Cold resistance molecular marker of potato and its application in breeding

Abstract

The application provides a potato cold resistance molecular marker and application of a combination thereof in breeding. Specifically relates to development of a potato cold resistance SNP marker and establishment of a molecular assisted selection system, and belongs to the field of plant molecular genetic breeding. Based on construction of a multi-parent hybrid population and analysis of field cold resistance of offspring, a pool separation analysis method (BSA) is used to locate the field cold resistance on chromosomes V and IX respectively; a target capture sequencing technology is used to genotype a candidate interval, and according to correlation analysis of genotypes and field cold resistance phenotypes, finally, six molecular markers (P<0.05) significantly related to the field cold resistance are obtained, and a cold resistance molecular marker assisted selection system is established. The SNP marker of the field cold resistance and the molecular marker assisted system provided by the application can be used for early molecular assisted selection of the field cold resistance of potatoes, and theoretically and in application, the advantage of using a multi-parent hybrid method to broaden and improve cold resistance genetic resources is highlighted.

Description

Technical Field

[0001] This invention relates to the field of plant molecular genetic breeding, specifically to molecular combination markers for cold resistance in potatoes and their application in breeding. Background Technology

[0002] Potato (Solanum tuberosum L.) is the world's fourth largest food crop and the most important tuber crop, widely cultivated and consumed worldwide. It plays a vital role in ensuring global food security and meeting the increasing food demands of future population growth. Frost damage is a major environmental stressor, significantly impacting plant growth, development, productivity, and geographical distribution. In my country's four major potato ecological zones, the southern winter-cropping region is more susceptible to frost damage because potato planting occurs primarily in late autumn, winter, or early spring. With the implementation of the potato staple food strategy, the majority of the planned increase in potato planting area is expected to come from the utilization of fallow winter fields in the south. However, common potato cultivars are not frost-resistant and lack cold acclimatization capabilities, severely hindering the development and utilization of fallow winter fields in the south and significantly impacting the further development of my country's potato industry. However, regarding the frost resistance of potato germplasm, common potato cultivars lack cold resistance and exhibit almost no genetic variation, suffering severe frost damage within just a few hours when temperatures drop below -3°C (Vega and Bamberg 1995; Luthra et al. 2007). Compared to the pest and disease problems in field production, cultivation or chemical treatments seem unlikely to help prevent frost damage; therefore, there is an urgent need to create new frost-resistant potato varieties through genetic improvement.

[0003] Although cold-resistant resources are scarce in common potato cultivars, wild potato species possess abundant resistance gene resources. Previous studies have evaluated the cold resistance of wild potato species using indoor electrical conductivity and field natural frost systems, finding that cold resistance is mainly distributed in germplasm of S. acaule, S. albicans, S. commersonii, S. demissum, and S. malmeanum (Chen and Li 1980; Vega and Bamberg 1995; Tu et al. 2021). These wild species are important resources for studying the genetic mechanisms of potato cold resistance and germplasm creation. However, due to ploidy and EBN-mediated interspecific hybridization barriers before and after zygosis, cold-resistant wild species cannot directly engage in sexual hybridization with common cultivars, thus preventing the exchange of resistance genes. Although previous researchers have made some progress in introducing the cold resistance of wild potatoes into potato cultivation through somatic cell fusion and ploidy breeding (Cardi et al 1993; Nyman and Waara 1997; Tu et al 2021), there is still a lack of new cold-resistant potato varieties suitable for large-scale planting and promotion.

