SSR marker detection primers for potato diploid identification and their uses

Abstract

The present invention relates to the field of biotechnology, and specifically relates to an SSR marker detection primer for potato diploid identification and its use, which can be used for rapid and accurate identification and analysis of the ploidy and genomic purity of the chromosome-doubled population of "Lishu No. 6", and has important application value in the research of potato breeding parent selection and hybrid offspring selection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an SSR marker detection primer for potato diploid identification and a use thereof. Background Art

[0002] Potato is a general term for some species with edible tubers in the genus Solanum of the Solanacea family, native to the Andes Mountains in South America. At present, 235 potato species have been discovered, including 7 cultivated species including common cultivated species and original cultivated species, and 228 wild species. The ploidy of these species is also relatively complex, including diploid (such as S. phureja), triploid (such as S. juzepczukii), tetraploid (such as S. tuberosum) and pentaploid (such as S. curtilobum), and most of them are diploid. It is difficult to hybridize between materials with different ploidy. In order to develop and utilize the abundant diploid resources, it is necessary to reduce the ploidy of the tetraploid common cultivated species S. tuberosum to diploid.

[0003] The commonly used potato ploidy identification methods in scientific research include: (1) Plant morphology identification: Compared with materials with lower ploidy, materials with higher ploidy have thicker leaves, thicker stems, larger flowers and fruits, etc. However, the role of genes controlling various traits is not very clear at present, and there are many exceptions. This method can only be used as an auxiliary means of ploidy identification. (2) Chloroplast counting in stomatal guard cells: The number of chloroplasts in the stomatal guard cells of a species is basically constant, and its number usually increases with the increase of ploidy. Therefore, the ploidy of the species can be roughly determined by the number of chloroplasts in stomatal guard cells. (3) Flow cytometry: Flow cytometry can directly measure the DNA content of cells, thereby identifying the ploidy of plants. However, this method cannot currently determine aneuploidy in organisms. (4) Chromosome counting: The ploidy of organisms is related to the number of chromosomes in cells. Selecting cells in the middle and late stages of cell division to count chromosomes is the most intuitive and accurate method for ploidy identification. However, this method has low work efficiency and requires high experimental skills of personnel. (5) Molecular marker identification: Molecular markers are usually a special DNA sequence in the genome. By detecting the differences in these DNA sequences, they can be used as auxiliary evidence to determine the gene (group) origin and ploidy of the plant. (6) Whole genome sequencing: The whole genome of the experimental material is sequenced and assembled, and the ploidy is determined based on the assembled chromosome sequence. However, this method is expensive and requires very high skills of the operator.

[0004] Zheng Yingzhuan et al. have studied the identification of triploid, tetraploid and diploid potatoes in the potato "Cooperation 88" chromosome-doubling population. In this study, 7 pairs of SSR primers were used to analyze the genetic relationship among diploid, triploid and tetraploid in the C88 chromosome-doubling population. According to the SSR detection results, the misdetection rate of ploidy in the chromosome-doubling population by these 7 pairs of primers was 12.0%. When these 7 pairs of primers were used to detect the chromosome-doubling population of "Lishu 6", the results were chaotic and could not be used for the ploidy detection of the parents and the chromosome-doubling population of this population. Therefore, it is necessary to find SSR marker primers suitable for the chromosome-doubling population of "Lishu 6" to quickly and accurately identify the ploidy of potatoes. Summary of the Invention

[0005] In order to overcome the above deficiencies in technical defects, the present invention provides an SSR marker detection primer for potato diploid identification and its use, which can be used to quickly identify the ploidy of the high-yield potato variety "Lishu 6" and its derived materials.

[0006] To achieve the above object, the present invention is achieved through the following solutions:

[0007] The female parent "Lishu 6" was genome sequenced on the Illumina xplus sequencing platform and the PacBio sequencing platform, and the second-generation and third-generation sequencing results were assembled by SOAPdenovo and HifiAsm for whole-genome sequence splicing. SSR scanning analysis was performed on the assembled genome of "Lishu 6", and primers were designed by extending 100 - 200 bp on both wings of the obtained SSR fragments to obtain corresponding detection primers. PCR amplification was performed with the detection primers in the chromosome-doubling population of "Lishu 6", and the amplification products were electrophoretically detected. According to the electrophoresis results, the inventors determined 6 pairs of primers that can be used to combine and distinguish the ploidy of this population (the screening process is shown in Figure 1 ), which can be used to quickly and accurately identify and analyze the ploidy and genomic purity of the chromosome-doubling population of "Lishu 6".

