Genome analysis methods for potato aneuploid interspecific hybrids and their application in the creation of cold-resistant addition lines

Abstract

The present invention provides a method for analyzing the genome of potato aneuploid interspecific hybrids and its application in the creation of cold-resistant addition line materials, belonging to the field of plant molecular genetic breeding. By sequencing the whole genome of aneuploid offspring and parents, the genome composition of potato aneuploid interspecific hybrids is accurately analyzed. Combined with the identification of interspecific hybrid cold resistance, cold resistance genetic mapping and fertility analysis, potato cold-resistant addition lines are finally successfully screened. By using colchicine-doubled cold-resistant addition lines, doubled addition lines with good growth conditions, good fertility and cold resistance are successfully obtained, which can be applied to potato cold-resistant breeding. The present invention expands the breeding approach for potato cold resistance and shortens the breeding process, providing a new breeding method system for potato breeding.

Description

Technical Field

[0001] The present invention relates to the field of plant molecular genetic breeding, and specifically provides a method for rapidly and accurately analyzing the genome composition of aneuploid interspecific hybrids and its application in the creation of cold-resistant additional line materials. Background Art

[0002] In higher plants, beneficial genes can be transferred from one species to another through interspecific hybridization and backcrossing. Heterologous addition lines (HLs) are lines in which one or a pair of heterologous chromosomes are attached to the chromosome set of one species. Heterologous addition lines are important building blocks for overcoming barriers to distant hybridization in plants and conducting genetic research and chromosome engineering breeding. They can be used for gene mapping, constructing introgression lines, and creating new germplasm resource collections. The bridge and backcross methods are commonly used to construct HLs and are widely used in crops such as tomato, maize, and rapeseed (Tan et al. 2018; Chetelat et al. 1998). The cultivation of HLs allows the introgression of beneficial genes from wild species into the genomes of cultivated species, thereby improving them and providing a new strategy for germplasm resource innovation. Potatoes are an important non-cereal crop that plays a crucial role in ensuring my country's food security. Like many other cultivated crop varieties, potato breeding has long been based on intraspecific hybridization, but it faces the problem of low genetic diversity of cultivated potatoes (Hardigan et al 2017), and the creation and application of potato addition lines have not been reported.

[0003] Compared to cultivated potato, wild potato populations are abundant and possess significantly greater genetic diversity than cultivated potato (Li et al. 2018). However, approximately 75% of wild resources are diploid, while most cultivated potato varieties are tetraploid. When most wild and cultivated potato species are hybridized, postzygotic hybridization barriers often arise due to differences in ploidy and endosperm balance numbers (EBN) (Spooner et al. 2014). Ploidy breeding, mediated by artificial doubling of parental chromosomes with colchicine and selection of parental 2n gametes, provides the technical and material foundation to address this challenge (Dong et al. 2020; Dewitte et al. 2011). Furthermore, when transferring wild genetic resources to cultivated varieties, in addition to overcoming interspecific hybrid incompatibility through ploidy level adjustment as described above, a third species can be used as a "bridge" to overcome interspecific incompatibility between cultivated and wild species. Previous studies have used Solanum chacoense as a typical "bridge species" in breeding practices (Ortiz and Ehlenfeldt 1992). Common cultivated potato varieties lack cold tolerance, but several wild species, including S. acaule (4X, 2EBN), S. commersonii (2X, 1EBN), and S. malmeanum (2X, 1EBN), exhibit strong cold tolerance (Vega and Bamberg 1995; Tu et al. 2021). Therefore, interspecific hybridization is the most effective way to improve the cold tolerance of potato varieties (lines).

[0004] The vast majority of existing potato varieties are autotetraploid cultivars (Solanum tuberosum). As an asexual crop, potato, lacking sexual reproduction, accumulates numerous deleterious mutations in its genome, leading to weak growth and reduced fertility in hybrid offspring. While interspecific hybridization promotes gene exchange, it also struggles to eliminate genetic linkage drag and deleterious mutations. Aneuploidy generated through interspecific hybridization offers a new and efficient breeding channel for eliminating genetic linkage drag. Some fertile gametes produced during meiosis in triploid plants can be transformed into different ploidy levels. The resulting aneuploid fertile gametes and 2n gametes can serve as a bridge to inducing chromosome number variation (Husband 2004). Interspecific aneuploidy is particularly valuable in cytogenetics and plant chromosome engineering breeding. Different types of aneuploid wheat lines (monosomic, disomic, and trisomic) have been successfully used to analyze chromosome function, map specific genes, and apply them to breeding practices (Law and Worland 1972). In potato, Carputo et al. (1997) used ploidy manipulation to produce aneuploids from 3X×2X hybrids, which were then doubled and backcrossed multiple times for potato breeding. However, due to the lack of further research on the genetic components of aneuploids, potato addition lines were not constructed. Therefore, it is practical to integrate chromosome component analysis and chromosome dosage variation of breeding materials with different chromosome compositions into potato breeding programs, providing a theoretical basis for the creation of cold-resistant potato addition lines.