[0004] Genetic studies in different plants have confirmed that frost resistance is a complex polygenic trait controlled by a few loci, resulting in most of the genetic variation (Cattivelli 2011). Previous studies have shown that non-domestication hardiness (NA) and cold acclimatization ability (ACC) are the two major genetic components of potato cold resistance, both exhibiting independent partial recessive inheritance (Stone et al. 1993). Subsequently, through genetic mapping and cold resistance localization, two quantitative trait loci (QTLs) for NA and ACC were preliminarily located on different segments of chromosome 5 (Vega et al. 2003). However, due to the limited population size, their initial localization requires further investigation. The complexity of potato cold resistance inheritance has led to a stagnation in its research, resulting in even fewer reports on the development of markers closely linked to potato cold resistance. Li Fei (2013) developed a CAPS marker for the SAD gene using diploid frost-resistant wild potato S. commersonii and frost-sensitive S. cardiophytlum, respectively, and validated it in backcross progeny of these two species.

[0005] Problems with existing technologies: There are currently no reported molecular markers for cold resistance in potato fields; previous studies mainly focused on developing cold-resistant SSR markers at the diploid level, and no cold-resistant SNP markers have been developed at the tetraploid level; there is a lack of molecular marker-assisted selection systems for potato frost resistance breeding. These are all major obstacles to the development of potato cold-resistance breeding. Current potato cold-resistance breeding mainly relies on indoor conductivity methods to determine the cold resistance of offspring, which involves long experimental cycles, increasing the breeding period and consuming significant human, material, and financial resources. Summary of the Invention

[0006] The key technical problem this invention aims to solve is to provide molecular combination markers for cold resistance in potatoes and their application in breeding. To solve the above technical problem, this invention adopts the following technical solution:

[0007] 1. SNP markers related to cold resistance in potato fields, wherein the SNP markers are identified by primers as shown in SEQ No. 1-12 of the sequence listing, and the sequences involved after primer amplification of the SNP markers are shown in SEQ No. 13-18 of the sequence listing.

[0008] 2. SNP combination markers related to cold resistance in potato fields, wherein the SNP combination markers include: Chr05V158+Chr09V26+Chr09V212, and the SNP combination markers are identified by the following primers: primers for identifying Chr05V158 are shown in SEQ No. 5 and SEQ No. 6 of the sequence listing; primers for identifying Chr09V26 are shown in SEQ No. 7 and SEQ No. 8 of the sequence listing; primers for identifying Chr09V212 are shown in SEQ No. 9 and SEQ No. 10 of the sequence listing.

[0009] 3. Application of potato SNP combination markers in field cold resistance identification, wherein the SNP combination markers include: Chr05V158+Chr09V26+Chr09V212, and the SNP combination markers are identified using the following primers: Chr05V158 is identified using primers as shown in SEQ No. 5 and SEQ No. 6 of the sequence listing; Chr09V26 is identified using primers as shown in SEQ No. 7 and SEQ No. 8 of the sequence listing; and Chr09V212 is identified using primers as shown in SEQ No. 9 and SEQ No. 10 of the sequence listing.

[0010] 4. Application of potato SNP combination markers in field cold resistance identification, the application is carried out through the following steps:

[0011] (1) Extract DNA from the cold-resistant material to be tested.

[0012] (2) SNP site target capture sequencing was performed using the combined marker primers SEQ No.5+SEQ No.6, SEQ No.7+SEQ No.8 and SEQ No.9+SEQ No.10.

[0013] (3) The SNP sites in the captured sequencing results are compared with sites 39 of SEQ No. 15, 37 of SEQ No. 16 and 158 of SEQ No. 17. If the base types of these three SNP sites are G / G, G / G or A / G and G / G or A / G respectively, the material can be considered to be a cold-resistant strain.

