A molecular marker based on chloroplast genome microsatellite and a method for identifying walnut germplasm and its genetic relationship

Abstract

The present application relates to a kind of molecular marker based on chloroplast genome microsatellite and the method for identifying walnut germplasm and its genetic relationship, belong to SSR molecular marker technical field.The present application is based on walnut chloroplast group gene screening and obtains the SSR molecular marker with the value of Juglans species identification, including SSR-P8 and SSR-P9, and synthesizes the primer with good polymorphism identification effect, can be used for the identification of eastern black walnut (Juglans nigra), Juglans major, north California black walnut (Juglans hindsii), small fruit black walnut (Juglans microcapa) and walnut (Juglans regia) 5 species germplasm, interspecific hybrid and genetic relationship.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of SSR molecular marker, and particularly relates to a SSR molecular marker based on chloroplast genome microsatellite, a kit and a method for identifying interspecific hybrid germplasm of Juglans and its genetic relationship based on the SSR fingerprint. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is publicly known.

[0003] In the 1930s, heart-shaped walnut (Juglans cordiformis), Jibao walnut (Juglans sieboldiana) and eastern black walnut (Juglans nigra), J. najor, J. hindsii and J. microcapa of Juglans nigra group were introduced into China. Chinese scientific researchers have made significant achievements in early fruiting, high yield, fast growth and disease-resistant walnut breeding by using rich walnut germplasm resources for interspecific and intraspecific hybridization breeding. Black walnut, which has high economic value, is an important timber and fruit-wood dual-purpose tree species, and is also one of the urban and rural greening tree species. It has the characteristics of cold tolerance, drought tolerance, salt and alkali tolerance, and disease and insect resistance. As one of the dominant tree species of fruit-wood dual-purpose, the wood structure of black walnut is extremely tight, with strong pressure and shock resistance, high mechanical strength, bright and beautiful color, and clear and beautiful texture, which is an ideal material for modern high-grade furniture, building decoration and handicraft carving.

[0004] The hybrid offspring obtained by hybridization often shows stronger growth rate and metabolic function than its parents, resulting in well-developed organs, increased body size, increased yield, or improved disease resistance, insect resistance, stress resistance, survival, reproductive ability, viability, etc. Interspecific hybridization belongs to distant hybridization, but mating is not easy to succeed. The interspecific hybridization of black walnut shows excellent performance, and analyzing its genetic background and genetic relationship can help to further improve the breeding efficiency.

[0005] Juglans is an outcrossing plant, long-term artificial selection and hybridization and natural hybridization in the same area lead to the complex genetic background of Juglans, and it is difficult to determine the real biological characteristics of Juglans by morphological classification method. Therefore, it is necessary to explore more effective methods for identification. SSR (Simple Sequence Repeat) molecular markers are designed based on highly conserved sequences, further fluorescent primers are synthesized, PCR amplification, capillary electrophoresis and other methods are used for large-scale detection, and the marker is a co-dominant marker, which shows Mendelian inheritance, good technical repeatability, easy operation, accuracy and reliability.

[0006] The chloroplast genome contains rich genetic information, but the structure and size are very conservative, and the genetic stability is high, and based on this, the information of the structure and sequence of the chloroplast genome has important value in revealing the origin, evolution and relationship of species.

[0007] The identification of Juglans varieties is more common in AFLP and SSR molecular markers, and the identification of Juglans interspecific hybrids and Juglans interspecific hybrids is less reported. Gao Yuna (2011) et al. found that Juglans microsatellite can be effectively amplified in Juglans relatives, which can be used for genetic diversity analysis and interspecific relationship research of Juglans. Chen Lingna (2011) used SSR method to construct the fingerprint of 35 main cultivars of Juglans regia, and Zhou Yubo also used SSR molecular markers to construct the fingerprint of 29 Sichuan Juglans varieties. With the progress of interspecific hybridization technology, more and more Juglans interspecific hybrids are obtained, but the research is relatively less, and the above SSR primers have not been applied to the identification of Juglans interspecific hybrids and genetic relationship. SUMMARY

[0008] Based on the above technical background, the purpose of the present application is to provide a SSR molecular marker which can be used for the identification of Juglans interspecific hybrids and genetic relationship. Based on the purpose, the present application takes plant tissue chloroplast genome as a screening object, and obtains a SSR molecular marker with good identification effect.

