High-throughput Specific Detection Methods for Transgenic Maize Based on the KASP Platform

doi:10.13560/j.cnki.biotech.bull.1985.2022-1191

Abstract

Abstract:

In order to promote the industrialization of transgenic maize and accelerate the transfer of excellent trait transformants and backbone inbred lines, high-throughput foreground selection methods for backcross population need to be developed urgently. In this study, the transgenic maize DBN9936 was used as the material, according to its exogenous insert fragment and its flanking sequence, 6 pairs of specific primers were designed using the primer5 software, combined with the internal standard gene zSSIIb primer for evaluation. After obtaining the optimal primer combination, further the specificity verification, detection limit testing and multi-sample verification were carried out. The results showed that the optimal primer combination was DBN9936-LB1*zSSIIb-k1; the results of KASP genotyping of 10 DBN9936 BC₁ seeds were completely consistent with those of agarose gel electrophoresis; the results of 10 transformants DBN9858, DBN9501, C0010.1.3, C0010.3.1, C0010.3.7, C0030.2.5, BT11, GA21, MIR162 and 2A-7 were negative on the dual platform. The detection limit of transgenic maize DBN9936 was stable at 10%; and the KASP genotyping results of 48 different DBN9936 hybrids were positive. In summary, this method can be used for the foreground selection in the process of backcrossing of transgenic maize DBN9936 with high sample throughput, which provides a technical reference for target gene detection of other crops in transgenic breeding.

Key words: transgenic maize, backcross breeding, foreground selection, KASP platform

ZHU Shao-xi, JIN Zhao-yang, GE Jian-rong, WANG Rui, WANG Feng-ge, LU Yun-cai. High-throughput Specific Detection Methods for Transgenic Maize Based on the KASP Platform[J]. Biotechnology Bulletin, 2023, 39(6): 133-140.

Figures/Tables 10

Table1 DBN9936 border sequences SEQ NO.ID 3 and SEQ NO.ID 4

编号No.

核苷酸序列Nucleotide sequence

SEQ NO.
ID 3

661 CCGCCGTATC TCCCGCCTCT CGCGCGCCAT CTCCCACACG CCGCCGCCGA
711 TCCTCTCTCTC GTCCTACGC CCACGAATGG CTCTCCAGCT GGAGAAAACC
761 GGCGGCCAGT GGATCCACCA TCAGGGGCAA GAAAACATCC CAAAACGCTA
811 GGCTCATGGG CACACAAGAC ACATTGTGGT GTAAACAAAT TGACGCTTAG
861 ACAACTTAAT AACACATTGC GGATACGGCC ATGCTGGCCG CCCCGCGCCG
911 ATCTAGTAAC ATAGATGACA CCGCGCGCGA TAATTTATCC TAGTTTGCGC
961 GCTATATTTT GTTTTCTATC GCGTATTAAA TGTATAATTG CGGGACTCTA

SEQ NO.
ID 4

8001 TACATTAAAA ACGTCCGCAA TGTGTTATTA AGTTGTCTAA GCGTCAATTG
8051 GATCCCGTGG TCCCGGTCCC GTTTAAACCT AATCAGTCAG TGCCGGTGAG
8101 AGCGTAGCTG CCTGGAAGCG GCCTCGTCAG CCAACACCGT GAGGCTAGTG
8151 CCATCAATAA GTTATCAGTC AAACGGTCC GTTTAAACTA TCAGTGTTTG
8201 AAAGCGAGGC GAGGCGATGG GCGCGCGCGC GGGTCTCCTT TCCTGTTTCC
8251 TATTTGCACG CCCCTCAGCA CAGTAGTACA AGACGATTAT TATTTTTAAA
8301 CAATTGGATC AAGATGAGGC GGGGGGGTTG GGGGGCACTC ACTTGACCGC