[0014] 5. The method for obtaining molecular markers of cold resistance in potato fields includes the following steps:

[0015] (1) The frost tolerance of 8 potato interspecific hybrids and 8 cultivated potatoes was evaluated under natural field frost conditions. Among them, 8 potato interspecific hybrids containing the lineage of S. commersonii, S. acaule and S. tuberosum (14FT04-25, 14FT04-44, 14FT04-63, 14FT04-71, 14FT24-10, 14FT43-25, 14FT51-08 and 14FT51-03) and 8 cultivated potatoes (Denali, Bora Valley, Pentland Crown, M1, M3, Plain, RH89-039-16 and Hua cai 1) were all breeding materials preserved in our laboratory. All of the above materials are available to the public and have been published in relevant literature (Dong Jianke et al., 2019). Based on weather forecasts from local meteorological stations in Luoyang (December 15, 2016, December 27, 2016, December 28, 2016) and Wuhan (January 21, 2017, February 11, 2017, January 9, 2018, January 11, 2018), three field frost experiments were conducted on the hybrid parents under low temperatures. Three field frosts at two experimental sites in Wuhan (January 21, 2017, January 9, 2018) and Luoyang (December 27, 2016) showed that eight cultivated potato varieties exhibited some degree of frost damage after natural field frosts, with average damage scores (AS) of 3.49±0.68 (January 21, 2017), 3.80±0.78 (January 9, 2018), and 5.18±0.80 (December 27, 2016), respectively. Meanwhile, eight interspecific hybrids showed extremely strong frost resistance in the field, with corresponding AS scores of 0.19±0.24, 0.58±0.52, and 0.10±0.20, respectively (Table 1). In addition, two other field frosts in Wuhan (February 21, 2017, and February 11, 2018) and the third frost in Luoyang (December 27, 2016) resulted in the death of all cultivated potatoes due to excessively low temperatures and prolonged duration. Figure 1 D), however, some interspecific hybrids exhibit strong frost resistance ( Figure 1 C), AS values ​​were 2.15±2.12, 2.20±1.24, and 2.39±0.98, respectively (Table 1). Field resistance comparisons of the cold-resistant and sensitive parents under different frost conditions showed that the average field resistance of the cold-resistant parent was significantly different from that of the sensitive parent under each frost condition. Figure 1 A, B).

[0016] (2) Complete diallel crosses were performed between 8 cold-resistant interspecific hybrids and 8 frost-sensitive cultivars from (1), resulting in 406 offspring from 23 combinations. Natural field frosts were conducted on the parents and offspring on February 11, 2017 and January 11, 2018. The field frost results of the offspring were statistically analyzed using the identification method of Vega and Bamberg (1995). The combination information and field cold resistance distribution of the 406 offspring are shown in Table 2. Based on two years of field frost data from 406 offspring, 30 extremely cold-resistant individual plants and 30 extremely sensitive individual plants were selected to construct mixed cold-resistant and extremely sensitive ponds, respectively. Comparative analysis of field cold resistance in the extremely cold-resistant and extremely sensitive ponds under different environmental conditions showed that the mean and coefficient of variation of cold resistance in the extremely cold-resistant ponds were 1.13±0.70, 61.51% (January 21, 2017) and 1.82±0.56, 0.63% (January 11, 2018), respectively, while the mean and coefficient of variation of cold resistance in the extremely sensitive ponds were 4.17±1.15, 27.60% (January 21, 2017) and 5.29±0.94, 17.74% (January 11, 2018), respectively. Under both environmental conditions, the field cold resistance of the cold-resistant ponds was significantly higher than that of the sensitive ponds. Figure 2 ).

[0017] (3) High-throughput sequencing of the extreme resistance pools was performed using the BSA-seq method. Candidate intervals were determined using both Δ(SNP-index) and G' values. To eliminate the influence of unreliable markers, screening was performed based on SNP-index. The screening criteria were: (1) the sequencing depth of all extreme resistance pools was greater than 20x; (2) the SNP-index values ​​of the extreme resistance pools could not be simultaneously greater than 0.8 or simultaneously less than 0.25. Based on the markers obtained from the screening, Δ(SNP-index) and G' were calculated using QTL-seqr software. The sliding window size was set to 2Mb. Finally, two candidate intervals were located, located on chromosome 5 from 18.3 to 42.74M and chromosome 9 from 12.4 to 46.35M, respectively. Figure 3 ).