[0005] To investigate the relationship between phenotypic variation and chromosome composition in the offspring of 3X×2X aneuploid hybrids, whole-genome genetic analysis is essential. Previous studies on the genetic composition of potato interspecific hybrids have primarily utilized potato genomic in situ hybridization (GISH) and related molecular marker techniques such as RAPD, AFLP, and SSR (Wang Haibo 2020). However, traditional cytological methods and molecular marker techniques have significant limitations in whole-genome genetic analysis of potato interspecific hybrids, particularly in detecting small fragments and exchange within interspecific hybrids. This leads to a certain degree of randomness and serendipity in genetic analysis. The advent of affordable high-throughput DNA sequencing methods, coupled with advanced computational analysis capabilities, has made detailed analysis of chromosome structure and function possible (Comai and Tan 2019).

[0006] Problems with existing technologies: Currently, due to the lack of accurate analysis of the whole-genome genetic components of potato aneuploid interspecific hybrids, there is a gap in the selection and application of potato addition lines; due to the characteristics of potato cultivars, there is a lack of effective methods to eliminate genetic linkage drags in the genome of potato cultivars. Summary of the Invention

[0007] The key technical problem to be solved by the present invention is: in view of the shortcomings of existing identification methods, a method for quickly and accurately analyzing the genome composition of interspecific hybrids of potato aneuploids and its application in the creation of cold-resistant additional line materials is provided.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] 1. A method for rapidly and accurately analyzing the genome composition of potato aneuploid interspecific hybrids, comprising the following steps:

[0010] (1) Construction of backcross population: The diploid wild species MLM266-2 was used as the female parent and the diploid CHC9701 was used as the male parent for hybridization. The cold resistance of the triploid hybrid offspring was screened. The offspring with the strongest cold resistance were backcrossed with CHC9701 to obtain the backcross population BC1.

[0011] (2) Chromosome ploidy analysis: Ploidy analysis of BC1 backcross progeny was performed using root tip chromosome counting, and the progeny with chromosome numbers distributed between 30 and 34 were obtained.

[0012] (3) DNA extraction of backcross progeny: The CTAB method was used to extract DNA from the BC1 backcross progeny and parental individual plants.

[0013] (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA samples with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 were subjected to whole genome sequencing.

[0014] (5) Quality control analysis of sequencing data.

[0015] (6) Mutation detection.

[0016] (7) Polymorphic SNP marker screening.

[0017] (8) Calculation of parental allele ratios in offspring.

[0018] (9) Analysis of genomic components of BC1 backcross progeny.

[0019] Furthermore, the above-mentioned library construction and sequencing steps were as follows: Paired-end (PE) library construction was performed according to the Illumina library construction method. One PE library was constructed for each sample DNA. Specifically, the genomic DNA was randomly fragmented into 300-500 bp fragments, and sequencing adapters were ligated. DNA clusters were prepared on a sequencing chip, and finally, PE150 sequencing was performed on an Illumina HiSeq sequencer. The sequencing depth was 30×, the library type was a 350 bp small fragment library of the whole plant and animal genome, and the sequencing strategy was HiSeq-PE150.

[0020] Furthermore, the sequencing data quality control analysis was detailed as follows: To obtain reliable reads, the raw reads were subjected to a series of quality control procedures using Cutadapt and Trimmomatic software to generate high-quality clean data. These quality control procedures included removing reads containing ≥10% unknown nucleotides; removing reads with >20% bases and Phred <5; removing reads aligned to adapters >10 nt, allowing for ≤10% mismatches; and removing PCR duplicates generated during PCR amplification during library construction. Ultimately, high-quality reads with Q20 ≥98.45% and Q30 ≥94.66% were obtained.

[0021] Furthermore, the specific analysis steps for the above-mentioned variation detection are as follows: ① Use the MEM algorithm of BWA software to align the sequencing data of each sample with the potato reference genome DMv4.04 to obtain the alignment results in SAM format; ② Use samtools software to convert the SAM format file into BAM format; ③ Use SortSam in Picard tool to sort the reads in the BAM file and remove PCR duplicates. The obtained BAM file can be used as a BAM file for variant calling. Finally, use the Haplotype Caller module of GATK for SNP and InDel variation detection.

[0022] Furthermore, the polymorphic SNP marker screening described above was performed as follows: VCF files were generated using GATK software to perform variant detection, and SNP and InDel markers meeting the requirements of the present invention were screened. First, sites that were homozygous and polymorphic in the parents MLM266-2 and CHC9701 were screened. The screening criteria included: sequencing depths of MLM266-2, CHC9701, and backcross progeny between 30-60×, and both parents were homozygous and polymorphic.

[0023] Furthermore, the method for calculating the parental allele ratio in the above-mentioned offspring is as follows: since the triploid mother of the backcross population BC1 may produce 1X, 1X-2X and 2X female gametes at the same time, and the father CHC9701 normally only produces 1X male gametes, a formation pattern diagram of the backcross offspring of the backcross population BC1 was proposed based on the distribution of chromosome number in the backcross offspring; in order to accurately study the composition of genetic components in the backcross offspring, genome resequencing was performed on 13 of the backcross offspring, and the polymorphic SNP sites of the parents MLM266-2 and CHC9701 were screened. For the screened SNP sites, the specific site proportion of MLM266-2 at the SNP site was calculated in each backcross offspring, and then the whole genome was slid with a sliding window of 500Kb, and the average value of the sliding window was calculated and plotted.

