A multiplex microsatellite pcr method for detection of Brassica L. a, b and c genome fragment introgressions upon interspecific hybridization

The genus Brassica L. is a source of oilseeds, vegetables, spices, fodder and ornamental crops widely cultivated around the world. The six most cultivated species of the genus Brassica comprise allotetraploid species B. juncea (L.) Czern . (2 n = 36, genome AABB), B. napus L. (2 n = 38, genome AACC) and B. carinata A. Braun (2 n = 34, genome BBCC), which are natural hybrids of corresponding diploid species B. rapa L. (2 n = 20, genome AA), B. nigra L. (2 n = 16, genome BB), and B. oleracea L. (2 n = 18, genome CC). An effective way to increase the genetic diversity and improve the agronomic traits of Brassica crops, such as high yields, resistance to diseases, and abiotic stresses is to introduce traits of interest by the interspecific hybridization. To control the introgression of genomic material upon the hybridization, the development and implementation of genetic markers are necessary. This paper proposes an effective approach for controlling the introgression of A, B, and C genomes of Brassica in intraspecific hybrids. The investigation aimed to develop a high-throughput technology based on multiplex PCR analysis of genome-specific microsatellite markers for controlling the introgression of A-, B-, and C-genomes in Brassica intraspecific hybrids. Control samples were obtained from the Center for Genetic Resources CGN (Netherlands) and the All-Russian Institute of Plant Genetic Resources N.I. Vavilov (VIR, St. Petersburg). Plant material for the genomic material introgression study were obtained from the Timofeev Breeding Station (Moscow). Genomic DNA was extracted by sorbent method. PCR was run with specific primers for the Na10-D09, Na12-A02, Na12-F12, Ni2-B02, Ni2-F02, Ni3-G04B, Ol12-A04, Ra2-E12, BRMS-043, BN6A2 loci. Fluorescently labelled PCR products were analyzed by high-resolution electrophoresis using a Nanofor-05 genetic analyzer (Syntol - The Institute for Analytical Instrumentation, Russia). The length of the amplified DNA fragments was determined using the DNA Fragment Analysis software (The Institute for Analytical Instrumentation, Russia). A multiplex PCR technique was developed based on the six microsatellite loci Na12-A02, BRMS-043, Na10-D09, Ol12-A04, Ni2-F02, BN6A2, allowing us to determine the markers of three Brassica genomes in one run. A, B, and C genome-specific markers were identified during multiplex PCR analysis of control samples of six Brassica species with known taxonomic attributions and genome compositions: B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. napus (AACC), B. juncea (AABB), and B. carinata (BBCC). The length of marker fragments was determined by high resolution electrophoresis using a genetic analyzer with an accuracy of one nucleotide. A-genome specific markers were identified at the loci Na12-A02 (178 bp, 180 bp, 182 bp), BRMS-043 (303 bp, 307 bp, 313 bp), and Na10-D09 (283 bp, 285 bp, 291 bp, 293 bp, 299 bp). B-genome specific markers were detected at the loci Na12-A02 (196 bp, 198 bp, 200 bp, 202 bp, 204 bp, 212 bp, 214 bp, 216 bp), Ol12-A04 (125 bp, 127 bp, 129 bp), Ni2-F02 (198 bp, 200 bp ., 202 bp, 204 bp, 208 bp), and BN6A2 (222 bp). C-genome specific markers were detected at the loci Na12-A02 (164 bp, 168 bp, 170 bp) and Ni2-F02 (164 bp, 166 bp, 168 bp, 186 bp). The developed multiplex PCR system reveals introgressions of fragments of genomes A, B and C in the genetic profiles of interspecific hybrids (Et2 ½ KK)2 ½ Tsv9, (Et2 ½ KK)1, Green ½ FBLM(1), JR ½ Agr2ki, BK ½ ZM1-1(6), BK ½ ZM1-1(8), BK, and KB. The method also confirmed the presence of the corresponding genomes in the studied samples with a known breeding history. Due to the automation, analysis allows the large-scale screening of plant samples. The proposed technology can be used in breeding practice as a tool for controlling the introgression of A, B and C genome material upon the interspecific hybridization, as well as controlling its inheritance in subsequent generations.