[0018] (4) Based on the sequencing results in (3), select differential SNP sites in the cold resistance candidate regions located on chromosomes V and IX. SNP site analysis and primer design: For SNP sites with a frequency difference greater than 0.25 between the two phenotype pools, search for the corresponding base information on the potato DM genome (http: / / solanaceae.plantbiology.msu.edu / pgsc_download.shtml) based on the selected SNP sites, and obtain the DNA sequence 150-200 bp to the left and right of the SNP site. Use the NCBI online primer design tool Prime-blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design primers to ensure that the primers do not contain SNP sites and that the product contains only the SNP sites of interest.

[0019] (5) Based on the SNP sites selected in (4), marker genotyping is performed using capture sequencing. The flowchart of the target capture sequencing technology is as follows: Figure 4 As shown. First, capture primers were used to capture DNA sequences at each target locus in the genotypes of two groups of resistant parents and offspring from an extremely cold-resistant mixed pool. Then, the products were recovered and quality-tested before the Paired-end (PE) sequencing library was constructed according to the Illumina library construction protocol. An equal amount of DNA from each sample was used to construct a PE library and PE150 sequencing was performed on an Illumina Hiseq sequencer.

[0020] (6) Based on the raw sequencing data in (5), data quality control was first performed to obtain high-quality clean data. Then, BWA software was used to break down the clean data into each target locus to obtain SAM format alignment results. Then, samtools software was used to convert the SAM format file into BAM format. Next, SortSam in Picard was used to sort the reads in the BAM file to obtain the final BAM file. Finally, GATK was used to determine the genotype and sequencing depth of each target locus.

[0021] (7) Based on the genotypes of each target locus in (6), and combined with the field resistance of the two groups of resistant and susceptible parents and the genotypes of the cold-resistant mixed offspring, the correlation between the two was analyzed to screen out candidate field cold resistance markers; then, the candidate SNP marker loci were target-captured and sequenced in the remaining offspring, and finally the genotypes of all offspring were obtained. Combined with the field resistance results of the hybrid offspring, the correlation between the marker genotype and field cold resistance was analyzed, and finally 6 SNP markers that were significantly associated with field resistance were obtained (Table 3).

[0022] Beneficial effects:

[0023] (1) This invention, building upon previous research methods using multi-parental hybrid populations for quantitative trait mapping, initially locates candidate regions associated with field cold resistance. Complete diallel crosses were performed between eight cold-resistant interspecific hybrids and eight frost-sensitive cultivars. Mixed-pool segregation analysis (BSA) was used to genetically map field cold resistance, finding it to regions 18.3–42.74M on chromosome V and 12.4–46.35M on chromosome IX, respectively. This lays an important foundation and is a necessary prerequisite for achieving molecular-assisted breeding for the genetic improvement of multiple genes controlling traits.

[0024] (2) Based on the identified candidate regions for field cold resistance in potatoes, this invention developed six SNP molecular markers that were significantly associated with field cold resistance. Genotypes of 330 progeny from 23 hybrid combinations were analyzed using these markers, and the correlations between genotype and field cold resistance were all highly significant (P < 0.01).

[0025] (3) This invention develops a method for detecting cold resistance in potatoes based on the located candidate regions for cold resistance in the field. This method constructs a marker-assisted selection system using the chromosome V marker Chr05V158 and chromosome IX markers Chr09V26 and Chr09V212. Target capture sequencing at these three specific loci is sufficient to obtain the field cold resistance of progeny materials. This method can rapidly and effectively evaluate the cold resistance of potato resources, shorten the breeding process, and provide a new breeding system for the development of potato breeding. Attached Figure Description

[0026] Figure 1 This is the result of field cold resistance analysis of multiple parents under different environments in Example 2 of the present invention. Eight cold-resistant interspecific hybrids and eight frost-sensitive cultivars were tested in four environments in Wuhan (…). Figure 1 A) and the three environments of Luoyang ( Figure 1 B) Field comparison of cold resistance, some representative cold-resistant interspecific hybrids ( Figure 1 C) and frost-sensitive varieties ( Figure 1 D) Field frost results.