[0008] Therefore, in the first aspect, the present invention provides an SSR marker detection primer for potato diploid identification, including the following primer pairs:

[0009] Primer pair A1: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;

[0010] Primer pair A2: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;

[0011] Primer pair A3: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively;

[0012] Primer pair B1: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively;

[0013] Primer pair G3: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively;

[0014] Primer pair G4: includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively.

[0015] The present invention uses these 6 pairs of primers for ploidy detection of the parents and the doubled haploid population of the potato tetraploid variety "Lishu No. 6", with an accuracy rate as high as 97.8%.

[0016] Furthermore, the nucleotide sequences of the primers are (a), (b), or (c) to achieve the same detection purpose;

[0017] (a) nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.12;

[0018] (b) nucleotide sequences that hybridize with the nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.12 under stringent conditions and the encoded nucleotide sequences;

[0019] (c) nucleotide sequences that have more than 80% homology with the nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.12 and the encoded nucleotide sequences.

[0020] In some specific embodiments, the nucleotide sequences of the SSR marker detection primer pair group provided by the present invention have 80% identity with the sequences shown in SEQ ID NO.1 - SEQ ID NO.12; preferably 85% identity, more preferably 90% identity, more preferably 95% identity, and most preferably 99% identity.

[0021] Exemplarily, the "stringent conditions" as described herein refer to conditions under which a probe will hybridize to its target sequence to a detectable extent that exceeds hybridization to other sequences (such as at least 2-fold over background). Stringent conditions are sequence-dependent and vary depending on the environment. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified. Optionally, the stringent conditions can be adjusted to allow for some sequence mismatches, such that lower degrees of similarity are detected. These nucleotide sequences that hybridize under stringent conditions can be used, for example, to express variant proteins of SEQ ID NO.1 or as primers, probes, exogenous donor sequences, guide RNAs, antisense RNAs, shRNAs, and siRNAs.

[0022] In a second aspect, the present invention provides a kit for identifying potato diploids, comprising the above primer pair set.

[0023] In a third aspect, the present invention provides the use of the SSR marker detection primer pair set in the ploidy identification of the progeny of potato distant hybridization.

[0024] Further, the potato is "Lishu No. 6".

[0025] Further, determine whether the DNA fragments shown in (a)-(n) are included in the sample to be tested;

[0026] (a) The nucleotide sequence shown in SEQ ID NO: 13;

[0027] (b) The nucleotide sequence shown in SEQ ID NO: 14;

[0028] (c) The nucleotide sequence shown in SEQ ID NO: 15;

[0029] (d) The nucleotide sequence shown in SEQ ID NO: 16;

[0030] (e) The nucleotide sequence shown in SEQ ID NO: 17;

[0031] (f) The nucleotide sequence shown in SEQ ID NO: 18;

[0032] (g) The nucleotide sequence shown in SEQ ID NO: 19;

[0033] (h) The nucleotide sequence shown in SEQ ID NO: 20;

[0034] (i) The nucleotide sequence shown in SEQ ID NO: 21;

[0035] (j) The nucleotide sequence shown in SEQ ID NO: 22;

[0036] (k) The nucleotide sequence shown in SEQ ID NO: 23;

[0037] (l) The nucleotide sequence shown in SEQ ID NO: 24;

[0038] (m) The nucleotide sequence shown in SEQ ID NO: 25;

[0039] (n) The nucleotide sequence shown in SEQ ID NO: 26.

[0040] In some specific embodiments, 6 pairs of primers (the nucleotide sequences of which are shown in SEQ ID NO.1 - SEQ ID NO.12) can amplify 14 DNA fragments (the nucleotide sequences of which are shown in SEQ ID NO.13 - SEQ ID NO.26) in the female parent. According to the presence or absence of these DNA fragments, they can be used to identify the ploidy of distant hybridization offspring.

[0041] In a third aspect, the present invention provides the use of the SSR marker detection primer pair group in the identification of the genomic purity of potato distant hybridization offspring.

[0042] Further, the potato is "Lishu No. 6".

[0043] Further, the uses include nucleic acid hybridization detection, molecular markers, preparation of gene chips, preparation of molecular probes, and preparation of detection kits.

