Impact of increased population size and three reference genomes on the development of linkage maps in alfalfa

Alfalfa ( Medicago sativa L.) is an important perennial forage crop and a highly heterozygous autotetraploid species (2 n = 4 x = 32), making high‐density linkage mapping challenging. This study evaluated the effects of reference genome choice and population size on the development of genetic linkage maps using genotyping‐by‐sequencing‐derived single nucleotide polymorphism (SNP) markers. Three F 1 populations were analyzed: PopA (RegenSY27x × UMN3988; n = 165), PopB (RegenSY27x × FL99; n = 374), and PopC (a subsample of PopB; n = 165). SNPs were called against three reference assemblies: Map1 based on the XinJiangDaYe homolog 1 (XJDY), Map2 based on the ZhongmuNo.1 (Zm1) consensus genome, and Map3 based on a graph‐based pangenome constructed from five alfalfa assemblies. The final maps contained 2635–2618 markers in PopA, 3222–3390 markers in PopB, and 1974–2239 markers in PopC across the three references. In the smaller populations, map length varied substantially depending on the reference genome, ranging from 1743.7 to 2701.1 cM in PopA and from 785.2 to 1109.3 cM in PopC. In contrast, the larger population (PopB) produced highly stable maps across references with lengths of 1361.5–1428.6 cM. Sequencing read depth strongly affected SNP discovery, with the UMN3988 cross producing substantially more markers than the FL99 cross. Although the pangenome improved read mapping and captured markers in structural variant regions absent from the linear reference, population size had the greatest influence on map length stability across different reference genomes for accurate linkage mapping in this highly heterozygous autopolyploid species.