[0027] Figure 2 This study compares the field cold resistance of two frost-prone ponds (extremely cold-resistant and extremely sensitive) in 2017 and 2018.

[0028] Figure 3The field cold resistance region localization results based on BSA-seq are shown in the figure. Candidate regions were determined using both Δ(SNP-index) and G' values. Based on the screened markers, using the potato genome DM_v4.03 as the reference genome, the Δ(SNP-index) and G' values ​​were calculated using QTL-seqr software, and plotted using R tools. Combining the two methods, two candidate regions were located, situated in the 18.3–42.74 Mb region on chromosome V and the 12.4–46.35 Mb region on chromosome IX, respectively.

[0029] Figure 4 This is a flowchart of the customized target capture sequencing technology in Embodiment 2 of the present invention.

[0030] Figure 5 This is a comparative analysis of field resistance of different marker combinations on chromosomes V and IX in Example 3 of the present invention. The vertical axis represents field cold resistance, and the horizontal axis represents different marker combinations on chromosomes V and IX, Chr05(0)+Chr09(1) (all three markers on chromosome 5 are '0', at least one of the three marker types on chromosome 9 is '1', and so on).

[0031] Figure 6 This is a comparative analysis of the genotyping results of different types of six markers and field resistance in Example 3 of the present invention. Note: The horizontal axis is labeled in the order of Chr05V42, Chr05V92, Chr05V158, Chr09V26, Chr09V212, Chr09V222), and '001011' indicates that the six SNP base types are Chr05V42 (G / G), Chr05V92 (A / A), Chr05V158 (G / G), Chr09V26 (A / A), Chr09V212 (G / G or A / G), and Chr09V222 (A / A or A / C); the vertical axis is field cold resistance.

[0032] Figure 7 Sequences 13 to 18 are shown, with SNP sites highlighted. Specific implementation methods

[0033] Unless otherwise specified, the methods and apparatus used in the following embodiments of this invention are conventional methods and apparatus; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions, and advantages of this invention clearer, the specific implementation methods of this invention are described in detail below with reference to specific embodiments. Examples of these preferred embodiments are illustrated in the specific embodiments. It should also be noted that, in order to avoid obscuring the technical solution of this invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solution according to this invention are shown in the embodiments, while other details that are not closely related are omitted.

[0034] Example 1

[0035] This embodiment provides three SNP markers located on chromosome V and three on chromosome IX that are associated with field cold resistance in potatoes. The key feature is that the clonal verification of the three SNP markers located on chromosome V can be achieved using the following primers:

[0036] 1. Forward primer sequence Chr05V42-F: 5'-GTGTCTGCGTCTGCTTCATTCA-3'; Reverse primer sequence Chr05V42-R: 5'-ATCGGCGGTGGAGCAGTC-3'.

[0037] 2. Forward primer sequence Chr05V92-F: 5'-TAGGGTTTCTTCTAGTCTTGGGGTATAGTA-3'; Reverse primer sequence Chr05V92-R: 5'-AGAACTTTTCCCGAATATTTGCTTTTCCTA-3'.

[0038] 3. Forward primer sequence Chr05V158-F: 5'-GCCCCGTTACAAACCCTGG-3'; Reverse primer sequence Chr05V158-R: 5'-TGCTTGCCTCAAATGCAAGTTCA-3'.

[0039] Clones of three SNP markers located on chromosome IX can be identified using the following primers:

[0040] 1. Forward primer sequence Chr09V26-F: 5'-ACTGGTCTGATACAAACAATTGATCCAAAA-3'; Reverse primer sequence Chr09V26-R: 5'-AGAGAAGTTACAACTCTATCAAGGTTGTCT-3'.

