AU2020104373A4

AU2020104373A4 - DNA Molecular Method for Identifying Yunrui Series of Maize varieties

Info

Publication number: AU2020104373A4
Application number: AU2020104373A
Authority: AU
Inventors: Junjiao Guan; Sheping Li; Xiaolin Li; Yangang Li; Furong Ma; Xiaohong Yang; Jianhua Zhang; Peng Zhang
Original assignee: Institute Of Grain Crops Yunnan Academy Of Agricultural Sciences; Institute of Quality Standards and Testing Technology of Yunnan Academy of Agricultural Sciences
Current assignee: Institute Of Grain Crops Yunnan Academy Of Agricultural Sciences; Institute of Quality Standards and Testing Technology of Yunnan Academy of Agricultural Sciences
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-03-18
Anticipated expiration: 2028-12-29

Abstract

The present invention provides a method for establishing a molecular label of Yunrui series of maize varieties, which is applied to variety molecular identification and variety management. The method is characterized by comprising the following steps: screening and identifying an SSR core primer of Yunrui series of maize, performing digital coding assignment of DNA information of varieties, forming a two-dimensional code representation, and marking the two-dimensional code representation on a packaging of commercial seeds for anti-counterfeiting and traceability of seeds of superior maize varieties.

Description

DNA Molecular Method for Identifying Yunrui Series of Maize varieties

TECHNICAL FIELD: The present invention belongs to the field of molecular biology, and specifically relates to the establishment of a DNA molecular method for identifying Yunrui series of maize varieties, which is applied to variety molecular identification and variety management. TECHNICAL BACKGROUND Yunnan belongs to the southwest and belongs to a mountainous maize growing area in southwest in China. Although Yunnan Province is not a major maize producing province in China, Yunnan Province has a unique natural environment and climate resources, which provides more possibilities for the selection of maize germplasm resources. The Yunrui series of new varieties (lines) are the most representative maize lines in Yunnan Province and have the characteristics of a high yield, a disease resistance, a strong stress resistance, an excellent quality, a good comprehensive trait and significant social and economic benefits. These series varieties have been promoted in more than 40 million mu in Yunnan, Sichuan, Guizhou, Guangxi and other surrounding provinces and are favored by the majority of farmers. With the continuous improvement of a breeding level in China, new breeds are constantly emerging, but the phenomenon of "variety homogeneity" is becoming more and more serious. The phenomena of "same-source and different names" and "deck varieties" are common in the market, which has caused a serious impact on China's variety management and has caused a lot of trouble, and has also caused great damage to the interests of China and farmers while cracking down on original breeding. Therefore, to solve this problem, it is necessary to establish a simple, fast and accurate variety identification and scientific seed management method. Variety identification and scientific seed management need to meet the requirements of uniqueness, identifiability (identification) and traceability. Molecular labeling can be used as one standard for variety-specific identification and is one of core technologies of variety digital management. The present invention takes a SSR fingerprint map of maize DNA as a core, carries out digital coding assignment of variety DNA information, forms a two-dimensional code representation, and marks the two-dimensional code representation on a packaging of commercial seeds, which can be used for key technology of information management technology of anti-counterfeiting and traceability of seeds of excellent maize varieties. SUMMARY: In order to be able to identify maize varieties accurately, quickly and conveniently, the present invention proposes a method for constructing a molecular label of maize varieties. The present invention realizes double anti-counterfeiting of varieties and seeds. The present invention also discloses a primer screening process and a two-dimensional code construction process of the method.

