Pea whole genome 20K SNP liquid phase breeding chip and application thereof

Abstract

The invention discloses a pea whole genome 20K SNP (Single Nucleotide Polymorphism) liquid phase breeding chip and application thereof, the liquid phase breeding chip comprises 21 and 659 SNP loci on a pea whole genome, the SNP loci comprise published 235 reliable functional loci obviously related to 57 characters, and examples for pea sample genetic typing are provided. The chip can be widely applied to important character analysis, germplasm resource genetic diversity analysis, variety identification analysis, auxiliary selective breeding, population structure analysis, heredity and evolution analysis, genetic relationship identification, whole genome association analysis or genome selection of peas, and is helpful for improving the pea breeding efficiency; the innovation and improvement of pea varieties are promoted.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology application and relates to a pea whole genome 20K SNP liquid phase breeding chip and application thereof. Background Art

[0002] pea( Pisum sativum L.), is an important food source, animal feed and industrial raw material, with outstanding nutritional and health functions. In recent years, pea has made significant progress in breeding and improvement of important agronomic traits, but due to its large and complex genome, complex phenotypic design breeding based on genomics faces many challenges, which seriously restricts the rapid development of basic research and breeding. With the rise of computational breeding and biological breeding, the breeding gap between peas and staple crops has further widened. In order to narrow this gap, it is urgent to develop breeding chips suitable for peas, which will help scientists more effectively identify and utilize potential genes, improve breeding efficiency, promote the innovation and improvement of pea varieties, and bring new growth points to agricultural production.

[0003] The development and application of gene chips have played an important role in accelerating the analysis of crop genetic characteristics, variety identification, resource protection and breeding. Through SNP chips, researchers can analyze population genetic structure, explore candidate genes related to economic traits, and implement various breeding studies such as genomic selection. Therefore, it is necessary to screen molecular markers related to important economic traits and accelerate the breeding of pea varieties through cutting-edge molecular breeding technologies.

[0004] Genome-wide association studies and genome-wide selection breeding are the foundation of modern breeding work. However, the high cost of resequencing and the low site density and flexibility of traditional solid-phase chips limit their application in large genome species such as pea and wheat. Therefore, the development of an efficient, fast, and low-cost large-scale genotype detection tool will promote the development of pea molecular breeding in my country.

[0005] Site information is the basis for gene chip probe design, and site selection is the core and key link of chip design, and its effect directly affects the practical value of the chip. For a long time, site selection has been mainly based on criteria such as allele frequency, deletion rate, and heterozygosity rate. With the deepening of pea genomics research, the site selection and evaluation of new chips have more reference information.

[0006] Therefore, it is necessary to develop a new breeding chip to provide new tools for pea breeding. Summary of the invention

[0007] The purpose of the present invention is to provide a pea whole genome 20K SNP liquid phase breeding chip and its application to solve the above existing technical problems. The pea 20K SNP liquid phase breeding chip can be widely used in the analysis of important traits of peas, analysis of genetic diversity of germplasm resources, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, kinship identification, whole genome association analysis or genome selection, which can improve the efficiency of pea breeding, promote the innovation and improvement of pea varieties, and bring new growth points to agricultural production.

[0008] The purpose of the present invention can be achieved through the following technical solutions: A pea whole genome 20K SNP liquid phase breeding chip, characterized in that the genotyping objects of the chip include 21,659 SNP sites on the pea whole genome.

[0009] Furthermore, the liquid phase chip is loaded with molecular probes or primers of the 20K SNP molecular marker combination of the whole pea genome; 21,659 SNP sites on the whole pea genome, and the reference genome of the SNP site combination is the pea genome " Pisum sativum cultivar Zhewan1 (PeaZW1)", the position and variation information of the SNP site are expressed in the form of chromosome: physical position: reference genotype / variant allele type.

[0010] The present invention also provides a kit for analyzing the pea genome, which comprises a reagent for detecting a 20K SNP molecular marker combination of the whole pea genome, or the above-mentioned molecular probe combination, or the above-mentioned liquid phase chip.

[0011] The present invention also provides the application of the above-mentioned liquid phase chip or kit in genetic diversity analysis of pea germplasm resources, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, kinship identification, whole genome association analysis or genome selection.

[0012] The application method is to use the pea whole genome 20K SNP liquid phase breeding chip to perform genotyping on pea samples.