[0044] Further, determine whether the test sample includes the DNA fragments shown in (a)-(i);

[0045] (a) The nucleotide sequence shown in SEQ ID NO: 27;

[0046] (b) The nucleotide sequence shown in SEQ ID NO: 28;

[0047] (c) The nucleotide sequence shown in SEQ ID NO: 29;

[0048] (d) The nucleotide sequence shown in SEQ ID NO: 30;

[0049] (e) The nucleotide sequence shown in SEQ ID NO: 31;

[0050] (f) The nucleotide sequence shown in SEQ ID NO: 32;

[0051] (g) The nucleotide sequence shown in SEQ ID NO: 33;

[0052] (h) The nucleotide sequence shown in SEQ ID NO: 34;

[0053] (i) The nucleotide sequence shown in SEQ ID NO: 35.

[0054] In some specific embodiments, 6 pairs of primers (the nucleotide sequences of which are shown in SEQ ID NO.1 - SEQ ID NO.12) can amplify 9 DNA fragments (the nucleotide sequences of which are shown in SEQ ID NO.29 - SEQ ID NO.36) in the male parent IVP101. According to the presence or absence of these DNA fragments, it can be inferred whether there is introgression of the male parent gene in the doubled haploid material, so as to be used for identifying the genomic purity of the distant hybridization offspring.

[0055] Further, the potato is "Lishu 6".

[0056] Further, the uses include nucleic acid hybridization detection, molecular marker, preparation of gene chip, preparation of molecular probe, and preparation of detection kit.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) Using the amplification results of 6 pairs of SSR marker primers to detect and judge the ploidy of the doubled haploid population. Compared with genome sequencing and flow cytometry, it does not require special dedicated instrument equipment and has a lower detection cost;

[0059] (2) Compared with methods such as embryo spot marker, plant morphology observation, and determination of physiological and biochemical indexes, the detection results are more objective, more accurate, and have good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a flow chart for screening SSR marker detection primers;

[0061] Figure 2 It is a ploidy detection chart of the "Lishu 6" hybrid-induced doubled haploid population material;

[0062] Figure 3 It is an agarose gel electrophoresis detection chart of SSR marker primers;

[0063] Figure 4 It is a cluster analysis chart of the SSR marker detection results of the "Lishu 6" hybrid-induced doubled haploid population. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0064] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are all obtained from regular biochemical reagent stores without special instructions. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0065] Based on the present invention, in order to develop the diploid resources of the tetraploid excellent potato variety "Lishu 6", the ploidy of "Lishu 6" was reduced, and a reduced ploidy population including diploid, triploid, and tetraploid materials was obtained. Therefore, it is very important to quickly and accurately identify the ploidy of potatoes.

[0066] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0067] Based on the present invention, in order to develop the diploid resources of the tetraploid excellent potato variety "Lishu 6", the ploidy of "Lishu 6" was reduced, and a reduced ploidy population including diploid, triploid, and tetraploid materials was obtained. After the inventor analyzed the genome of "Lishu 6", SSR marker primers suitable for the reduced ploidy population of "Lishu 6" were developed, which can be used to better judge the ploidy of the reduced ploidy population materials and estimate the flow of parental genes.

[0068] Example 1

[0069] The ploidy of the tetraploid potato variety "Lishu 6" was reduced. For the obtained materials, leaves of some materials were cut, quickly frozen in liquid nitrogen and stored at -80°C. Flow cytometry was used to determine the ploidy of the reduced ploidy materials (see Figure 2 ), as Figure 2 shown in, a: Lishu 6 (tetraploid, female parent); b: IVP101 (diploid, male parent); c: diploid offspring; d: triploid offspring; e: tetraploid offspring.

[0070] Example 2

[0071] The tetraploid potato variety "Lishu 6" was subjected to second-generation and third-generation genome sequencing on the Illumina xplus sequencing platform and the PacBio sequencing platform respectively. The SOAPdenovo and HifiAsm were used to splice and assemble the whole-genome sequences of the second-generation and third-generation sequencing results.