The key technical problems that the present invention solves for establishing maize molecular labels are: 1. A method for collecting data of characteristic DNA fingerprint information of Yunrui series of maize varieties is constructed to realize authenticity identification and seed traceability of a molecular label of Yunrui maize varieties. 2. The DNA fingerprint information of the maize varieties refers to DNA fingerprint data obtained by an optimized SSR molecular marker analysis, and expressed by a size of a capillary electrophoresis fragment. 3. The difference from the current SSR standard for the maize varieties (NY/T 1432-2007) is mainly in an optimization of PCR technical parameters, which is specifically as follows: a reaction system is optimized from 12.35 PL to pL, 2 pL buffer optimized to 0.6 pL, 2 pL MgCl2 optimized to 0.6 MgCl2 or not added, dNTP solution optimized from 1.2 pL to 0.8pL, Taq DNA polymerase optimized from 0.2 pL to 0.25pL, and a detection sample DNA optimized from 2 pL to 1pL. If MgCl2 is not added, replacement is performed with an equivalent amount of sterile water. 4. A primer used for the SSR marker is a primer screened from 200 maize varieties through an SSR molecular marker. This set of the primers may identify Yunrui series varieties with the least primers with a wide genome coverage, may distinguish the Yunrui series varieties from other main maize varieties on the Yunnan Plateau, and may distinguish from the Yunrui series varieties one by one. 5. A method for constructing a QR code system of identification of a molecular label of Yunrui series varieties. DESCRIPTION OF THE INVENTION 1. Collection of basic commodity information data of maize varieties: Crop type: maize Variety Botany Type: Maize Breeding types of varieties: conventional variety, an inbred line, a single cross variety, a double cross variety and a triple cross variety. Variety name: Name of production operator: Unit identification code: Trackback URL: Approval area and year of approval: 2 Collection of DNA fingerprint data 2.1 Primer screening of SSR marker DNA fingerprint information was data obtained by an SSR mark analysis. For the Yunrui series of maize varieties, an SSR primer with a high polymorphism, a good discrimination, easy data statistics, a good amplification repeatability, and primer sites evenly distributed on a chromosome was selected as a core primer. More than 40 pairs of maize SSR primers were screened using 200 Yunnan maize varieties with different genetic backgrounds, and 4 pairs of maize SSR primers with a high polymorphism, a good discrimination, easy statistics, a good amplification repeatability, and primer sites evenly distributed on the chromosome were screened out. 30 pairs of primers were used to amplify and detect 200 Yunnan maize varieties to obtain a DNA fingerprint map. Through data analysis, the fingerprints map of 9 Yunrui series of the maize varieties were determined for analysis, and finally 4 pairs of primers were determined as core primers to identify Yunrui series of the maize varieties. Maize is diploid, and the DNA fingerprint information is represented by 10 Arabic numerals in total. DNA fingerprint data is read by capillary fluorescence electrophoresis. Table 2. Information of core primer of maize

Chro Annealing Common No. Prmer moso temperatur Primer sequence (5'--3') variation mes e°C bp

179 186 189 191 193 196 198 200 Forward direction: 202 AATCATGCGTAGGCGTAG 205 CT 207 bnIg11 Reversedirection: 210 1 91 9.03 60 GCCAGAGGAAAAAGAAG 213 GCT 215 217 223 225 227 229 231 233 235 244 246 Forward direction: 337 CGTTTAAGAACGGTTGAT 339 2.08 60 TGCATTCC 341 2 bnIg19 40k7 Reversedirection: 344 GCCTTTATTTCTCCCTTGC 346 TTGCC 348

350 352 354 356 358 360 362 364 368 370 375 377 379 382 385 387 238 240 246 248 250 252 254 256 Forward direction: 258 TTACCAACGCAACACGAG 260 GC 262 3 umc20 2.04 60 Reverse direction: 266 07y4 GCTATAGGCCGTAGCTTG 273 GTAGACAC 275 277 279 281 283 285 287 289 291 293 248 Forward direction: 252 CCCCTCTTCCTCAGCACC 254 TTG 256 bnlg23 507 60 Reversedirection: 258 05k4 CGTCTTGTCTCCGTCCGT 260 GTG 264 270 272