[0024] Furthermore, the above-mentioned genomic component analysis method of the BC1 backcross offspring is as follows: based on bioinformatics and genetic analysis, the specific site proportion of the parent MLM266-2 in each polymorphic SNP site in the backcross offspring can be divided into four types, corresponding to 2 / 3:Ⅰ, 1 / 3:Ⅱ:, 1 / 2:III, and 0:Ⅳ respectively. Based on this analysis of the specific site proportion of MLM266-2 in the backcross offspring, the genetic composition of chromosomes 1-12 in each backcross offspring can be calculated.

[0025] 2. A method for screening potato cold-resistant addition lines using aneuploid interspecific hybrid material, comprising the following steps:

[0026] (1) Construction of backcross population: The diploid parent MLM266-2 was hybridized with CHC9701, and the cold resistance of the triploid hybrid offspring was screened. The offspring with the strongest cold resistance were backcrossed with CHC9701 to obtain the backcross population BC1.

[0027] (2) Chromosome ploidy analysis: The ploidy of the backcross progeny BC1 was analyzed by counting the chromosomes at the root tip, and the progeny with a chromosome number distribution between 30 and 34 were obtained.

[0028] (3) DNA extraction of backcross progeny: The CTAB method was used to extract DNA from the BC1 backcross progeny and parental individual plants.

[0029] (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA samples with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 were subjected to whole genome sequencing.

[0030] (5) Quality control analysis of sequencing data.

[0031] (6) Mutation detection.

[0032] (7) Polymorphic SNP marker screening.

[0033] (8) Calculation of parental allele ratios in offspring.

[0034] (9) Analysis of genomic components of BC1 backcross progeny.

[0035] (10) Identification and genetic positioning of cold resistance of backcross offspring: The cold resistance of backcross offspring was evaluated by the conductivity leakage method. Then, the sequencing results of the four strains with the strongest and four strains with the weakest domestication cold resistance in the backcross offspring were subjected to whole-genome selective scanning. The Δ(SNP-index) value of each variant site was calculated using QTLseqr software, and its distribution on the genome was examined and mapped using a 2Mb sliding window. The results of the whole-genome selective scanning showed that there were four obvious cold resistance segments of domestication cold resistance, which were anchored on chromosomes 4, 9, 11 and 12 respectively.

[0036] (11) Fertility identification of backcross offspring: The parents and backcross offspring were planted in a greenhouse, and the flowering characteristics and fertility were counted. During the peak flowering period, the parents and backcross offspring that could flower normally were subjected to acetic acid carmine pollen vitality analysis. The pollen grain size of the parents and backcross offspring was observed, and 2n pollen grains in the backcross offspring were screened and the frequency of 2n pollen grains was counted.

[0037] (12) Screening of cold-resistant additional lines for potato: Based on the overall comparison of the genomic composition, cold-resistant genetic location and fertility of the above-mentioned backcross offspring, the materials with the following genomic composition in the BC1 backcross offspring were finally selected as cold-resistant additional lines: the number of chromosomes was 31, and the number of chromosomes 1-12 from CHC9701 was 2, 1, 2, 2, 1, 2, 2, 1, 1, 1, 1, and the number of chromosomes 1-12 from MLM266-2 was 0, 1, 1, 1, 1, 1, 1, 2, 1, 1, 2.

[0038] 3. Use of an aneuploid interspecific hybrid material in breeding cold-resistant potato varieties, the material being obtained by the following steps:

[0039] (1) Construction of backcross population: The cold resistance of the triploid hybrid offspring was screened, and the offspring with the strongest cold resistance were backcrossed with CHC9701 to obtain the backcross population BC1.

[0040] (2) Chromosome ploidy analysis: Ploidy analysis of BC1 backcross progeny was performed using root tip chromosome counting, and the progeny with chromosome numbers distributed between 30 and 34 were obtained.

[0041] (3) DNA extraction of backcross progeny: The CTAB method was used to extract DNA from the BC1 backcross progeny and parental individual plants.

[0042] (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA samples with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 were subjected to whole genome sequencing.

[0043] (5) Quality control analysis of sequencing data.

[0044] (6) Mutation detection.

[0045] (7) Polymorphic SNP marker screening.

[0046] (8) Calculation of parental allele ratios in offspring.

[0047] (9) Analysis of genomic components of BC1 backcross progeny.

[0048] (10) Identification and genetic positioning of cold resistance of backcross offspring: The cold resistance of backcross offspring was evaluated by the conductivity leakage method. Then, the sequencing results of the four strains with the strongest and four strains with the weakest domestication cold resistance in the backcross offspring were subjected to whole-genome selective scanning. The Δ(SNP-index) value of each variant site was calculated using QTLseqr software, and its distribution on the genome was examined and mapped using a 2Mb sliding window. The results of the whole-genome selective scanning showed that there were four obvious cold resistance segments of domestication cold resistance, which were anchored on chromosomes 4, 9, 11 and 12 respectively.

[0049] (11) Fertility identification of backcross offspring: The parents and backcross offspring were planted in a greenhouse, and the flowering characteristics and fertility were counted. During the peak flowering period, the parents and backcross offspring that could flower normally were subjected to acetic acid carmine pollen vitality analysis. The pollen grain size of the parents and backcross offspring was observed, and 2n pollen grains in the backcross offspring were screened and the frequency of 2n pollen grains was counted.

[0050] (12) Screening of cold-resistant additional lines for potato: Based on the overall comparison of the genomic composition, cold-resistant genetic location and fertility of the above-mentioned backcross offspring, the materials with the following genomic composition in the BC1 backcross offspring were finally selected as cold-resistant additional lines: the number of chromosomes was 31, and the number of chromosomes 1-12 from CHC9701 was 2, 1, 2, 2, 1, 2, 2, 1, 1, 1, 1, and the number of chromosomes 1-12 from MLM266-2 was 0, 1, 1, 1, 1, 1, 1, 2, 1, 1, 2.