[0009] In a first aspect, the present application provides a walnut SSR molecular marker based on chloroplast genome microsatellite, wherein the walnut varieties are Juglans nigra, Juglans major, Juglans hindsii, Juglans microcapa and Juglans regia, and the SSR molecular marker comprises SSR-P8 and SSR-P9, wherein the SSR-P8 is amplified by primers with sequences shown in SEQ ID NO: 1-2, and the SSR-P9 is amplified by primers with sequences shown in SEQ ID NO: 3-4, and the amplification object is chloroplast genome.

[0010] In a second aspect, the present application provides a kit comprising the primers shown in SEQ ID NO: 1-4.

[0011] Preferably, the kit further comprises a PCR amplification system, which at least comprises buffer, dNTP, DNA polymerase and ddH2O; further, the DNA polymerase is Taq enzyme.

[0012] In a third aspect, the present application provides the primers with sequences shown in SEQ ID NO: 1-4, the walnut SSR molecular marker of the first aspect, and the kit of the second aspect for use in any one of the following aspects:

[0013] (1) for walnut germplasm genetic diversity analysis or seed quality identification;

[0014] (2) for walnut variety identification, kinship analysis or maternal tracing;

[0015] (3) for walnut genetic map or fingerprint map establishment.

[0016] In a fourth aspect, the present application provides a method for constructing an SSR fingerprint map of black walnut and common walnut varieties, wherein the black walnut is Juglans nigra, Juglans major, Juglans hindsii and Juglans microcapa, and the method comprises the following steps:

[0017] Extracting chloroplast genome of walnut to be identified, and performing PCR amplification by using the primers shown in SEQ ID NO: 1-4 or the kit of the second aspect, and separating and detecting the PCR amplification product by electrophoresis to construct the SSR fingerprint map of the walnut varieties.

[0018] The SSR fingerprint map constructed based on the SSR-P8 and SSR-P9 is shown in the following table.

[0019]

[0020] Preferably, the extraction method of the chloroplast genome can be achieved based on the conventional method in the art, for example, extraction by a commercially available kit, and in one embodiment verified by the present application, the chloroplast genome is extracted by a modified CTAB method.

[0021] Preferably, the PCR amplification procedure is as follows: 92-96℃ pre-denaturation for 3-6min; 92-96℃ denaturation for 30s, 50-55℃ (the annealing temperature fluctuates around 54℃) recombination for 35s, 70-74℃ extension for 40s, a total of 30-35 cycles; and finally 70-74℃ extension for 3min.

[0022] Preferably, the electrophoretic separation mode includes but is not limited to agarose gel electrophoresis detection, polyacrylamide gel electrophoresis or capillary electrophoresis.

[0023] In the fifth aspect of the present application, a variety identification method of black walnut and ordinary walnut is provided, the identification method comprising: establishing the SSR fingerprint of the walnut variety to be identified according to the method of the fourth aspect, and identifying the walnut variety according to the information of two DNA fragments of SSR-P8 and SSR-P9.

[0024] In the sixth aspect of the present application, a kinship identification method of hybrid offspring of black walnut and ordinary walnut is provided, the identification method comprising: establishing the SSR fingerprint of the variety to be identified according to the method of the fourth aspect, and the SSR fingerprint of the hybrid is consistent with that of the female parent.

[0025] The beneficial effects of one or more of the above technical solutions are:

[0026] 1. The SSR molecular marker provided by the present application is amplified based on the chloroplast genome of plants, and since the chloroplast genome belongs to a sequence with high conservation degree, the corresponding SSR molecular marker provided by the present application also has good identification effect.