[0041] 2. Forward primer sequence Chr09V212-F: 5'-CCAAGATTATTGAATTGATGGTCTATGGGT-3'; Reverse primer sequence Chr09V212-R: 5'-ACAAGTTGGGTTCTTTACTTGATAACGATT-3'.

[0042] 3. Forward primer sequence Chr09V222-F: 5'-ACATTAAACAACTTGATTGTTGCGCT-3'; Reverse primer sequence Chr09V222-R: 5'-TCATGTATCAGCAACTGTGATTGTCT-3'.

[0043] Example 2

[0044] This embodiment provides a method for obtaining molecular markers of cold resistance in potato fields, specifically including the following steps:

[0045] (1) The frost tolerance of 8 potato interspecific hybrids and 8 cultivated potatoes was evaluated under natural field frost conditions. The 8 interspecific hybrids containing *S. commersonii*, *S. acaule*, and *S. tuberosum* lineages and the 8 cultivated potatoes were all breeding materials preserved in our laboratory (Dong Jianke et al., 2019), and all of these materials are publicly available. Based on weather forecasts from local meteorological stations in Luoyang (December 15, 2016, December 27, 2016, December 28, 2016) and Wuhan (January 21, 2017, February 11, 2017, January 9, 2018, January 11, 2018), three field frost tests were conducted on the hybrid parents under low-temperature conditions. Three field frosts at two experimental sites in Wuhan (January 21, 2017, January 9, 2018) and Luoyang (December 27, 2016) showed that eight cultivated potato varieties exhibited some degree of frost damage after natural field frosts, with average damage scores (AS) of 3.49±0.68 (January 21, 2017), 3.80±0.78 (January 9, 2018), and 5.18±0.80 (December 27, 2016), respectively. Meanwhile, eight interspecific hybrids showed extremely strong frost resistance in the field, with corresponding AS scores of 0.19±0.24, 0.58±0.52, and 0.10±0.20, respectively (Table 1). In addition, two other field frosts in Wuhan (February 21, 2017, and February 11, 2018) and the third frost in Luoyang (December 27, 2016) resulted in the death of all cultivated potatoes due to excessively low temperatures and prolonged duration. Figure 1 D), however, some interspecific hybrids exhibit strong frost resistance ( Figure 1C), AS values ​​were 2.15±2.12, 2.20±1.24, and 2.39±0.98, respectively (Table 1). Field resistance comparisons of the cold-resistant and sensitive parents under different frost conditions showed that the average field resistance of the cold-resistant parent was significantly different from that of the sensitive parent under each frost condition. Figure 1 A, B).

[0046] (2) Complete diallel crosses were performed between 8 cold-resistant interspecific hybrids and 8 sensitive cultivars from (1), resulting in 406 offspring from 23 combinations. Natural field frosts were conducted on the parents and offspring on February 11, 2017 and January 11, 2018. The field frost results of the offspring were statistically analyzed using the identification method of Vega and Bamberg (1995). The combination information and field cold resistance distribution of the 406 offspring are shown in Table 2. Based on two years of field frost data from 406 offspring, 30 extremely cold-resistant individual plants and 30 extremely sensitive individual plants were selected to construct mixed cold-resistant and extremely sensitive ponds, respectively. Comparative analysis of field cold resistance in the extremely cold-resistant and extremely sensitive ponds under different environmental conditions showed that the mean and coefficient of variation of cold resistance in the extremely cold-resistant ponds were 1.13±0.70, 61.51% (January 21, 2017) and 1.82±0.56, 0.63% (January 11, 2018), respectively, while the mean and coefficient of variation of cold resistance in the extremely sensitive ponds were 4.17±1.15, 27.60% (January 21, 2017) and 5.29±0.94, 17.74% (January 11, 2018), respectively. Under both environmental conditions, the field cold resistance of the cold-resistant ponds was significantly higher than that of the sensitive ponds. Figure 2 ).