278 290 292 294 296 2.2 Record of SSR fingerprint data The SSR fingerprint data was based on capillary fluorescence electrophoresis to read the allelic variation data of each site and each sample, the allelic variation of a homozygous site was recorded as X/X, and the allelic variation of a heterozygous site was recorded as X/Y, where X and Y are the sizes of two different allelic variation fragments at the site, and a small fragment was at front, a large fragment was at rear, recording is performed in a size of two fragments, arrangement is performed in an order of primers. 3. Through the information entry and marking of maize varieties, the SSR fingerprint data is arranged from front to back to form a data string of numbers and letters to achieve an object of distinguishing differences between varieties simply and clearly. 4. A QR code standard was used to design the two-dimensional code, and the two-dimension uses a counter principle to realize that the user may quickly identify the authenticity of the seeds with ordinary equipment (a mobile phone). 5. The DNA fingerprint data was represented by 10 Arabic numerals. The fingerprint information was the data obtained by analyzing 4 pairs of SSR markers. 6. Arrangement was performed from front to back by inputting and marking information of the maize varieties to form a data string of text, numbers and letters. The formed data string of the text, the numbers and the letters is converted into a two-dimensional code by a data converter. Examples are given below for further details. Example: Construction of a seed molecular label of Yunrui 508 (Dianshen maize 2015036) of maize varieties: (1) Genomic DNA extraction About 200-300 mg of maize seedlings or leaves were placed into a 2.0 mL centrifuge tube, added with liquid nitrogen and ground thoroughly. 700 pL of a CTAB extracting solution preheated at 65 °C was added to each tube, mixed thoroughly, and inverted a plurality of times in a water bath at 65 °C for 60 min. An equal volume of chloroform/isoamyl alcohol (24:1) mixture solution was added to each tube, was mixed well, stood still for 10 minutes, and was centrifuged at 12,000 rpm for 15 minutes. A supernatant was absorbed and transferred to a new tube, was added with an equal volume of pre-chilled isopropanol, was mixed upside down, was placed at -20 °C for 30 min, was centrifuged at 4 °C at 12,000 rpm for 10 min, discarded the supernatant, and was added with 70% ethanol, discarded the ethanol solution after rotating a plurality of times, and stood upside down on an experimental table with a filter paper, and was placed at room temperature for more than 10 mins. 100 pL of ultrapure water or TE buffer was added and fully dissolved for later use. CTAB extraction solution: 81.7 g of sodium chloride and 20.0 g of CTAB ware dissolved in an appropriate amount of water, then added with 100 mL of 1 mol/L Tris-HCI, and 40 mL of 0.5 mol/L EDTA, diluted to 1000 mL and stored at 4 °C.

(2) PCR amplification 4 pairs of SSR primers were used to perform amplification analysis on Yunrui 508. A reaction system: 10 pL of a reaction volume, including 1 tL of 10xPCR reaction buffer, 0.6 tL of 25 mmol/L MgCl2, 0.8tL of 2.5 mmol/L dNTP solution, 0.25 pL of 5 mol/L forward primer, 0.25 tL of 5 mol/L reverse primer, 0.25ptL of 2U/L Taq DNA polymerase, 5.15ptL of ultrapure water, and 1pL of sample DNA. If the buffer in Table 2 contained MgCl2, an equal volume of sterile water was added instead, without adding MgCl2 solution.

A reaction process: pre-denaturation was performed at 94 °C for 5 mins with one cycle; and denaturation was performed at 94 °C for 40s according to Appendix B of Table B. One recommended primer was annealed at an annealing temperature for 35 s, extended at 72 °C for 45 mins with 30 cycles in total, then extended at 72 °C for 5 min, and stored at 4 °C.