[0027] 2. For the identification of five species of Juglans, i.e., eastern black walnut (Juglans nigra), giant walnut (Juglans major), northern California black walnut (Juglans hindsii), small fruit black walnut (Juglans microcapa) and walnut (Juglans regia), and interspecific hybrids, the present application provides two groups of SSR molecular markers, and the five species of Juglans can be distinguished by simultaneously using the two groups of SSR molecular markers, which has lower amplification cost and faster detection speed.

[0028] 3. In view of the problem that the genetic relationship of hybrid species obtained through interspecific hybridization is unknown, the application provides a method for identifying the genetic relationship of a hybrid offspring of a black walnut and a common walnut, which can identify the female parent of the hybrid species, and the identification result is efficient and accurate. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the application, and their

[0030] Figure 1 Corresponding to the peak values of eastern black walnut (Juglans nigra), giant walnut (Juglans major), northern California black walnut (Juglans hindsii), small fruit black walnut (Juglans microcapa) and walnut (Juglans regia). DETAILED DESCRIPTION

[0031] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains.

[0032] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, they indicate the presence of a feature, step, operation, device, component and / or combination thereof.

[0033] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0034] Example 1 primer obtaining method

[0035] The hybrid species obtained by distant hybridization of black walnut and common walnut with clear parent were used to extract chloroplast genome:

[0036] 1. Extraction of total DNA from plant tissue

[0037] The improved CTAB method was used to extract the chloroplast genome DNA.

[0038] 2. Quality and concentration detection of genomic DNA

[0039] After extraction, the quality of DNA was detected by 1% agarose gel electrophoresis, and the concentration was detected by Qubit 3.0 (Thermo Fisher Scientific, USA) fluorescence quantifier.

[0040] 3. Gene sequencing

[0041] After quality inspection, Illumina HiSeq X Ten platform was used for double-end sequencing, the sequencing read length was 150 bp, and the sequencing depth was 5G.

[0042] 4. Chloroplast genome sequence assembly and annotation

[0043] After obtaining the sequencing raw data (Raw Data), the low-quality sequences in the raw data were filtered out using Trimmomatic. software to obtain effective data (Clean Data). Using the walnut chloroplast genome published by NCBI as a reference (NC_028617), the sequence of the chloroplast genome was extracted from the obtained Clean Data, and ABySS v2.1.5 software was used for assembly, with Kmer Size set to 127; the assembled chloroplast genome was annotated using Plann 1.1.2 software based on the reference genome, and the annotation results were corrected using Apollo v2.3.1 software. Using OGDRAW tool, the positions of IR (Inverted Repeat Region), LSC (Large Single Copy-Region) and SSC (Small Single Copy-Region) of the chloroplast genome were marked to generate a complete circular chloroplast genome physical map.

[0044] 5. Chloroplast genome repeat sequence structure analysis

[0045] Scattered repeat sequences were analyzed by REPuter software to identify forward, reverse, complementary and palindrome repeats in the genome. The parameters were set as follows: Hamming distance = 3 (sequence consistency ≥ 90%), repeat base unit n ≥ 30 bp; MISA software was used for simple sequence repeat (SSR) prediction, and the parameters were set as follows: minimum SSR sequence length was 10 bp, and minimum repeat number threshold for single nucleotide to six nucleotides was set to 10, 5, 4, 3, 3 and 3, respectively.

[0046] 6. Primer design and synthesis

[0047] Based on the above step 5, the SSR sequence is initially selected, and the primer is designed and synthesized by using Primer3 software. After PCR amplification, the product is detected by 1% agarose electrophoresis to detect the amplification condition. The product with good amplification is used for subsequent experiments.

[0048] 7. Polyacrylamide gel electrophoresis detection

[0049] Based on the above step 6, the product with good amplification is detected by polyacrylamide gel electrophoresis, the primer with obvious polymorphism is screened, and the size of the amplified fragment is preliminarily counted, so as to carry out the next step of capillary electrophoresis fluorescence primer combination.