[0047] (3) High-throughput sequencing of the extreme resistance pools was performed using the BSA-seq method. Candidate intervals were determined using both Δ(SNP-index) and G' values. To eliminate the influence of unreliable markers, screening was performed based on SNP-index. The screening criteria were: (1) the sequencing depth of all extreme resistance pools was greater than 20x; (2) the SNP-index values ​​of the extreme resistance pools could not be simultaneously greater than 0.8 or simultaneously less than 0.2. Based on the markers obtained from the screening, Δ(SNP-index) and G' were calculated using QTLseqr software. The sliding window size was set to 2Mb. Finally, two candidate intervals were located, located on chromosome 5 from 18.3 to 42.74M and chromosome 9 from 12.4 to 46.35M, respectively. Figure 3 ).

[0048] (4) Based on the sequencing results in (3), select differential SNP sites in the cold resistance candidate regions located on chromosomes V and IX. SNP site analysis and primer design: For SNP sites with a frequency difference greater than 0.25 between the two phenotype pools, search for the corresponding base information on the potato DM genome (http: / / solanaceae.plantbiology.msu.edu / pgsc_download.shtml) based on the selected SNP sites, and obtain the DNA sequence 150-200 bp to the left and right of the SNP site. Use the NCBI online primer design tool Prime-blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design primers, ensuring that the primers do not contain SNP sites and that the product contains only the SNP sites of interest.

[0049] (5) Based on the SNP sites selected in (4), marker genotyping is performed using capture sequencing. The flowchart of the target capture sequencing technology is as follows: Figure 4 As shown. First, capture primers were used to capture DNA sequences at each target locus in the genotypes of two groups of resistant parents and offspring from an extremely cold-resistant mixed pool. Then, the products were recovered and quality-tested before the Paired-end (PE) sequencing library was constructed according to the Illumina library construction protocol. An equal amount of DNA from each sample was used to construct a PE library and PE150 sequencing was performed on an Illumina Hiseq sequencer.

[0050] (6) Based on the raw sequencing data from (5), data quality control was first performed to obtain high-quality clean data. Then, BWA software was used to break down the clean data into each target locus, obtaining alignment results in SAM format. The SAM format files were then converted to BAM format using samtools software. Next, SortSam in the Picard tool was used to sort the reads in the BAM file, obtaining the final BAM file. Finally, GATK was used to determine the genotype and sequencing depth of each target locus.

[0051] (7) Based on the genotypes of each target locus in (6), and combined with the field resistance of the two groups of resistant and susceptible parents and the genotypes of the cold-resistant mixed offspring, the correlation between the two was analyzed to screen out candidate field cold resistance markers; then, the candidate SNP marker loci were target-captured and sequenced in the remaining offspring, and finally the genotypes of all offspring were obtained. Combined with the field resistance results of the hybrid offspring, the correlation between the marker genotype and field cold resistance was analyzed, and finally 6 SNP markers that were significantly associated with field resistance were obtained (Table 3).

[0052] Table 1. Field cold resistance of multiple parents under different environments

[0053]

[0054] Table 2. Offspring number and field cold resistance distribution of multi-parent hybrid populations

[0055]

[0056] Table 3. Six SNP molecular markers significantly associated with field cold resistance

[0057]

[0058] Example 3

[0059] This embodiment describes a method for establishing a molecular-assisted breeding system using the six SNP markers related to field cold resistance obtained in the above embodiments; the specific verification steps are as follows:

[0060] (1) To further clarify the effect of the combination of the three markers of chromosome V and the three markers of chromosome IX on field cold resistance, genotyping was performed on each offspring (marker order: Chr05V42, Chr05V92, Chr05V158, Chr09V26, Chr09V212, Chr09V222). The offspring genotypes can be divided into four types: Chr05(0)+Chr09(0) (all six marker types of chromosomes 5 and 9 are '0'), Chr05(0)+Chr09(1) (all three markers of chromosome 5 are '0', and at least one of the three marker types of chromosome 9 is '1', and so on), Chr05(1)+Chr09(0) and Chr05(1)+Chr09(1). The above four types correspond to 21, 52, 8 and 245 genotypes, respectively. The field hardiness results of different marker combinations showed that, in the field frosts of 2017 and 2018, the offspring of the Chr05(1)+Chr09(1) type had the strongest field hardiness, which was significantly different from the other three types. Figure 5 ).