[0051] (13) Chromosome doubling of cold-resistant potato addition lines: The chromosomes of the selected addition line materials were doubled using colchicine, resulting in a line with 62 chromosomes. This line has strong cold resistance and fertility. In addition, this line can be directly hybridized with ordinary cultivated potatoes as a new cold-resistant breeding resource for wild germplasm incorporation.

[0052] 4. The application of potato diploid materials MLM266-2 and CHC9701 in breeding cold-resistant addition lines and cold-resistant varieties is specifically achieved by steps (1) to (13) in the above 3.

[0053] 5. Use of a cold-resistant potato addition line in breeding cold-resistant varieties, the cold-resistant addition line having the following genomic composition: 31 chromosomes, with chromosomes 1-12 derived from CHC9701 in the following order: 2, 1, 2, 2, 2, 1, 2, 2, 1, 1, 1, 1; and chromosomes 1-12 derived from MLM266-2 in the following order: 0, 1, 1, 1, 1, 1, 1, 1, 2, 1, 1, 2.

[0054] Beneficial effects:

[0055] The present invention provides a method for rapidly and accurately analyzing the genomic composition of potato aneuploid interspecific hybrids. The method uses a genome resequencing method to sequence aneuploid offspring and parents, and screens for homozygous and polymorphic sites in both parents. For each SNP site that meets the requirements screened out in the parents, the proportion of the parent-specific site at that site in the offspring is calculated. The present invention also provides a method for screening potato cold-resistant addition lines and its application, based on the above-mentioned precise analysis of the genomic composition of aneuploid interspecific hybrids, combined with the mapping of cold resistance of aneuploid interspecific hybrids and somatic cell doubling. The present invention uses whole-genome sequencing technology combined with bioinformatics analysis to quickly and accurately analyze the chromosome number and genomic composition of aneuploid offspring, avoiding the need for extensive root tip chromosome counting, in situ hybridization, molecular marker detection, and other means to explore the genetic composition of aneuploid interspecific hybrids. In addition, based on the precise analysis of the genomic composition of aneuploid interspecific hybrids by whole-genome sequencing, the present invention effectively creates potato cold-resistant addition lines by combining aneuploid genotypes with cold-resistant phenotypes. This method expands the breeding ideas for potato cold resistance from another perspective and shortens the breeding process, providing a possible new breeding method system for the development of potato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the pedigree diagram of the backcross population BC1 (FT059) in Example 1 of the present invention.

[0057] Figure 2Chromosome counts for root tips of BC1 progeny and parents. A and B represent the backcross parents FT040-7 and CHC9701, and CP represents the backcross progenies FT059-1, FT059-3, FT059-4, FT059-6, FT059-7, FT059-9, FT059-10, FT059-11, FT059-13, FT059-14, FT059-15, FT059-17, FT059-18, and FT059-19, respectively. Numbers represent chromosome number. Scale bar: 5 μm.

[0058] Figure 3 Diagram showing the pattern of aneuploidy in BC1 offspring. The gray chromosomes represent the genome of the parent, MLM266-2, and the white chromosomes represent the genome of the parent, CHC9701. A and a indicate homozygous polymorphic sites in the parents, MLM266-2 and CHC9701, respectively.

[0059] Figure 4 The distribution of specific loci from parent MLM266-2 in some BC1 offspring. The horizontal axis represents chromosomes 1-12, and each point in the graph represents the percentage of specific loci from parent MLM266-2 at that locus.

[0060] Figure 5 Chromosome composition of 13 BC1 offspring. The horizontal axis represents the 13 backcross offspring, and the vertical axis represents the number of chromosomes from MLM266-2 and CHC9701 in each offspring.

[0061] Figure 6 Identification and positioning of cold resistance of BC1 offspring. (A) shows the cold resistance results of backcross offspring before (NA) and after (CA) cold acclimation. The horizontal axis represents BC1 offspring and parents, and the vertical axis represents the cold resistance determination index LT. 50 (℃); (B) is the genetic location of cold acclimation resistance (CA).

[0062] Figure 7 Flowering characteristics and fertility analysis of BC1 offspring and their parents. (A) shows the flowering status and pollen grains of some BC1 offspring and their parents, stained with aceto-Yang red. Arrows indicate 2n pollen grains. (B) shows the pollen viability of the parents and backcross offspring. (C) shows the frequency of 2n pollen in backcross offspring.

[0063] Figure 8 The genetic composition and specific pattern of the additional line material FT059-17. Among them, (A) is the distribution diagram of the MLM266-2 allele ratio in the additional line material, in which I, II, III and IV represent Figure 3There are 4 different types; (B) is a schematic diagram of the genetic composition of the additional line material, and the arrows indicate the heterologous additional chromosomes.

[0064] Figure 9 Analysis of ploidy, plant, and pollen characteristics of the doubling addition line FT059-17. (A) Field plant after doubling; (B) Root tip chromosome count after doubling; (C) Pollen viability after doubling; (D) Cold resistance analysis after doubling. Specific implementation methods

[0065] The methods and apparatus used in the following embodiments of the present invention are conventional methods and apparatus unless otherwise specified; the equipment and reagents used are conventional equipment and reagents purchased from a reagent company. In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments. Examples of these preferred implementations are illustrated in the specific embodiments. It should also be noted that in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solutions of the present invention are shown in the embodiments, and other details that are not very relevant are omitted.