[0072] Perform SSR scanning analysis on the assembled "Lishu 6" genome, extend 100 - 200 bp on both wings of the obtained SSR fragments for primer design. First, remove the fragments with low GC% content at both ends that cannot be used for designing amplification primers; then, select the amplified fragments that are specific to a certain chromosome and different on different chromatids of the same chromosome; then, align the expected amplified fragments with the published diploid potato Phureja DM 1 - 3 516R44 v8.1 genome, and perform ePCR on the primers for this genome, retaining the fragments and primers that can distinguish the parents; verify the primers retained by screening through PCR detection in the "Lishu 6" doubled haploid population, and perform electrophoresis detection on the amplification products, and screen according to the electrophoresis results to determine 6 pairs of SSR marker detection primers that can be used for ploidy screening of the doubled haploid population, namely primer pair A1 (its nucleotide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2), primer pair A2 (its nucleotide sequence is shown in SEQ ID NO: 3, SEQ ID NO: 4), primer pair A3 (its nucleotide sequence is shown in SEQ ID NO: 5, SEQ ID NO: 6), primer pair B1 (its nucleotide sequence is shown in SEQ ID NO: 7, SEQ ID NO: 8), primer pair G3 (its nucleotide sequence is shown in SEQ ID NO: 9, SEQ ID NO: 10) and primer pair G4 (its nucleotide sequence is shown in SEQ ID NO: 11, SEQ ID NO: 12).

[0073] Example 3

[0074] Extract the total DNA of "Lishu 6", IVP101 and leaves respectively by the modified CTAB method, and perform PCR amplification detection and verification on the designed 6 pairs of InDel marker detection primers in the two parents and the doubled haploid population identified as diploid, triploid and tetraploid by flow cytometry. The amplification system and amplification program are shown in Table 1 and Table 2:

[0075] Table 1 Amplification system

[0076]

[0077] Table 2 Amplification program

[0078]

[0079] The amplification products are detected by electrophoresis with 2% agarose (see Figure 3 ), and it is found that 6 pairs of primers can be used to distinguish the parents. Figure 3 Among them, M is Maker, L is LS6, I is IVP101, and the other lanes are the LS6×IVP101 doubled haploid offspring population; from Figure 3It can be seen that in female parent LS6, 6 pairs of designed primers can amplify a total of 14 fragments. Among them, primer pair A1 can amplify 3 target fragments, namely A1-1 (whose nucleotide sequence is shown in SEQ ID NO: 13), A1-2 (whose nucleotide sequence is shown in SEQ ID NO: 14), and A1-3 (whose nucleotide sequence is shown in SEQ ID NO: 15), with lengths of 238bp, 232bp, and 214bp respectively; primer pair A2 can also amplify 3 target fragments, namely A2-1 (whose nucleotide sequence is shown in SEQ ID NO: 16), A2-2 (whose nucleotide sequence is shown in SEQ ID NO: 17), and A2-3 (whose nucleotide sequence is shown in SEQ ID NO: 18), with lengths of 184bp, 178bp, and 160bp respectively; primer pair A3 can amplify 2 target fragments, namely A3-1 (whose nucleotide sequence is shown in SEQ ID NO: 19) and A3-2 (whose nucleotide sequence is shown in SEQ ID NO: 20), with lengths of 207bp and 196bp respectively; primer pair B1 can amplify 3 target fragments, namely B1-1 (whose nucleotide sequence is shown in SEQ ID NO: 21), B1-2 (whose nucleotide sequence is shown in SEQ ID NO: 22), and B1-3 (whose nucleotide sequence is shown in SEQ ID NO: 23), with lengths of 167bp, 162bp, and 141bp respectively; primer pair G3 can only amplify 1 target fragment B3-1 (whose nucleotide sequence is shown in SEQ ID NO: 24), with a length of 232bp; primer pair G4 can amplify 2 target fragments, namely G4-1 (whose nucleotide sequence is shown in SEQ ID NO: 25) and G4-2 (whose nucleotide sequence is shown in SEQ ID NO: 26), with lengths of 343bp and 298bp respectively. According to the presence or absence of these DNA fragments, they can be used to identify the ploidy of distant hybridization offspring.