[0013] The beneficial effects of the present invention are: The pea 20K SNP liquid phase breeding chip of the present invention is based on association analysis of 57 agronomic traits of 237 pea samples and whole genome resequencing data. In addition, resequencing data of 77 varieties previously studied are added, from which 21,659 high-quality and representative SNP sites are screened and obtained, including 235 published reliable functional sites that are significantly correlated with 57 traits.

[0014] Based on high-throughput sequencing technology, the one-time output result information is large and nearly a thousand samples of materials can be tested simultaneously. It is suitable for mainstream second-generation sequencing platforms such as Illumina and MGI and has wide platform adaptability.

[0015] The pea 20K SNP liquid phase breeding chip of the present invention can be widely used in important pea trait analysis, germplasm resource genetic diversity analysis, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, kinship identification, whole genome association analysis or genome selection, which can improve pea breeding efficiency and promote the innovation and improvement of pea varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Picture 1 The figure is a distribution map of the SNP sites of the present invention on the pea genome; Picture 2 The figure is the number and spacing of the SNP sites of the present invention on each chromosome; Picture 3 This is a graph showing the probe capture rates of the pea 20K SNP liquid-phase breeding chip sites in 14 representative samples of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further explained with reference to the detailed description of specific embodiments below, but the examples described below are merely illustrations of the technical solutions described in the present invention and do not limit the technical solutions described in the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0018] The SNP referred to in the present invention refers to single nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism at the genome level caused by a single nucleotide variation in the nucleotide sequence, including single base deletion, insertion, transversion and conversion.

[0019] Example 1 is as follows:

[0020] Design of pea 20K SNP liquid phase breeding chip: The present invention provides a SNP site combination for pea variety identification, comprising 21,659 SNP sites on the whole genome of pea, wherein the reference genome of the SNP site combination is the pea genome " Pisum sativumcultivarZhewan1 (PeaZW1)", the position and variation information of the SNP site are expressed in the form of chromosome: physical position: reference genotype / variant allele type.

[0021] The specific process of preparing the 21,659 SNP molecular marker site combination liquid phase breeding chip is as follows: 1. Germplasm resource collection A diversity panel of 237 cultivars was planted at the Yangdu Research Base of Zhejiang Academy of Agricultural Sciences (120.21551° E, 30.25308° N), which were planted on November 12, 2020 and 2021, respectively. In addition, resequencing data of 77 cultivars previously studied were added. The cultivar categories are shown in Table 1. Young leaves were collected two weeks after planting and quickly frozen in liquid nitrogen for DNA extraction. A total of 57 traits were investigated. Two experimental replicates were performed in 2020 and 2021. Five independent experimental measurements were performed for each trait and the average was calculated. The pea cultivars used are listed below.