[0066] Example 1

[0067] This embodiment provides a method for rapidly and accurately analyzing the genomic composition of interspecific hybrids of potato aneuploids. The method utilizes whole-genome resequencing to sequence the aneuploid interspecific hybrids and their parents, screening for homozygous and polymorphic SNPs in both parents. For the selected SNPs that meet the requirements, the proportion of parent-specific SNPs at that site in the offspring is calculated to accurately analyze the genomic composition of the interspecific hybrids. The method specifically includes the following steps:

[0068] (1) Backcross population construction: The diploid wild species MLM266-2 was used as the female parent and the diploid CHC9701 was used as the male parent for hybridization. Both MLM266-2 and CHC9701 are existing breeding materials (Tu Wei et al. 2015). Based on the EBN theory, the hybrid offspring of MLM266-2×CHC9701 are all triploid. The cold resistance and pollen fertility of the triploid hybrid offspring were screened. The offspring with the strongest cold resistance and good fertility, FT040-7, was backcrossed with CHC9701 to obtain the backcross population BC1, which was named FT059 ( Figure 1 ).

[0069] (2) Ploidy analysis of the backcross population progeny was performed using root tip chromosome counts. For specific methods, refer to Wang Haibo, 2020. The experimental results are shown in Figure 2 The results showed that all the backcross offspring were aneuploid ( Figure 2), the chromosome number is distributed between 30-34.

[0070] (3) DNA from offspring and parental individual strains of the BC1 population was extracted using the CTAB method (Dellaporta et al 1983).

[0071] (4) Library construction and sequencing: The concentration and purity of the DNA extracted in (3) were detected by UV spectrophotometer. DNA with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 was identified as qualified DNA and then subjected to whole genome sequencing. Paired-end (PE) library construction was performed according to the Illumina library construction method. A PE library was constructed for each sample DNA, that is, the genomic DNA was randomly broken into fragments of 300bp to 500bp and connected to sequencing adapters. DNA clusters were prepared on the sequencing chip, and finally PE150 sequencing was performed on the Illumina HiSeq sequencer. The sequencing depth was 30×, the library type was a small fragment library (350bp) of the whole genome of animals and plants, and the sequencing strategy was HiSeq-PE150.

[0072] (5) Sequencing data quality control analysis: To obtain reliable reads, the raw reads (FASTQ format files) obtained in (4) were subjected to a series of quality control procedures (Cutadapt and Trimmomatic software) to obtain high-quality clean data. Quality control included the following steps: removing reads containing ≥10% unknown nucleotides (N); removing reads with bases >20% and Phred <5; removing reads >10nt aligned to the adapter, allowing ≤10% mismatches; and removing PCR duplicates generated by PCR amplification during library construction. Finally, high-quality reads were obtained (Q20 ≥98.45% and Q30 ≥94.66%).

[0073] (6) Variant detection. The specific analysis steps are as follows: ① Use the MEM algorithm of BWA software (version 0.7015-r1140) to align the sequencing data of each sample with the reference genome (http: / / solanaceae.plantbiology.msu.edu / pgsc_download.shtml, DMv4.04) to obtain the alignment results in SAM format; ② Use samtools software (version 1.3.1) to convert the SAM format file into BAM format; ③ Use SortSam in Picard tools (version 1.91) to sort the reads in the BAM file and remove PCR duplicates. The resulting BAM file can be used for variant calling. Finally, use the Haplotype Caller module of GATK to perform SNP and InDel variant detection.

[0074] (7) Polymorphic SNP Marker Screening: VCF files were obtained by performing mutation detection using GATK software, and SNP and InDel markers that met the requirements of the present invention were screened. First, sites that were homozygous and polymorphic in the parents MLM266-2 and CHC9701 were screened (for example, a SNP base site on MLM266-2 was AA, while in CHC9701 it was CC, TT, or GG). The screening criteria included: ① The sequencing depth of the parents and progeny was between 30-60×; ② Both parents were homozygous and polymorphic.

[0075] (8) Calculation of allele ratios of offspring parents: Since the triploid female parent FT040-7 in the backcross population may produce 1X, 1X-2X and 2X female gametes at the same time, while the male parent CHC9701 normally only produces 1X male gametes, combined with the distribution of chromosome numbers in the backcross offspring in (2), a pattern diagram of the formation of aneuploid backcross offspring of BC1 was proposed ( Figure 3 In order to accurately study the genetic composition of backcross offspring, genome resequencing was performed on 13 of the backcross offspring. The parental polymorphic SNP sites screened in (7) were used to calculate the specific site proportion of the parent MLM266-2 at the SNP site in each backcross offspring. Then, a sliding window of 500 Kb (100 Kb step size) was slid across the entire genome, and the average value of the sliding window was calculated and plotted ( Figure 4 ).