(3) Capillary electrophoresis fluorescence detection of a PCR product The fluorescently labeled PCR product was diluted 30 times with ultrapure water. 1 tL of the PCR product was drawn from a mixed solution and added to holes of a deep-well plate dedicated to a DNA analyzer. 0.1 tL of LIZ500 molecular weight internal standard and 8.9 tL of deionized formamide were added respectively to each hole in the plate. The sample was denatured on a PCR machine at 950C for 5 mins, taken out, immediately placed on crushed ice, and cooled for more than 10 mins, centrifuged briefly for 10 s and placed on a DNA analyzer for capillary electrophoresis. Data analysis software was used to analyze collected data. The allelic variation size data for each sample was read at each site. (4) DNA fingerprint data The sizes of the amplified fragments of Yunrui 508 at 4 sites were as follows respectively (arranged in the order of primers): 189/189, 196/196, 348/348, 360/360, 258/258, 264/264, 278/278 Molecular marker data were replaceable or expandable items, and there were more polymorphic sites that can be replaced or supplemented. (5) Molecular label data of variety seeds DNA fingerprint information: 189/189, 196/196, 348/348, 360/360,

258/258, 264/264, 278/278 6. QR code production The QR code standard was adopted to design a two-dimensional code, and the two-dimensional use counter principle. The QR code of Yunrui 508 seed molecular label was shown in FIG. 1. Crop type: maize Variety Botany Type: Maize Breeding type: conventional variety Variety name: Yunrui 508 Name of producer and operator: Institute of Food Crops, Yunnan Academy of Agricultural Sciences Unit identification code: 1 Trackback URL: Approved area and year of approval: Yunnan, 2015 DNA fingerprint information: 189/189, 196/196, 348/348, 360/360, 258/258, 264/264, 278/278 Brief Description of the drawings Fig. 1 is a two-dimensional code of a molecular label corresponding to different varieties of seeds in a specific embodiment of the present invention.

Claims

Claims 1. A method for collecting data of characteristic DNA fingerprint information of Yunrui series of maize varieties is constructed to realize authenticity identification and seed traceability of a molecular label of Yunrui maize varieties.
2. The DNA fingerprint information of the maize varieties refers to DNA fingerprint data obtained by an SSR molecular marker analysis, and expressed by a size of a capillary electrophoresis fragment.
3. A primer used for the SSR marker is a primer screened from 200 maize varieties through an optimized SSR method. This set of the primers may identify Yunrui series varieties with the least primers with a wide genome coverage, may distinguish the Yunrui series varieties from other main maize varieties on the Yunnan Plateau, and may distinguish from the Yunrui series varieties one by one. The information of this set of the primer is as follows: . Chro Annealing No. Pnmer moso temperatur Primer sequence Common allelic s me e°C (5'-3') variation bp 179 186 189 191 193 196 198 200 202 Forward direction: 205 AATCATGCGTAGGC 207 bnlgll 9.03 60 GTAGCT 210 1 91 Reversedirection: 213 GCCAGAGGAAAAA 215 GAAGGCT 217 223 225 227 229 231 233 235 244 246 Forward direction: 337 CGTTTAAGAACGGT 339 2 4k79 2.08 60 TGATTGCATTCC 341 Reverse direction: 344 GCCTTTATTTCTCC 346

CTTGCTTGCC 348 350 352 354 356 358 360 362 364 368 370 375 377 379 382 385 387 238 240 246 248 250 252 254 256 Forward direction: 258 TTACCAACGCAACA 262 CGAGGC 266 3 mc20 2.04 60 Reverse direction: 268 07y4 GCTATAGGCCGTAG 273 CTTGGTAGACAC 275 277 279 281 283 285 287 289 291 293 248 Forward direction: 254 CCCCTCTTCCTCA 256 4 bnIg23 GCACCTTG 258 05k4 Reversedirection: 260 CGTCTTGTCTCCGT 264 CCGTGTG 270

275 278 290 292 294 296
4. The DNA fingerprint data is based on capillary fluorescence electrophoresis to read the allelic variation data of each site and each sample, the allelic variation of a homozygous site is recorded as X/X, and the allelic variation of a heterozygous site is recorded as X/Y, where X and Y are the sizes of two different allelic variation fragments at the site, and a small fragment is at front, a large fragment is at rear, recording is performed in a size of two fragments, arrangement is performed in an order of primers.
5. A molecular identification QR code of the Yunrui series varieties is to convert variety commodity information and variety DNA fingerprint information into a two-dimensional code through a data converter, and set a unit identification code using a counting principle.