Multiplex PCR Detection for Late Blight Resistant Genes R8,RB and Virus Resistant Genes Rx1,Ryadg in Potato

doi:10.16420/j.issn.0513-353x.2020-0318

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (2): 389-396.doi: 10.16420/j.issn.0513-353x.2020-0318

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Multiplex PCR Detection for Late Blight Resistant Genes R8,RB and Virus Resistant Genes Rx1,Ry_adg in Potato

LIU Cheng^1,*, WANG Shiyao^1,*, SHI Weiling¹, SONG Yuhao¹, JIANG Rui¹, ZHAO Yong¹, MO Shichun², LÜ Dianqiu¹, WANG Jichun¹, LIU Xun¹^,^**()

¹Chongqing Key Laboratory of Biology and Genetic Improvement in Tuber and Root Crops,College of Agronomy and Biotechnology,Southwest University,Chongqing 400715,China
²Chongqing Wuxi Agricultural and Rural Committee,Chongqing 405899,China

Received:2020-04-26 Revised:2020-06-01 Online:2021-02-25 Published:2021-03-09
Contact: LIU Xun E-mail:liuxun828@swu.edu.cn

Abstract

Abstract:

Based on defined RB and R8 genes for late blight resistance in 218 germplasm resources,Rx1 and Ry_adg for potato virus X（PVX）and potato virus Y（PVY）extreme resistance were screened in the present study,respectively. The mixed DNA from two screened potato genotypes containing Rx1,RB and Ry_adg,R8 was used as templates. The multiplex PCR system was developed with the reported markers for Rx1 and Ry_adg and designed markers for RB and R8. In addition,the amplification of the PCR reaction system was monitored by the potato GBSS gene markers. The crucial factors of multiplex PCR system including extension temperature and primers concentration were optimized. Five specific fragments were simultaneously amplified in one PCR reaction. This detection method was used to detect these R genes from advanced breeding lines. The results revealed that four breeding lines containing four above mentioned R genes were screened. In conclusion,the established multiplex PCR detection system will be used for molecular marker-assisted selection,providing a simple and efficient technical method for late blight and viral diseases multi-R genes aggregation breeding,and new resources for the new varieties selection with four R genes in potato.

Key words: potato, late blight, potato virus X, potato virus Y, multiplex PCR

CLC Number:

S532

LIU Cheng, WANG Shiyao, SHI Weiling, SONG Yuhao, JIANG Rui, ZHAO Yong, MO Shichun, LÜ Dianqiu, WANG Jichun, LIU Xun. Multiplex PCR Detection for Late Blight Resistant Genes R8,RB and Virus Resistant Genes Rx1,Ry_adg in Potato[J]. Acta Horticulturae Sinica, 2021, 48(2): 389-396.

Figures/Tables 6

Table 1 Primers sequences for R8,RB,Rx1 and Ryadg markers

基因 Gene	标记名称 Marker	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	产物/bp Product
Rx1	RxSP-1230	F：ATCTTGGTTTGAATACATGG;R：CACAATATTGGAAGGATTCA	55	1 230
Ry_adg	RYSC3-321	F：ATACACTCATCTAAATTTGATGG;R：AGGATATACGGCATCATTTTTCCGA	55	321
R8 RB	R8-1832 RB-629	F：GGAGAACACTGAATCTGGGAGA;R：ACTCTGTATTCGGAGGGGACT F：GAATCAAATTATCCACCCCAACTTTTAAAT; R：CAAGTATTGGGAGGACTGAAAGGT	55 55	1 832 629
GBSS	GBSS-981	F：GTGCGAAGAATATAGTCTGGC;R：TCAGCAAAGCTAAGTGGGATC	55	981

Fig. 1 Detection results of Rx1（A）,Ryadg（B）and marker composition（C）in potato germplasm M：DNA marker DL2000 plus;1-12：Parts of genotypes.

Fig. 2 Amplification of individual R and GBSS in potato

Fig. 3 Optimization of extension temperature（A）and primers concentration of R8（B）for the multiplex PCR

Fig. 4 The PCR results of multiplex PCR（A）and the marker composition for RB,R8,Rx1 and Ryadg（B）in advanced breeding lines M：DNA Marker;1-12：Parts of breeding lines.

Fig. 5 The PCR results of multiplex PCR using rude DNA from selected advanced breeding lines N：Negative control.

References 18

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Multiplex PCR Detection for Late Blight Resistant Genes R8,RB and Virus Resistant Genes Rx1,Ry_adg in Potato

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