 PCR amplification system

[0047]

[0048]

[0049] Table 2 PCR reaction conditions:

[0050]

[0051] The amplification products were subjected to agarose gel electrophoresis for preliminary primer screening. Details are as follows:

[0052] Prepare a 1% agarose gel, add 3 μL of the PCR amplification product and mix with 0.5 μL of bromophenol blue, then incubate at 120V for 30 minutes. Observe the electrophoresis results under UV light and take pictures.

[0053] 4. Capillary electrophoresis to detect band size

[0054] (1) Mix ABI HiDi Formamide and the internal standard of ABI GeneScan 500LIZ Size Standard at a ratio of 99:1 to prepare a mixture.

[0055] (2) Dispense 10 μl of the mix into each well of a 96-well reaction plate. Place the 96-well plate in a plate centrifuge and centrifuge at 1200 rpm for 15 s.

[0056] (3) Add 1 μl of PCR sample to each well of a 96-well plate, place it in a plate centrifuge, and centrifuge at 1200 rpm for 15 seconds.

[0057] (4) Seal the 96-well plate with sealing film and place it in a PCR instrument. The denaturation program is 98°C for 5 min, without heating the hot cap. After the program is completed, immediately place the 96-well plate on an ice-water mixture for rapid cooling.

[0058] (5) Place the 96-well plate in a flat plate centrifuge and centrifuge at 1200 rpm for 15 seconds.

[0059] (6) Load the sample into the 3730 sequencer for capillary electrophoresis.

[0060] (7) Use Genemapper software to analyze SSR data.

[0061] The final two primer pairs exhibited good amplification performance and strong stability, and their sequences are as follows:

[0062] The Chr12-SSR1 includes:

[0063] Upstream primer: 5′-CCCCACTCCCCCAGATTTTC-3′

[0064] Downstream primer: 5′-AGGAGCTCAACATGAGTGCC-3′;

[0065] The Chr6-SSR2 includes:

[0066] Upstream primer: 5′-CAACAAGCAACACCCTCAGC-3′

[0067] Downstream primer: 5′-AAGGAAAGCGGCATCGAGAA-3′.

[0068] The SSR loci it targets (genome version: Cillinoinensis_573_v1.1) are as follows:

[0069] SSR12-1 is located at Chr12:22685469bp-22685482bp, with CT repeat sequence, repeated 7 times; SSR6-2 is located at Chr06:7785735bp-7785761bp, with CTT repeat sequence, repeated 9 times.

[0070] This invention employed the primers Chr12-SSR1 and Chr6-SSR2 to perform the above-described procedure for extracting plant genomic DNA from 18 different thin-shelled pecan varieties, followed by capillary electrophoresis detection. Thirty samples were collected from each pecan variety for testing, and the results were consistent, indicating good reproducibility. One example of the results is shown below: Agarose gel electrophoresis results are as follows... Figure 1 As shown; capillary electrophoresis results are as follows Figure 2 and Figure 3 As shown, Figure 2These are the capillary electrophoresis results for primer Chr12-SSR1. Figure 3 The capillary electrophoresis results for primer Chr6-SSR2 are shown in the table below. The band sizes of the amplified products are as follows:

[0071] Table 3. Band sizes of amplified products from 18 different thin-shelled pecan varieties.

[0072]

[0073]

[0074] In Table 3, "-" indicates that no fragment was amplified.

[0075] The results above show that, based on the SSR molecular marker typing results, it can be determined whether the sample belongs to the Caddo thin-shelled pecan variety, as detailed below:

[0076] (1) After amplification using Chr12-SSR1 primers, Golby, Barton, Caddo and Stuart all had two bands of about 237bp and 239bp, which can distinguish them from the 18 varieties.

[0077] (2) After amplification using Chr6-SSR2 primers, all five varieties—Mcmillan, Mohawk, Caddo, Gloriu Grande, and Rosey—showed two bands of approximately 160 bp and 163 bp. Therefore, the variety that could amplify two bands both before and after the amplification was identified as Caddo.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting thin-shelled pecan varieties, characterized in that, include: Genomic DNA was extracted from the thin-shelled pecans to be tested; the genomic DNA was amplified by PCR using primer pairs; If both primer pairs amplify two bands, then the thin-shelled pecan can be identified as the Caddo variety. The primer pairs include: Chr12-SSR1, Chr6-SSR2; The Chr12-SSR1 includes: Upstream primer: 5′-CCCCACTCCCCCAGATTTTC-3′ Downstream primer: 5′-AGGAGCTCAACATGAGTGCC-3′; The Chr6-SSR2 includes: Upstream primer: 5′-CAACAAGCAACACCCTCAGC-3′ Downstream primer: 5′-AAGGAAAGCGGCATCGAGAA-3′.

2. The method according to claim 1, characterized in that, If both primer pairs amplify two bands, it includes: Primer pair Chr12-SSR1 amplification yielded two bands of 237bp and 239bp, while primer pair Chr6-SSR2 amplification yielded two bands of 160bp and 163bp.

3. The method according to any one of claims 1-2, characterized in that, The reaction conditions for the PCR amplification include: 95~97℃, 5~10min; 94~98℃ denaturation 30~45s, 54~58℃ annealing 45~60s, 72~75℃ extension 1~2min, 29~32 cycles; 72~75℃ extension 7~10min.

4. Application of primer pairs or kits in the detection of the Caddo pecan variety; The primer pair includes: Chr12-SSR1, Chr6-SSR2; The Chr12-SSR1 includes: Upstream primer: 5′-CCCCACTCCCCCAGATTTTC-3′ Downstream primer: 5′-AGGAGCTCAACATGAGTGCC-3′; The Chr6-SSR2 includes: Upstream primer: 5′-CAACAAGCAACACCCTCAGC-3′ Downstream primer: 5′-AAGGAAAGCGGCATCGAGAA-3′; The kit includes the primer pair.

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