JIA-2019-11

2454 CHEN Bing-ru et al. Journal of Integrative Agriculture 2019, 18(11): 2446–2456 Fig. 4 Kompetitive allele specific PCR (KASP) verification of significant single nucleotide polymorphism (SNP) related to starch content. A, scatter plots of KASP marker A009649 in 100 accessions. Red dots represent the dominant homozygote, green dots represent the heterozygote, blue dots represent the recessive homozygote, black dots represent the NTC (non-template control), and pink dots represent the missing or failed data. B, the variance of starch content of 100 accessions with different significant SNP (A009649) alleles. Table 3 Association analysis of seven scatter plots of Kompetitive allele specific PCR (KASP) makers and starch content in Tassel 5 1) Marker Chr Position marker_ F P -value marker_Rsq add_ F add_ P A009645 1 51073817 0.46827 0.6279 0.01233 0.93387 0.33693 A009646 1 74870574 1.41525 0.2486 0.0326 2.84913 0.09509 A009647 4 46965686 0.57735 0.56365 0.01389 1.15233 0.28617 A009648 5 11190345 1.27645 0.28438 0.0295 0.76287 0.38489 A009649 6 12508174 3.41938 0.03759 0.07875 6.47771 0.01282 A009650 6 57714612 2.97397 0.05741 0.07631 1.38132 0.2437 A009651 6 57714862 2.97397 0.05741 0.07631 1.38132 0.2437 1) marker_ F , F -value from the F test on marker; P -value, P -value from the F test on marker; marker_Rsq, R -squared for the marker after fitting other model terms (population structure); add_ F , F -value from the F test on the additive model; add_ P , P -value from the F test on the additive model. phosphate in cytoplasm, which may compete with starch synthesis and have a negative effect. Another SNP, rs8773750, can explain 8.2% of the variation in the trait. The gene Sobic.009G071800 was 149 kb away from it; this gene encodes 6-phospho-fructokinase (PFK), which is involved in the embden-meyerhof pathway (EMP). The EMP is important to the energy cycle as it converts fructose to glucose-1-phosphate. PFK is an allosteric enzyme (PFK-1 and PFK-2) and is the most important rate-limiting enzyme in the EMP; the homologous enzyme is on Chr2 in maize (Liu et al. 2016). The inheritance of AM contents in sorghum grain starch adhere to Mendel’s Laws (Karper 1933) and it is well known that amylose production is regulated by the waxy ( Wx ) gene on Chr10 which encodes GBSS I (McIntyre et al. 2008). The waxy gene located on Chr10 was not detected with GWAS in this natural population. We speculated that this is due to two reasons: 1) GWAS methodology has some limitations due to missing genotypes, genetic heterogeneity, unexpected LD, small effect size, low allele frequency, or complex genetic architectures and may fail to uncover the causative loci we sought. 2) Wx may not be the only gene involved in determining AM content, as observed in rice (Ayres et al. 1997; Olsen and Purugganan 2002). The significant SNPs of starch andAM were not identical, starch content may not just add AM and AP contents and may be a more complicated process. 5. Conclusion In this study, a total of 260000 SNPmarkers and the panel of 634 accessions of S. bicolor were performed GWAS of starch, AM, andAM/AP of grain. Twelve candidate genes associated with starch metabolism were located, KASP markers were developed to validate the GWAS results. The C allele was correlated with a high starch content, while the T allele was linked with a low level of starch content. The results of KASP markers indicated that C and T alleles were associated with high starch content and low starch content, respectively. Acknowledgements This work was supported by the earmarked fund for China 78 74 70 Starch content (%) 66 62 58 T C Allele A B