[0050] 8. Fluorescent capillary electrophoresis detection

[0051] Through gel electrophoresis verification, two pairs of primers with good polymorphism are designed in step 7, and capillary electrophoresis is carried out. After mixing formamide and molecular weight marker in a volume ratio of 100:1, 15uL is added to the loading plate, 1uL of 10-fold diluted PCR product is added, and then 3730XL sequencer is used for capillary electrophoresis to obtain the fingerprint of the sample. After the combination of the two pairs of primers, five species of eastern black walnut (Juglans nigra), giant walnut (Juglans major), northern California black walnut (Juglans hindsii), small fruit black walnut (Juglans microcapa) and walnut (Juglans regia) can be accurately distinguished. The primer sequences are shown in the following table 1:

[0052] Table 1

[0053]

[0054] Based on the primers in table 1, the fingerprint of five species of eastern black walnut (Juglans nigra), giant walnut (Juglans major), northern California black walnut (Juglans hindsii), small fruit black walnut (Juglans microcapa) and walnut (Juglans regia) is established:

[0055] Table 2

[0056]

[0057] According to the fingerprint constructed according to the above primers, the fingerprint of the hybrid is consistent with that of the mother, and the genetic relationship of the hybrid offspring of black walnut can be identified according to the polymorphism results of the above fingerprint.

[0058] Example 2

[0059] Select 3 accessions of each of J. nigra, J. regia, J. major, J. hindsii, J. hindsii, J. nigra x J. regia and J. major x J. regia, 50 accessions of each of J. nigra x J. regia and J. major x J. regia, and 112 accessions in total, with the female parent of the hybrid accessions being clear, and using P8 and P9 primers to perform capillary electrophoresis on the 112 accessions, with the specific steps being as follows:

[0060] 1. DNA extraction

[0061] Genomic DNA was extracted from the leaves of the 112 collected Juglans accessions by using a modified CTAB method.

[0062] 1) Preheat the CTAB extraction solution in a 65°C water bath;

[0063] 2) Grind the sample in liquid nitrogen, transfer the powdered material to a 2 mL centrifuge tube, and add the preheated CTAB extraction solution (3-5 ml of extraction solution per gram of sample), incubate at 65°C for 30-60 min, and mix gently every 10 min;

[0064] 3) Centrifuge at 11000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube;

[0065] 4) Add an equal volume of phenol / chloroform (1:1), mix thoroughly, centrifuge at 11000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube;

[0066] 5) Add an equal volume of chloroform, mix thoroughly, centrifuge at 11000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube;

[0067] 6) Repeat steps 4 and 5;

[0068] 7) Add 2 / 3 volume of isopropanol and mix, let stand at room temperature for 15 min;

[0069] 8) Centrifuge at 11000 rpm for 6 min, discard the supernatant;

[0070] 9) Rinse the precipitate with 70% ethanol once, centrifuge at 11000 rpm for 2 min at room temperature, discard the supernatant, and repeat the washing once;

[0071] 10) Add 40 ul of 1x TE solution (sterilized) to the precipitate, mix first, then let stand for 30 min, and mix 1-2 times during the process.

[0072] 11) Take 2-3 ul of the extraction product for 2.0% agarose gel electrophoresis detection, and store the rest at -20°C for later use.

[0073] 2. Perform PCR amplification on the test samples using the two selected primer pairs cpSSR-P8 and cpSSR-P9

[0074] 1)PCR reaction system:

[0075] SSR primer system (20ul) : ddH2O 14.8ul, dNTP 0.4ul, Buffer 2ul, F 0.3ul (20uM), R 0.3ul (uM), DNA template 2ul, Taq 0.2ul.