[0076] (9) Analysis of genomic components of BC1 backcross offspring: Based on bioinformatics and genetic analysis, the specific site ratio of each polymorphic SNP site in the backcross offspring MLM266-2 can be divided into four types, corresponding to 2 / 3: I, 1 / 3: II, 1 / 2: III, 0: IV ( Figure 3 ), the genetic composition of chromosomes 1-12 in each backcross progeny can be calculated based on the specific loci ratio analysis of the parent of the backcross progeny MLM266-2 (Table 1, Figure 5 ). Statistical analysis results showed that the total number of chromosomes in the 13 backcross progenies was Figure 2 The results are consistent, with a distribution range of 30 to 34 and an average of 32 ± 1.15. In addition, the number of chromosomes from the parent MLM266-2 in the offspring was statistically analyzed, and the results showed that its distribution range was 10 to 18, with an overall average of 14.08 ± 2.40; the number of chromosomes from the father CHC9701 in the 13 offspring was also statistically analyzed, and its average was 17.92 ± 1.80. In addition, the number of chromosomes 1 to 12 in the 13 offspring was statistically analyzed, and the results showed that the genetic components of chromosomes 4, 6, 9 and 12 of MLM266-2 were relatively more complete than those of the other chromosomes. The theoretical number of chromosomes in the BC1 backcross offspring in the present invention is 30 (12 of which come from MLM266-2 and 18 from CHC9701). Based on the analysis of the chromosome components of the offspring, it was found that the number of chromosomes from MLM266-2 in the offspring was slightly higher than the theoretical value, which provides a material basis for the creation of potato addition lines.

[0077] Table 1 Chromosome composition of BC1 offspring from parents MLM266-2 and CHC9701

[0078]

[0079]

[0080] Example 2

[0081] This embodiment provides a method for screening potato cold-resistant addition lines using aneuploid interspecific hybrid materials. The specific verification steps are as follows:

[0082] (1) Construction of backcross population: The diploid wild species MLM266-2 was used as the female parent and the diploid CHC9701 was used as the male parent for hybridization. Based on the EBN theory, the hybrid offspring were all triploid. The cold resistance and fertility of the triploid hybrid offspring were screened. The offspring with the strongest cold resistance and better fertility, FT040-7, was backcrossed with CHC9701 to obtain the backcross population BC1.

[0083] (2) Ploidy analysis of BC1 backcross progeny was performed using root tip chromosome counting, and the progeny with chromosome numbers distributed between 30 and 34 were obtained.

[0084] (3) The CTAB method was used to extract DNA from BC1 backcross progeny and parental individual plants.

[0085] (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 was considered to have passed the quality inspection and then whole genome sequencing was performed.

[0086] (5) Quality control analysis of sequencing data.

[0087] (6) Mutation detection.

[0088] (7) Polymorphic SNP marker screening.

[0089] (8) Calculation of parental allele ratios in offspring.

[0090] (9) Analysis of genomic components of BC1 backcross progeny.

[0091] (10) Identification and positioning of cold resistance of backcross offspring: The half-lethal temperature (LT) was obtained by combining the conductivity leakage value of plant tissues at different low temperatures with the Logistic equation. 50 ) can be used as a means of evaluating cold resistance, using LT 50 To evaluate the cold resistance of potato has become the main method for studying the cold resistance of potato (Tu Wei et al. 2015). The present invention uses the conductivity leakage method to measure the cold resistance of BC1 progeny. The results show that there is no significant difference in the non-acclimated cold resistance (NA) of the backcross progeny, but there is a very significant difference in the acclimated cold resistance (CA) between the backcross progeny ( Figure 6 A). Combining the sequencing results in Example 1 with the Bulked Segregant Analysis method, the sequencing results of the four strains with the strongest cold resistance and the four strains with the weakest cold resistance in the backcross progeny were subjected to whole-genome selective scanning ( Figure 6 A), the Δ(SNP-index) value of each variant site was calculated using QTLseqr software, and its distribution on the genome was investigated and mapped using a 2Mb sliding window. The results of the genome-wide selective scan showed that there were four distinct cold-resistant segments of domestication cold resistance, which were anchored on chromosomes 4, 9, 11, and 12, respectively ( Figure 6 B).

[0092] (11) Fertility identification of backcross offspring: The parents and offspring were planted in the greenhouse of the base and the flowering habits were counted. It was found that 9 backcross offspring were able to bloom ( Figure 7 A), the parents and 9 backcross offspring were stained with acetic acid carmine at the peak flowering stage. The results showed that all the offspring lines had pollen viability, and there were certain differences between the different genotypes of the offspring ( Figure 7 B). Observation of pollen grain sizes of the parents and backcross offspring revealed that some of the backcross offspring had 2n pollen grains ( Figure 7 A), the 2n pollen grain frequency statistics showed that there were two offspring materials with higher 2n pollen frequency ( Figure 7 C).

[0093] (12) Screening of potato cold-resistant additional lines: The theoretical number of chromosomes in the BC1 backcross offspring in this example is 30 (12 of which come from MLM266-2 and 18 from CHC9701). Based on the whole genome sequencing method in Example 1, the chromosome number and genome composition of the aneuploid offspring were accurately analyzed. The total number of chromosomes in the 13 BC1 offspring ranged from 30 to 34, the number of chromosomes from the parent MLM266-2 ranged from 10 to 18, and the overall mean was 14.08±2.40; the mean number of chromosomes from the parent CHC9701 was 17.92±1.80. An overall comparative analysis was conducted on the number of chromosomes 1-12 of MLM266-2 in the 13 offspring, as well as the genomic composition, cold resistance positioning, and fertility of the 13 offspring. Materials with the following genomic composition of the BC1 backcross offspring were selected: the number of chromosomes was 31, and the number of chromosomes 1-12 from CHC9701 was 2, 1, 2, 2, 2, 1, 1, 1, 1, and the number of chromosomes 1-12 from MLM266-2 was 0, 1, 1, 1, 1, 1, 1, 2, 1, 1, 2 (Table 1, Figure 8 A). This additional line material has strong cold resistance and pollen fertility ( Figure 6 A, Figure 7 B), and two chromosomes from MLM266-2 are present on chromosomes 9 and 12 ( Figure 8 B), so this potato cold-resistant additional line can lay the material foundation for breeding cold-resistant potato varieties.

