US 11,851,718 B2
InDel marker genotype database of Malus and use in identity fingerprinting of germplasm resources
Xinzhong Zhang, Beijing (CN); Xuan Wang, Beijing (CN); Fei Shen, Beijing (CN); Ruiting Chen, Beijing (CN); Zhenhai Han, Beijing (CN); Yi Wang, Beijing (CN); Ting Wu, Beijing (CN); Xuefeng Xu, Beijing (CN); Wei Li, Beijing (CN); Changpeng Qiu, Beijing (CN); and Xi Zhang, Beijing (CN)
Assigned to CHINA AGRICULTURAL UNIVERSITY, Beijing (CN)
Filed by China Agricultural University, Beijing (CN)
Filed on Dec. 29, 2020, as Appl. No. 17/136,209.
Claims priority of application No. 202010966745.9 (CN), filed on Sep. 15, 2020.
Prior Publication US 2022/0090213 A1, Mar. 24, 2022
Int. Cl. C12Q 1/6895 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6888 (2018.01)
CPC C12Q 1/6895 (2013.01) [C12Q 1/686 (2013.01); C12Q 1/6888 (2013.01); C12Q 2600/13 (2013.01); C12Q 2600/156 (2013.01)] 7 Claims
 
1. A method for constructing an InDel marker genotype database for Malus, comprising the following steps:
genotyping InDel markers of genomic DNA samples of Malus germplasm resources by multiplex PCR;
recording the results of the genotyping for each Malus germplasm resource;
assembling the recorded results into an InDel marker genotype database;
wherein the markers genotyped correspond to the loci represented by SEQ ID Nos. 1-102;
wherein upstream primers used for PCR of the loci represented by SEQ ID Nos. 1-102 are SEQ ID Nos. 103-204, respectively;
wherein downstream primers used for PCR of the loci represented by SEQ ID Nos. 1-102 are SEQ ID Nos. 205-306, respectively;
wherein the multiplex PCR reactions are performed according to the following, wherein G1-G9 represent nine multiplex PCR reactions, each containing primers for amplifying the indicated combination of markers:
 
 
 
 Fluore-
 
Group Marker scent
 
number number label Marker combination
 
 
G1 24 HEX C06039, C08054, C11078, C15109,
 
 
 
 C15115, C16124
 
 
 PET C03012, C04022, C04024, C07044,
 
 
 
 C09064, C13096
 
 
 NED C03011, C05028, C08056, C08059,
 
 
 
 C10073
 
 
 FAM C02009, C05026, C06037, C13101,
 
 
 
 C14104, C15110, C15114
G2 15 HEX C01001, C04019, C09068, C15116,
 
 
 
 C16120
 
 
 PET C01002, C03017, C07043, C17127
 
 
 NED C01003, C06040, C11081, C17126
 
 
 FAM C05030, C09063
G3 9 HEX C05032, C07049, C12089
 
 
 PET C17128
 
 
 NED C13094
 
 
 FAM C03018, C08052, C12086, C13093
G4 11 HEX C01004, C07047, C10070, C12090,
 
 
 
 C13097
 
 
 NED C04020, C06038, C08058, C16122
 
 
 FAM C05029, C15111
G5 10 HEX C02008, C05031, C12092
 
 
 NED C11082, C12088
 
 
 FAM C06041, C07050, C12087,
 
 
 
 C13100, C13102
G6 13 HEX C08051, C08057, C14108
 
 
 NED C01005, C02006, C03014, C04023
 
 
 FAM C09062, C11077, C11080,
 
 
 
 C13098, C15119, C16123
G7 9 HEX C07045, C11079, C17129
 
 
 NED C07048
 
 
 FAM C04021, C08060, C12091,
 
 
 
 C14105, C16125
G8 8 HEX C05135, C10136
 
 
 NED C12132, C04133, C06137
 
 
 FAM C09067, C04134, C07138
G9 3 HEX C08055
 
 
 NED C10071
 
 
 FAM C10072.