[0022] Table 1 ordinal number Product name ordinal number Product name ordinal number Product name ordinal number Product name 1 Zhewan No.1 80 Dongfang No.4 159 CS5008 238 L 1256 2 ZW009 81 Dongfang N0.1 160 SW1107 239 AGG1705 3 Zhongwan No.4 82 802 161 A4-3 240 Unknown 4 Xiangwan No.1 83 801 162 S5007 241 AGG116 5 Cuizhu 84 Dongfang No.11 163 W1003 242 AGG3308 6 Zhongwan No.9 85 Dongfang No.3 164 CW1106 243 AGG3315 7 19 86 806 165 S2008-1 244 G1890 8 Zhognwan No.10 87 805 166 RSW1106 245 G3787 9 Chaoxiangwan 88 166 167 WD04-03 246 G1803 10 CH-KSKT 89 Wuxuwan 168 A4-11 247 G2686 11 I523 90 G0534 169 CH1101 248 AGG1663 12 51 91 DX28 170 WD03-09 249 Wild 13 750 92 175 171 Bawan No.1 250 AGG3344 14 J29 93 Longwan No.6 172 BW01 251 AGG2309 15 J16 94 MP1835 173 LB1202 252 PI 344010 16 Xiangwan No.1 95 DX17 174 S1001 253 G758 17 Ku 96 ZW182 175 WD04-08 254 K-2820 18 J14 97 ZW183 176 WD04-07 255 PI 344538 19 J210 98 Terese 177 WD03-05 256 AGG3343 20 GS26 99 Tordag rms3 178 WD04-05 257 AGG858 21 Mizhu No.9 100 Tordag 179 ZWNJ003 258 AGG1493 22 JP2 101 Baihuaxianfeng 180 ZWNJ004 259 PIG 275 23 Early onward 102 ZJ00613 181 ZWNJ005 260 AGG1555 24 Onward 103 ZJ00683 182 ZWNJ007 261 AGG3318 25 Ambassador 104 ZJ00611 183 ZWNJ008 262 AGG3310 26 Valverde 105 ZJ00610 184 ZWNJ009 263 AGG2537 27 D8341 106 ZJ00609 185 ZWNJ011 264 AGG1665 28 65 107 ZJ00407 186 ZWNJ013 265 AGG1664 29 Zhognwan 9-1 108 ZW286 187 ZWNJ016 266 AGG105 30 Shiquanshimei 109 P330481019 188 ZWNJ022 267 AGG1430 31 ZW074 110 P330481020 189 ZWNJ026 268 AGG1429 32 P330328004 111 P330681001 190 ZWNJ027 269 G5092 33 2017332041 112 Wuzuijian 191 ZWNJ046 270 AGG894 34 Maidouzi 113 Dalihei 192 ZWNJ048 271 AGG36 35 2017335024 114 P330327019 193 ZWNJ050 272 AGG3382 36 ZW086 115 P330305004 194 ZWNJ057 273 AGG2384 37 Tuwandou 116 P330305004-1 195 ZWNJ070 274 AGG2380 38 2018333221 117 P331102021 196 ZWNJ071 275 AGG1651 39 2018331454 118 P331081024 197 ZWNJ073 276 W.J. 40 2018335478 119 P325700004 198 ZWNJ074 277 ZW6 41 2018334148 120 Dongfang No.129 199 ZWNJ079 278 PI 250439 42 P330326026 121 1717 200 ZWNJ082 279 PI 242028 43 P331082025 122 178 201 ZWNJ084 280 G5670 44 2017334051 123 ZW183-1 202 ZWNJ085 281 AGG4064 45 2018333247 124 GS28 203 ZWNJ087 282 AGG1724 46 2018331246 125 D8341 204 ZWNJ092 283 AGG190 47 2018332244 126 ZW61-1 205 ZWNJ093 284 G3315 48 P331102020 127 Longwan No.6-1 206 ZWNJ093-1 285 G3045 49 2018335252 128 WD03-03 207 ZWNJ097 286 No. 9705 50 2018332073 129 WD04-11 208 ZWNJ107 287 G2620 51 2018331002 130 Zhengao 209 ZWNJ108 288 Matar 52 2018332112 131 S2005 210 ZWNJ109 289 G 24180 53 P331124010 132 7-6052-2 211 ZWNJ120 290 Moshong 54 2018332045 133 CH01 212 ZWNJ123 291 G5670 55 2017334052 134 Longwan No.4 213 ZWNJ125 292 G2295 56 P330604020 135 Dingwan No.1 214 ZWNJ126 293 G2704 57 2017334051 136 Tianshanbai 215 ZWNJ130 294 G2001 58 2018335451 137 6001 216 ZWNJ131 295 G917 59 2018335449 138 Afila-1 217 ZWNJ138 296 G759 60 298335207 139 Graf 218 ZWR 297 G754 61 2018335437 140 Liangza No.1 219 ZWR1-2-1 298 G1552 62 2017334023 141 9129 220 ZWR1-2-2 299 G1246 63 2018335450 142 Mb1201 221 ZWR07 300 G626 64 2018334412 143 S2006 222 ZWR11 301 G317 65 P331022014 144 RW09 223 ZWR12 302 G5669 66 2018333642 145 SG-106 224 ZWR13 303 K-1898 67 129 146 SW1102 225 ZWR15 304 AGG3098 68 ZW133 147 WD04-09 226 ZWR19 305 AGG1497 69 P330324016 148 LB09 227 ZWR27 306 AGG1496 70 2019335014 149 WD04-10 228 ZWR30 307 AGG1495 71 P330324029 150 CS6007 229 Baiheima 308 AGG1723 72 2019333675 151 Sn100 230 Maziliangjia 309 AGG1494 73 2019333677 152 SG1105 231 ZWR34 310 PI 560066 74 2019335002 153 WD04-10 232 Baiziliangjia 311 PI 560064 75 2019335001 154 WG07 233 ZWR039 312 AGG3731 76 2018331246 155 S2004 234 Zhewan No.3 313 AGG3342 77 76 156 S6008 235 Zhewan No.4 314 AGG3104 78 Xinpin005 157 S5002-7-2 236 Zhewan No.5 79 ZW155 158 WD04-04 237 Zhewan No.6 2. Data quality control Genomic DNA was extracted from fresh leaves using the CTAB method. DNA libraries for Illumina sequencing were constructed using NEBNext Ultra DNA LibraryPrep Kits. DNA was randomly sheared using a Covaris ultrasonic disruptor, and end repair and A-tail adapters were added. The purified DNA was amplified by PCR, and the library was evaluated and sequenced using DNBSEQ-T7 in PE150 mode. The obtained raw image data files were converted into raw sequencing sequences by base recognition analysis, called Raw Reads, and the results were stored in FASTQ file format. In order to ensure the quality of information analysis, Raw Reads were filtered, and the default parameters of fastp (v.0.12.2) were used to filter the raw DNA reads, remove sequencing adapters and low-quality bases, and obtain Clean Reads for subsequent information analysis.