[0061] (2) Based on the four types in (1), the genotypes of the offspring of the Chr05(1)+Chr09(1) type were further classified. The classification results showed that there were a total of 12 types. Figure 6Among the various markers, the '111111' combination had the most offspring, accounting for 40.74% (99 / 243). Comparative analysis of the field cold resistance among different combinations showed no significant difference in field resistance during the 2017 field frost. However, during the 2018 field frost, some differences in resistance emerged, with the '001011' combination exhibiting the worst field resistance and the '111111' combination showing the strongest. Apart from these two markers, there were no significant differences in resistance among combinations with other markers. The offspring of the '001011', '100011', and '100111' combinations showed relatively poor field resistance, while the offspring of Chr05V158, Chr09V26, and Chr09V212 combinations, all showing a marker type of '1', exhibited stronger field resistance. Based on the above analysis results, and considering the correlation and concordance between SNP markers and resistance phenotypes, we believe that combining the Chr05V158 marker on chromosome 5 with the Chr09V26 and Chr09V212 markers on chromosome 9 can effectively select for resistance in offspring (Chr05V158 + Chr09V26 + Chr09V212).

[0062] Example 4

[0063] This embodiment is an application of potato SNP combination markers in the field cold resistance identification described in the above embodiments. The specific verification steps are as follows:

[0064] (1) Extract DNA from the cold-resistant material to be tested.

[0065] (2) SNP site target capture sequencing was performed using the combined marker primers SEQ No.5+SEQ No.6, SEQ No.7+SEQ No.8 and SEQ No.9+SEQ No.10.

[0066] (3) Figure 7 As shown, the SNP sites in the captured sequencing results were compared with sites 39 of SEQ No. 15, 37 of SEQ No. 16, and 158 of SEQ No. 17, and then further compared with the cold-resistant SNP sites in Table 3. If the base types of these three SNP sites are G / G, G / G(A / G), and G / G(A / G), the material can be considered a cold-resistant strain.

[0067] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0068] References and cited experimental methods:

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[0079]

[11] Vega SE,Bamberg JB.Screening the US potato collection for frosthardiness[J].American Potato Journal,1995,72(1):13-21.

Claims

1. A combination of SNP molecular markers for identifying field cold resistance in potatoes, characterized in that... The sequences of the SNP molecular marker combinations are shown in SEQ ID No. 15 to SEQ ID No. 17; wherein, the 39th nucleotide of the sequence shown in SEQ ID No. 15 is G or A, the 37th nucleotide of the sequence shown in SEQ ID No. 16 is G or A, and the 158th nucleotide of the sequence shown in SEQ ID No. 17 is G or A.

2. The application of the SNP molecular marker combination as described in claim 1 in the field identification of cold resistance in potatoes, characterized in that... The application is implemented through the following steps: (1) Extract DNA from the cold-resistant material to be tested; (2) SNP site target capture sequencing was performed using primers SEQ ID No.5+SEQ ID No.6, SEQ ID No.7+SEQ ID No.8 and SEQ ID No.9+SEQ ID No.10; (3) The SNP sites in the captured sequencing results are compared with site 39 of SEQ ID No. 15, site 37 of SEQ ID No. 16 and site 158 of SEQ ID No.

17. When the genotypes of these three SNP sites are G / G, G / G or A / G, G / G or A / G respectively, the material is a cold-resistant strain.

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