[0076] 2) PCR reaction using the following cycle parameters:

[0077] SSR PCR amplification program: 94℃ pre-denaturation 5min; 94℃ denaturation 30S, 54℃ (annealing temperature fluctuates above and below 54℃) 35s, 72℃ extension 40S, a total of 35 cycles; final 72℃ extension 3min.

[0078] 3, agarose gel electrophoresis detection

[0079] Take 3ul of PCR product for detection in 1% agarose gel electrophoresis, and select the good amplification effect for subsequent PAGE detection.

[0080] 4, polyacrylamide gel electrophoresis detection

[0081] 1) Assemble glass plate

[0082] Wash the glass plate thoroughly with dishwashing liquid and drain. Rub the short glass plate with affinity silane twice, and the long glass plate with release silane once. Place the two glass plates neatly with the edges aligned, and fix them on the base with clamps. Insert the matching comb and mark the glue pouring position 1-2 cm below the comb. Remove the comb.

[0083] 2) Preparation and pouring of separation gel and concentration gel

[0084] Take 75ml 5% acrylamide gel in 30℃ warm water, add 80ul TEMED, 160ul ammonium persulfate, mix well, pour into the two glass plates, check for air bubbles, insert the comb, clamp it, and let it solidify for more than an hour.

[0085] 3) Sample processing

[0086] Take 4ul of the preliminary selected PCR product, add 4x loading buffer, mix well. Denature at 95℃ or in boiling water bath for 5min, then immediately put it on ice for standby.

[0087] 4) Assemble electrophoresis tank and pre-electrophoresis

[0088] After the glass plate is taken off, it is placed vertically against the power supply frame in the electrophoresis tank, with the concave edge of the gel plate facing the power supply frame. The gel plate is fixed in the power supply tank as required. 1x TBE electrophoresis buffer is added, and the power supply frame in the middle of the two gel plates (the upper tank) needs to be filled with electrophoresis buffer. The electrophoresis buffer in the upper tank is not in communication with the electrophoresis buffer added in the electrophoresis tank (the lower tank). The comb in the gel plate is gently pulled out. The power supply is started (the voltage is adjusted to 3000v, the current is adjusted to 200mA, and the power is adjusted to 90W. In fact, it is electrophoresis at constant power), and pre-electrophoresis is performed for about half an hour.

[0089] 5) Spotting

[0090] The red comb is inserted, and the treated sample liquid and 20bp marker are slowly added into the comb hole in the glass plate, and attention should be paid not to disperse the sample.

[0091] 6) Electrophoresis

[0092] The power supply is turned on, and electrophoresis is started. After about an hour, when the sample bromophenol blue strip on the gel plate is observed to move close to the bottom end, the electrophoresis is stopped, the power supply is turned off, and the glass plate is taken out.

[0093] 7) Staining

[0094] The two glass plates are gently pried apart with a spatula, and the red edge strip is taken off. The glass plate with the attached gel is placed in clean water for about 3 minutes (shaking), then placed in silver nitrate solution for 3 minutes of shaking, taken out, rinsed in clean water, and placed in developing solution until the strip is stained, then immediately taken out and rinsed in clean water.

[0095] 8) Scanning

[0096] After the gel on the glass plate is dry, the gel electrophoresis result is scanned with a scanner, the primer polymorphism is observed, and the size of the amplified fragment is preliminarily counted, so as to facilitate the next step of capillary electrophoresis fluorescence primer combination.

[0097] 6、Capillary electrophoresis

[0098] After the formamide and the molecular weight marker are mixed at a volume ratio of 100:1, 15uL is taken and added to the loading plate, 1uL of 10-fold diluted PCR product is added, and then capillary electrophoresis is performed using a 3730XL sequencer to obtain the fingerprint of the sample.

[0099] The results show that the selected primer can accurately identify the four species of eastern black walnut, northern California black walnut, giant walnut, and small fruit black walnut, and the 50 hybrid seedlings (J. nigra x J. regia) and the 50 hybrid seedlings (J. major x J. regia) are consistent with their maternal species.