[0023] 3. Design of pea 20K SNP liquid phase breeding chip loci Clean reads were aligned to the reference genome PeaZW1 using the mem algorithm in BWA (v.0.7.17-r1188) software. After alignment, PCR duplicates were removed using the Picard tool (v.1.123). A single BAM file was generated for each sample for SNP detection. Subsequently, the SAM file format was converted, the BAM file was sorted, and the alignment quality was filtered using the 'q30' parameter using Samtools (v.1.9). The HaplotypeCaller module of the Genome Analysis Toolkit (GATK, v.4.1.9.0) was used to extract raw SNPs. Individual SNP extraction was performed using hard filtering of mapping quality ≤20.0, minimum sequencing coverage ≤5, and maximum sequencing coverage ≥200. Then, the variants were merged into a single variant call file using GATK Combine Variants. Possible false SNPs were further filtered using VCFtools (v.0.1.16), and the criteria included: (1) If a marker is not detected in more than 20% of the samples, the marker is filtered out, that is, the genotype missing rate is ≤ 20%, and the parameter is vcftools --max-missing 0.8; (2) If the minimum allele frequency of a marker is lower than 0.01, it is filtered out, that is, the minimum allele frequency (MAF) is <0.01, and the parameter is vcftools --maf 0.01; (3) Extract 100 bp sequences upstream and downstream of the SNP marker and perform blast comparison on the whole genome sequence. If the similarity is greater than 60% and the alignment length is greater than 60% (about 120 bp), it is considered to be a copy and only single-copy sites are retained; (4) Extract the 100 bp sequence upstream and downstream of the SNP marker, calculate the GC content, and retain sites with a GC content greater than 40% and less than 60%; (5) Extract the 100 bp sequence upstream and downstream of the SNP marker, analyze whether it contains the SSR sequence, and retain the sites without SSR sequences; (6) Analyze the InDels within 100 bp upstream and downstream of the SNP marker, and retain the SNPs without InDels of more than 5 bp within 100 bp upstream and downstream; (7) Extract the 100 bp sequence upstream and downstream of the SNP marker, analyze whether the sequence contains N, and retain the sequence without N within 100 bp upstream and downstream of the SNP site.

[0024] (8) Addition of chip functional sites A large-scale GWAS was performed using the filtered SNPs. Association analysis was performed using the Efficient Mixed-Model Association eXpedited (EMMAx) (v.2012-021-0) program with default parameters. The first three principal components were used as covariates. The effective number of independent SNPs and the recommended P value were estimated using the Genetic type 1 Error Calculator (gec, v.0.2) software. In the mixed model, the recommended P value was P<4.63 × 10^-7. Finally, 235 reliable SNP functional loci significantly associated with 57 traits were obtained.

[0025] The above methods ultimately screened 21,659 high-quality, representative SNP loci, including 235 published reliable functional loci significantly associated with 57 traits. The distribution of loci on chromosomes is shown in Figure 2. Picture 1 , the number and spacing of each chromosome are as follows Picture 2 .

[0026] 4. Development of pea 20K SNP liquid phase breeding chip The 21,659 SNP sites finally screened were designed and synthesized by Tianjin Jizhi Gene Technology Co., Ltd., and the 20K SNP liquid phase breeding chip of pea was developed using TargetSeq liquid phase hybridization capture technology. TargetSeq liquid phase hybridization capture technology is a technology that designs capture probes for the target region genome based on a multi-factor algorithm, synthesizes effective specific probes at high throughput, and then performs liquid phase hybridization with the interrupted genomic DNA. After capturing and enriching the target region sequence, high-throughput sequencing is finally performed using mainstream sequencing platforms such as Illumina and Life Technology.