Table2 PCR primer names and sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物长度 Product length/bp
zSSIIb-1F	CTCCCAATCCTTTGACATCTGC	151
zSSIIb-2R	TCGATTTCTCTCTTGGTGACAGG	151
C0030.3.5-F	CTCGTCAGCCAACACCGTGA	202
C0030.3.5-R	CCCCGC CTCATCTTGATCCA	202

Table3 KASP primer names and sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
DBN9936-LB1	F: FAM-CCACCATCAGGGGCAAGA	84
	R: AAGCGTCAAATTTGTTTACACCAC
DBN9936-LB2	F: FAM-GATCCACCATCAGGGGCAAGA R: CATGGCCGTATCCGCAATGTG	122
DBN9936-LB3	F: FAM-CCACCATCAGGGGCAAGA R: AGCATGGCCGTATCCGCA	121
DBN9936-RB1	F: FAM-CGTGAGGCTAGTGCCATCAAT R: AAACAGGAAAGGAGACCCGC	110
DBN9936-RB2	F: FAM-CCAACACCGTGAGGCTAGTG R: AAACAGGAAAGGAGACCCGC	117
DBN9936-RB3	F: FAM-CGTGAGGCTAGTGCCATCAA R: AGGGGCGTGCAAATAGGAAA	121
zSSIIb-k1	F: HEX-CAAAGTCAGAGCGCTGCAATGCAAA R: CGCTGCTGCCCCCACTCGTT	74
zSSIIb-k2	F: HEX-CCTTTGACATCTGCTCCGAAGCAAA R: CAGGAGCTGATGGATGATCAGCTTT	119

Fig. 1 Results of single primer amplification typing A: The scan results of single primer amplification of DBN9936-LB1; B: the scan results of single primer amplification of DBN9936-RB1; C: the scan results of single primer amplification of DBN9936-RB2; D: the scan results of single primer amplification of DBN9936-RB3; E: the scan results of single primer amplification of DBN9936-LB2; F: the scan results of single primer amplification of DBN9936-LB3. Each point in the figure indicates the test results of one sample. The dots of the upward arrow indicate the DBN9936 standard, and the dots of the downward arrow indicate Jingke 968. Blue dots: positive homozygous genotypes; red dots: negative homozygous genotypes; black dots: NTC. The same below

Fig. 2 Composite primer amplification typing results A: The composite amplification scan results of primer DBN9936-LB1 * zSSIIb-k1; B: the composite amplification scan results of primer DBN9936-LB1 * zSSIIb-k2. Green dots: Heterozygous genotypes. The same below

Fig. 3 Target gene detection genotyping map The cycle dots indicates the BC1 sample. The 10 right semicircle dots indicate transgenic maize samples containing transformants（DBN9858, DBN9501, C0010.1.3, C0010.3.1, C0010.3.7, C0030.2.5, BT11, GA21, MIR162 and 2A-7）

Table 4 Comparison of double platform detection results

样品 Sample	KASP检测结果 Results of KASP detection	普通PCR检测结果 Results of ordinary PCR detection
1	-	-
2	+	+
3	+	+
4	+	+
5	+	+
6	+	+
7	-	-
8	+	+
9	-	-
10	+	+
11	-	-
12	-	-
13	-	-
14	-	-
15	-	-
16	-	-
17	-	-
18	-	-
19	-	-
20	-	-

Fig. 4 Agarose electrophoresis results of PCR products A: The amplification product band of primer C003.3.5 transformant specific sequence; B: the amplification product band of the primer for internal standard gene zSSIIB. M: Molecular weight standard; P1 and P2: DBN9936 standard; P3 and P4: Jingke 968; 1-10: 10 DBN9936 backcross BC1 samples; 11-20: 10 transgenic maize containing transformants（DBN9858, DBN9501, C0010.1.3, C0010.3.1, C0010.3.7, C0030.2.5, BT11, GA21, MIR162 and 2A-7）

Fig. 5 Detection limit test A: The sensitivity test results; B: the detection limit test results

Fig. 6 Target gene detection genotyping map The cycle dots indicates the transgenic hybrid

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