[0100] Table 3

[0101]

[0102]

[0103] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the scope of the present application.

Claims

1. A chloroplast genome microsatellite-based SSR molecular marker, characterized in that, The SSR molecular markers are SSR-P8 and SSR-P9, wherein the SSR-P8 is amplified by the primers shown as SEQ ID NO: 1-2, the SSR-P9 is amplified by the primers shown as SEQ ID NO: 3-4, the amplification object is a walnut chloroplast genome, and the walnut variety is selected from the group consisting of eastern black walnut Juglans nigra , giant walnut Juglans major , northern California black walnut Juglans hindsii , small fruit black walnut Juglans microcarpa , and walnut Juglans regia .

2. The application of the combination of primer pairs SEQ ID NO: 1-2 and SEQ ID NO: 3-4 or the SSR molecular marker of claim 1 in any one of the following aspects: (1) walnut germplasm genetic diversity analysis and seed quality identification; (2) walnut variety identification or fingerprint establishment; (3) walnut genetic relationship analysis or maternal tracing application; For applications (1)-(2), the walnut variety is selected from the group consisting of Eastern Black Walnut Juglans nigra , Chandler Walnut Juglans major , Northern California Black Walnut Juglans hindsii , Stuart Walnut Juglans microcarpa , and English Walnut Juglans regia ; For use (3) the walnut variety is selected from the group consisting of hybrids Juglans nigra × Juglans regia and Juglans major × Juglans regia .

3. A black walnut and common walnut seed variety Juglans regia The method for constructing the SSR fingerprint of the black walnut and common walnut seed variety, characterized in that, The black walnut variety is selected from the group consisting of eastern black walnut Juglans nigra , kwakiut walnut Juglans major , northern California black walnut Juglans hindsii , and small fruit black walnut Juglans microcarpa , and the fingerprint construction method comprises the following steps: The chloroplast genome of the walnut to be identified is extracted, the microsatellite molecular marker SSR-P8 is amplified by PCR using the primer of SEQ ID NO: 1-2, and the microsatellite molecular marker SSR-P9 is amplified by PCR using the primer of SEQ ID NO: 3-4, the PCR amplification product is separated and detected by capillary electrophoresis, and the SSR fingerprint of the walnut variety is constructed.

4. The method of claim 3, wherein the SSR fingerprint is constructed by: The extraction method of the chloroplast genome is improved CTAB method.

5. The method of claim 3, wherein the SSR fingerprint is constructed by: The PCR amplification program is as follows: 92-96℃ pre-denaturation for 3-6min; 92-96℃ denaturation for 30s, annealing temperature is 50-55℃ for 35s, 70-74℃ extension for 40s, a total of 30-35 cycles; finally 70-74℃ extension for 3min.

6. A method for identifying a variety of black walnut and English walnut, characterized by, The identification method comprises establishing the SSR fingerprint of the walnut variety to be identified according to the method of any one of claims 3-5, and identifying the walnut variety according to the SSR fingerprint of the two molecular markers SSR-P8 and SSR-P9, and the fingerprint information is shown in the following table: 。 7. A method for identifying the kinship of hybrid offspring of black walnut and common walnut, characterized in that, The chloroplast genome of the walnut to be identified is extracted, the microsatellite molecular marker SSR-P8 is amplified by PCR using the primers shown as SEQ ID NO: 1-2, the microsatellite molecular marker SSR-P9 is amplified by PCR using the primers shown as SEQ ID NO: 3-4, the PCR amplification products are separated and detected by capillary electrophoresis, the SSR fingerprint of the walnut variety is constructed, and the female parent of the hybrid offspring is traced according to the SSR fingerprint of the two molecular markers SSR-P8 and SSR-P9, the SSR fingerprint of the hybrid offspring is consistent with that of the female parent, and the hybrid variety is selected from Juglans nigra × Juglans regia and Juglans major × Juglans regia , and the fingerprint information is shown in the following table. 。

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