Development and application of KASP markers for functional sites of the early flowering gene BnCRY2 in Brassica napus L. spring rapeseed

Cloning of the candidate gene <italic>BnCRY2</italic>, associated with the early-flowering locus <italic>BnaC08cqDTF</italic>, identified an A/G single-nucleotide polymorphism (SNP) within the third exon of the corresponding alleles. Based on this SNP, a KASP marker named BN900449 was developed. This marker, along with four additional markers—multiplex PCR markers PB05 and PB06, and KASP markers A015348 and K000272—tightly linked to the early-flowering loci <italic>qFT.A02-1</italic> and <italic>qFTA10</italic>, was used to genotype a natural population of 497 <italic>Brassica napus</italic> L. accessions. Genotypic analysis revealed that each of the three loci effectively divided the population into two groups with significantly different flowering times. Notably, accessions carrying two of the early-flowering loci flowered, on average, earlier than those carrying only one. Three representative accessions, each harboring a different single-locus allele (<italic>BnaC08cqDTF</italic>, <italic>qFTA10</italic>, and <italic>qFT.A02-1</italic>), were selected for diallel crosses aimed at pyramiding early-flowering alleles. Marker-assisted selection subsequently yielded 41 extremely early-flowering lines, which exhibited flowering times 3-4 days earlier than their respective parents. Among these, 195 lines were identified that combined extreme earliness with double-low seed quality. Multi-location yield trials conducted at five high-altitude sites over two consecutive years showed that the average yield of selected lines exceeded that of <italic>Brassica rapa</italic> L. cultivar Haoyou 11—the predominant spring-sown variety in high-altitude regions—by 23.07%. This study not only expands the genetic framework for developing extremely early- flowering <italic>B. napus</italic> spring-type germplasm but also provides elite parental resources for breeding early-maturing, high-yielding, and high-quality rapeseed cultivars.