An updated molecular toolkit for genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) Moench]

Several mutations of the sorghum [Sorghum bicolor (L.) Moench] GRANULE-BOUND STARCH SYNTHASE (GBSS) gene [Sobic.010G022600; commonly known as Waxy (Wx)] result in a low amylose:amylopectin starch ratio. Recessive waxy (wx) alleles improve starch digestibility in ethanol production, human foods and beverages, and animal feed. However, breeding waxy sorghum is challenging due to reliance on traditional PCR markers for genotyping, which are not amenable to next-generation sequencing (NGS). Most commercial breeding programs use high-throughput genotyping and genomic selection in large, segregating populations prior to flowering. This study provides the first published NGS markers for the two most commonly used waxy (wx) alleles of sorghum and is the first to fully sequence the large insertion that is causal of the wx^a allele. In the absence of a pangenome including wx^a genotypes, we constructed an in silico B.Tx623 wx^a genome assembly from the B.Tx623 reference genome (v3.1.1) including the insertion, a ~ 5-kb-long terminal repeat (LTR) retrotransposon of the copia superfamily. The in silico wx^a assembly improved read mapping at Sobic.010G022600 in wx^a individuals, identified 78 new uniquely mapped reads, and made it possible to distinguish different Waxy genotypes using short-read sequencing data. Functional PACE-PCR markers, suitable for marker-assisted selection and multiplexed, low-to-mid-density genomic selection, were developed for Wx, wx^a, and wx^b alleles. The PACE markers were validated in segregating populations of three public and private breeding programs. These new molecular breeding resources comprise a toolkit that will improve the efficiency of developing commercial waxy sorghum hybrids using genomics-assisted approaches.