[0027] Example 2 is as follows:

[0028] The method of using the pea 20K SNP liquid phase breeding chip of the present invention to perform genotyping on pea samples: First, the genomic DNA is fragmented to form small fragments of 200-300bp. The DNA fragments are repaired at the ends and connected to the pre-PCR amplification library. The probe is hybridized with the target region, and then the target fragment is enriched and eluted with streptavidin affinity-labeled magnetic beads. Post-PCR amplification is performed after capture, and finally the target region is subjected to second-generation sequencing. The raw image data file obtained by the high-throughput sequencing DNBSEQ-T7 sequencing platform is converted into the original sequencing sequence through base recognition analysis, which is called RawData or Raw Reads. The results are stored in the FASTQ file format, which contains the sequence information of the sequencing sequence and its corresponding sequencing quality information. The original sequencing sequence or Raw Reads obtained by sequencing contains low-quality Reads with connectors. In order to ensure the quality of information analysis, Raw Reads are filtered to obtain Clean Reads for subsequent information analysis.

[0029] The main steps of data filtering are as follows: (1) Remove reads with adapters.

[0030] (2) Filter reads with N content exceeding 10%.

[0031] (3) Remove reads with bases with quality values ​​lower than 10 accounting for more than 50% of the bases.

[0032] The sequencing reads obtained by resequencing need to be relocated to the reference genome before subsequent variation analysis can be performed. The bwa software is mainly used to align the short sequences obtained by the second-generation high-throughput sequencing DNBSEQ-T7 sequencing platform with the reference genome. By aligning and locating the position of Clean Reads on the reference genome, the sequencing depth, genome coverage and other information of each sample are counted, and the variation is detected. Subsequently, Samtools (v.1.9) was used to convert the SAM file format, sort the BAM file, and filter the alignment quality with the 'q30' parameter. The HaplotypeCaller module of the Genome Analysis Toolkit (GATK, v.4.1.9.0) was used to extract the original SNPs. Individual SNP extraction uses hard filtering of mapping quality ≤20.0, minimum sequencing coverage ≤5, and maximum sequencing coverage ≥200. Then, GATK Combine Variants is used to merge the variants into a single variant call file. VCFtools (v.0.1.16) is used to further filter possible false SNPs, and the criteria include: (1) If a marker is not detected in more than 20% of the samples, the marker is filtered out, that is, the genotype missing rate is ≤ 20%, and the parameter is vcftools --max-missing 0.8; (2) If the minimum allele frequency of a marker is lower than 0.01, it is filtered out, that is, the minimum allele frequency (MAF) is <0.01, and the parameter is vcftools --maf 0.01; Finally, the genotyping results of each target SNP in a specific pea material are obtained, achieving high-throughput SNP genotyping.

[0033] Example 3 is as follows:

[0034] To verify the genotyping effect of the pea 20K SNP liquid phase breeding chip, the pea 20K SNP liquid phase breeding chip designed in Example 1 was used to perform genotyping detection on 14 pea samples. The specific method is as described in Example 2.

[0035] The probe capture efficiency analysis of the sequencing data of 14 representative materials showed that 98.46% of the reads were aligned to the reference genome, of which the average capture rate of the probes was 44.47%, and the off-target reads accounted for 53.98%. Picture 3 As shown, when the pea 20K SNP liquid phase breeding chip is used to perform genotyping on the test materials, the target site detection rate is high, the typing results are accurate and reliable, and it can be used for SNP typing detection of different pea samples.

[0036] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A pea whole genome 20K SNP liquid phase breeding chip, characterized in that: The genotyping targets of this chip include 21,659 SNP sites in the whole pea genome.

2. The pea whole genome 20K SNP liquid phase breeding chip according to claim 1, characterized in that: The liquid phase chip is loaded with molecular probes or primers of the 20K SNP molecular marker combination of the whole pea genome. The genotyping objects of the chip include 21,659 SNP sites on the whole pea genome, and the reference genome of the SNP sites is the pea genome. Pisum sativum cultivar Zhewan1 (PeaZW1)”.

3. A kit for analyzing pea genome, characterized in that: The kit comprises reagents for detecting a 20KSNP molecular marker combination of the whole pea genome.

4. An application of the pea 20K SNP liquid phase breeding chip as claimed in claim 2 in genetic diversity analysis of pea germplasm resources, variety identification analysis, assisted selection breeding, population structure analysis, genetic and evolutionary analysis, kinship identification, whole genome association analysis or genome selection.

5. The use according to claim 4, characterized in that: The application method comprises the following steps: using the pea whole genome 20K SNP liquid phase breeding chip to perform genotyping on pea samples.

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