[0094] Example 3

[0095] This embodiment provides an application of using aneuploid interspecific hybrid materials to cultivate cold-resistant potato varieties. The specific verification steps are as follows:

[0096] (1) Construction of backcross population: The diploid wild species MLM266-2 was used as the female parent and the diploid CHC9701 was used as the male parent for hybridization. Based on the EBN theory, the hybrid offspring were all triploid. The cold resistance of the triploid hybrid offspring was screened, and the offspring with the strongest cold resistance, FT040-7, was backcrossed with CHC9701 to obtain the backcross population BC1.

[0097] (2) Ploidy analysis of BC1 backcross progeny was performed using root tip chromosome counting, and the progeny with chromosome numbers distributed between 30 and 34 were obtained.

[0098] (3) The CTAB method was used to extract DNA from BC1 backcross progeny and parental individual plants.

[0099] (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 was considered to have passed the quality inspection and then whole genome sequencing was performed.

[0100] (5) Quality control analysis of sequencing data.

[0101] (6) Mutation detection.

[0102] (7) Polymorphic SNP marker screening.

[0103] (8) Calculation of parental allele ratios in offspring.

[0104] (9) Analysis of genomic components of BC1 backcross progeny.

[0105] (10) Identification and localization of cold resistance of backcross offspring: The cold resistance of backcross offspring was evaluated by the conductivity leakage method. Then, the sequencing results of the four strains with the strongest and four strains with the weakest domesticated cold resistance in the backcross offspring were subjected to whole-genome selective scanning. The Δ(SNP-index) value of each variant site was calculated using QTLseqr software, and its distribution on the genome was examined and mapped using a 2Mb sliding window. The results of the whole-genome selective scanning showed that there were four obvious cold-resistant segments of domesticated cold resistance, which were anchored on chromosomes 4, 9, 11 and 12 respectively.

[0106] (11) Fertility identification of backcross offspring: The parents and offspring were planted in the greenhouse of the base and the flowering characteristics were counted. The pollen vitality of the parents and backcross offspring was checked by acetic acid magenta staining during the flowering period. The pollen vitality and pollen grain size of the parents and backcross offspring were observed. The individual plants with good pollen vitality and 2n pollen were selected among the backcross offspring.

[0107] (12) Screening of potato cold-resistant addition lines: Based on the retention of the number of chromosomes of MLM266-2 in chromosomes 1-12 in the 13 offspring and the overall comparative analysis of the genomic composition, cold-resistant positioning and fertility of the 13 offspring, the BC1 backcross offspring were screened for materials with the following genomic composition: 31 chromosomes, and the number of chromosomes 1-12 from CHC9701 was 2, 1, 2, 2, 1, 2, 2, 1, 1, 1, 1, and the number of chromosomes 1-12 from MLM266-2 was 0, 1, 1, 1, 1, 1, 1, 2, 1, 1, 2 (Table 1, Figure 8 A). This additional line material has strong cold resistance and pollen fertility ( Figure 6 A, Figure 7B), and two chromosomes from MLM266-2 are present on chromosomes 9 and 12 ( Figure 8 B), so this potato cold-resistant additional line can lay the material foundation for breeding cold-resistant potato varieties.

[0108] (13) Application of cold-resistant potato additional lines: The backcross offspring constructed in (1) are theoretically 2EBN, while the common potato cultivars are 4EBN. According to the EBN theory (Johnston et al 1980), the backcross offspring cannot be directly backcrossed with the common cultivars. Given that the technology of doubling potatoes using colchicine is already very mature (Dong Jianke et al 2020), this chromosome doubling technology was used to double the additional line in Example 2. The results after colchicine doubling showed that the additional line was successfully doubled, with a chromosome number of 62 ( Figure 9 B). The doubled additional lines were planted in the field and their pollen fertility was counted. The results showed that they had good growth potential and could flower normally ( Figure 9 A, C), pollen grains can be stained with acetic acid red, indicating pollen fertility; the cold resistance test results of the doubled addition line showed that it still had strong cold resistance, significantly stronger than the cultivated variety Redsen ( Figure 9 D) This example shows that the doubled addition line can be used as a new cold-resistant breeding resource (4EBN) and can be directly hybridized with cultivated potato for wild germplasm introduction.

[0109] References and cited experimental methods:

[0110] [1] Dong Jianke, Tu Wei, Wang Haibo, Ying Jingwen, Du Juan, Zhao Xijuan, Zhao Qinghao, Huang Wei, Cai Xingkui, Song Botao (2020). Establishment of efficient chromosome doubling method and creation of cold-resistant resources for potato. Acta Agronomica Sinica, 46(11): 1659-1666.