[0080] Moreover, it was found that in the male parent IVP101, 9 fragments could be amplified by 6 pairs of designed primers in total. Among them, primer pair A1 amplified 1 target fragment, namely IVP101-A1-1 (the nucleotide sequence of which is shown in SEQ ID NO: 27), with a length of 226 bp; primer pair A2 amplified 1 target fragment, namely IVP101-A2-1 (the nucleotide sequence of which is shown in SEQ ID NO: 28), with a length of 172 bp; primer pair G3 amplified 1 target fragment, namely IVP101-G3-1 (the nucleotide sequence of which is shown in SEQ ID NO: 29), with a length of 214 bp; primer pair A3 amplified 2 target fragments, namely IVP101-A3-1 (the nucleotide sequence of which is shown in SEQ ID NO: 30) and IVP101-A3-2 (the nucleotide sequence of which is shown in SEQ ID NO: 31), with lengths of 199 bp and 198 bp respectively; primer pair B1 amplified 2 target fragments, namely IVP101-B1-1 (the nucleotide sequence of which is shown in SEQ ID NO: 32) and IVP101-B1-2 (the nucleotide sequence of which is shown in SEQ ID NO: 33), with lengths of 148 bp and 134 bp respectively; primer pair G4 amplified 2 target fragments, namely IVP101-G4-1 (the nucleotide sequence of which is shown in SEQ ID NO: 34) and IVP101-G4-2 (the nucleotide sequence of which is shown in SEQ ID NO: 35), with lengths of 331 bp and 325 bp respectively. Whether there is introgression of the male parent gene in the reduced ploidy material can be inferred based on the presence or absence of these fragments.

[0081] Based on the PCR detection results, ploidy analysis was carried out. The detection results of 6 pairs of SSR marker primers in 269 "Lishu 6" reduced ploidy population materials were marked, and the sorted results were subjected to cluster analysis by the UPGMA method (Unweighted Pair-Group Method with Arithmetic means) (see Figure 4 ). As Figure 4 shown, the part with yellow background represents LS6 (female parent), the part with red background represents IVP101 (male parent), the part with green background represents diploid, and the part with purple background represents triploid and tetraploid; it was found that among 188 diploid materials, 2 diploid materials were classified into triploid and tetraploid materials, and 4 tetraploid materials were classified into diploid materials, with an accuracy rate of 97.8%.

[0082] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without violating the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. A primer set for detecting SSR markers for potato diploid identification, characterized in that It includes primer pairs as shown below: Primer pair A1: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; Primer pair A2: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively; Primer pair A3: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively; Primer pair B1: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively; Primer pair G3: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively; and Primer pair G4: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively.

2. Use of the primer set according to claim 1 in ploidy identification of distant hybridization offspring of "Lishu 6" potato.

3. The use according to claim 2, characterized in that: Determine whether the DNA fragments shown in (a)-(n) are included in the sample to be tested; (a) Nucleotide sequence shown in SEQ ID NO: 13; (b) Nucleotide sequence shown in SEQ ID NO: 14; (c) Nucleotide sequence shown in SEQ ID NO: 15; (d) Nucleotide sequence shown in SEQ ID NO: 16; (e) Nucleotide sequence shown in SEQ ID NO: 17; (f) Nucleotide sequence shown in SEQ ID NO: 18; (g) Nucleotide sequence shown in SEQ ID NO: 19; (h) Nucleotide sequence shown in SEQ ID NO: 20; (i) Nucleotide sequence shown in SEQ ID NO: 21; (j) Nucleotide sequence shown in SEQ ID NO: 22; (k) Nucleotide sequence shown in SEQ ID NO: 23; (l) Nucleotide sequence shown in SEQ ID NO: 24; (m) Nucleotide sequence shown in SEQ ID NO: 25; (n) Nucleotide sequence shown in SEQ ID NO:

26.

4. Use of the primer set according to claim 1 in genomic purity identification of distant hybridization offspring of "Lishu 6" potato.

5. The use according to claim 4, wherein: Determine whether the DNA fragments shown in (a)-(i) are included in the sample to be tested; (a) Nucleotide sequence shown in SEQ ID NO: 27; (b) Nucleotide sequence shown in SEQ ID NO: 28; (c) Nucleotide sequence shown in SEQ ID NO: 29; (d) Nucleotide sequence shown in SEQ ID NO: 30; (e) Nucleotide sequence shown in SEQ ID NO: 31; (f) Nucleotide sequence shown in SEQ ID NO: 32; (g) Nucleotide sequence shown in SEQ ID NO: 33; (h) The nucleotide sequence shown in SEQ ID NO: 34; (i) The nucleotide sequence shown in SEQ ID NO:

35.

6. The use according to claim 2 or 3, characterized in that: The uses include nucleic acid hybridization detection, molecular markers, preparation of gene chips, preparation of molecular probes, and preparation of detection kits.

7. Use according to claim 4 or 5, characterized in that: The uses include nucleic acid hybridization detection, molecular markers, preparation of gene chips, preparation of molecular probes, and preparation of detection kits.

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