[0111] [2] Tan Chen, Zhu Kaiyuan, Xiang Yi, Li Zaiyun (2018). Establishment of an efficient identification system for B genome-specific addition line materials in Brassica. Molecular Plant Breeding, 16(10): 3227-3233.

[0112] [3] Tu Wei, Zhao Xijuan, Kou Shuang, Kang Li, Chen Lin, Song Botao (2015). Establishment and application of direct evaluation system for cold resistance of potato seedlings. China Potato, 29(01): 1-7.

[0113] [4] Wang Haibo. Genomic composition of potato + eggplant somatic hybrid and the genetic basis of bacterial wilt resistance[D]. Huazhong Agricultural University, 2020.

[0114] [5]Carputo D,Barone A,Cardi T,Sebastiano A,Frusciante L,PeloquinSJ.1997.Endosperm balance number manipulation for direct in vivo germplasmintrogression to potato from a sexually isolated relative(Solanum commersoniiDun.).Pro Nat Acad Sci,USA,94,12013-12017.

[0115] [6]Chetelat RT,Rick CM,Cisneros P,Alpert KB,DeVernaJW.1998.Identification,transmission,and cytological behavior of Solanumlycopersicoides Dun.monosomic alien addition lines in tomato(Lycopersiconesculentum Mill.).Genome,41,40-50

[0116] [7]Comai L,Tan EH.2019.Haploid induction and genomeinstability.Trends in Genetics,35,791-803.

[0117] [8]Dellaporta,SL,Jonathan,W,Hicks,JB.1983.A Plant DNAminipreparation:version II.Plant Mol.Biol.Rep,1,19-21.

[0118] [9]Dewitte A,Van Laere K,Van Huylenbroeck J.2011.Use of 2n Gametes inPlant Breeding.Plant Breeding,59-86.

[0119]

[10] Hardigan MA,Laimbeer FPE,Newton L,Emily C,John PH,Brieanne V,Krystle W,Joshua CW,David SD,Eva MF,Richard E V,Robin B.2017.Genome diversityof tuber-bearing Solanum uncovers complex evolutionary history and targets ofdomestication in the cultivated potato.Pro Nat Acad Sci,USA,114,E9999-E10008.

[0120]

[11] Husband BC.2004.The role of triploid hybrids in the evolutionarydynamics of mixed-ploidy populations.Biological Journal of the LinneanSociety,82,537-546.

[0121]

[12] Law C,Worland A.1972.Aneuploidy in wheat and its uses in geneticanalysis.In:Lupton FGH,editor.Wheat Breeding:Its Scientific Basis.London:Chapman and Hall;1987.pp.71–108.Annual report.

[0122]

[13] Li YP,Colleoni C,Zhang JJ,Liang QQ,Hu YF,Ruess H,Simon R,Liu YH,Liu HM,Yu GW,Schmitt E,Ponitzki C,Liu GJ,Huang HH,Zhan FL,Chen L,Huang YB,Spooner DM,Huang BQ.2018.Genomic analyses yield markers for identifyingagronomically important genes in potato.Molecular Plant,11,473-484.

[0123]

[14] Johnston SA,Den Nijs TPM,Peloquin SJ,Hanneman RE.1980.Thesignificance of genic balance to endosperm development in interspecificcrosses.Theor Appl Genet,57,5-9

[0124]

[15] Ortiz R,Ehlenfeldt MK.1992.The importance of endosperm balancenumber in potato breeding and the evolution of tuber-bearing Solanumspecies.Euphytica,60,105-113.

[0125]

[16] Spooner DM,Ghislain M,Simon R,Jansky SH,GavrilenkoT.2014.Systematics,Diversity,Genetics,and Evolution of Wild and CultivatedPotatoes.Botanical Review,80,283-383.

[0126]

[17] Tu W,Dong J,Zou Y,Zhao Q,Wang H,Ying J,Wu J,Du J,Cai X,SongB.2021.Interspecificpotato somatic hybrids between Solanum malmeanum andS.tuberosum provide valuableresources for freezing-tolerance breeding.PlantCell Tiss.Org.Cult.147,73-83.

[0127]

[18] Vega SE,Bamberg JB.1995.Screening the US potato collection forfrost hardiness.Am Potato J,72,13-21.

Claims

1. A method for rapid and accurate analysis of the genome composition of interspecific hybrids of potato aneuploids, characterized in that The method specifically comprises the following steps: (1) Backcross population construction: The diploid wild species MLM266-2 was used as the female parent and the diploid CHC9701 was used as the male parent to hybridize. The cold resistance of the triploid hybrid offspring was screened. The offspring with the strongest cold resistance were backcrossed with CHC9701 to obtain the backcross population BC1. (2) Chromosome ploidy analysis: Ploidy analysis of BC1 backcross progeny was performed using root tip chromosome counting, and progeny with a chromosome number distribution between 30 and 34 were obtained; (3) DNA extraction of backcross progeny: DNA of BC1 backcross progeny and parental plants was extracted using the CTAB method; (4) Library construction and sequencing: The concentration and purity of the extracted DNA were detected by UV spectrophotometer. DNA samples with a significant absorption peak at OD260 and an OD260 / OD280 ratio of 1.7-1.9 were subjected to whole genome sequencing; (5) Sequencing data quality control analysis; (6) Mutation detection; (7) Polymorphic SNP marker screening; (8) Calculation of parental allele ratios in offspring; (9) Analysis of genomic components of BC1 backcross progeny.

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