Genome-wide association analysis provides molecular insights into natural variation in watermelon seed size

Abstract

Watermelons used for seed consumption tend to have larger seeds, whereas watermelons used for flesh consumption often require relatively small seeds. Therefore, watermelon seed size has received extensive attention from consumers and breeders. However, the natural variation and genetic mechanism of watermelon seed size remain unclear. In the present study, 100-seed weight, seed hilum length, seed length, seed width, and seed thickness were examined in 197 watermelon accessions. Furthermore, association analysis was performed between seed size traits and high-quality SNP data. The results revealed that there were strong correlations among the five seed traits, and seed enlargement was an important feature during watermelon seed size domestication. The seed-consumed biological species Citrullus mucosospermus and the edible seed watermelon Citrullus lanatus had significantly larger seeds than the other species. Eleven non-repeating significant SNPs above the threshold line were obtained from GWAS analysis. Four SNPs on chromosome 5 were considered to be closely associated with seed size traits (S5:32250307, S5:32250454, S5:32256177, and S5:32260870) and could be used as potential molecular markers for the breeding of watermelon cultivars with a target seed size. In addition, based on gene annotation information and previous reports, five genes near the four significant SNPs may regulate seed size. qRT-PCR analysis showed that two genes that may be involved in abscisic acid metabolism, Cla97C05G104360 and Cla97C05G104380, may play an important role in regulating watermelon seed size. Our findings provide molecular insights into natural variation in watermelon seed size and valuable information for molecular marker-assisted breeding.

References

[1] Guo S, Zhang J, Sun H et al. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet. 2013; 45: 51, 2470-8.

[2] Renner SS, Sousa A, Chomicki G . Chromosome numbers, sudanese wild forms, and classification of the watermelon genus Citrullus, with 50 names allocated to seven biological species. Taxon. 2017; 66: 1393-405.

[3] Chomicki G, Renner SS . Watermelon origin solved with molecular phylogenetics including Linnaean material: another example of museomics. New Phytol. 2015; 205: 526-32.

[4] Guo S, Zhao S, Sun H et al. Resequencing of 414 cultivated and wild watermelon accessions identifies selection for fruit quality traits. Nat Genet. 2019; 51: 1616-1623.

[5] Wang Y, Wang J, Guo S et al. CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon. Hortic Res. 2021; 8: 70.

[6] Ge L, Yu J, Wang H et al. Increasing seed size and quality by manipulating BIG SEEDS1 in legume species. Proc Natl Acad Sci U S A. 2016; 113: 12414.

[7] Jofuku KD, Omidyar PK, Gee Z et al. Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proc Natl Acad Sci U S A. 2005; 102: 3117.

[8] Cheng ZJ, Zhao XY, Shao XX et al. Abscisic acid regulates early seed development in Arabidopsis by ABI5-mediated transcription of SHORT HYPOCOTYL UNDER BLUE1. Plant Cell. 2014; 26: 1053-68.

[9] Ishimaru K, Hirotsu N, Madoka Y et al. Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield. Nat Genet. 2013; 45: 707-11.

[10] Feng Z, Wu C, Wang C et al. SLG controls grain size and leaf angle by modulating brassinosteroid homeostasis in rice. J Exp Bot. 2016; 67: 4241-53.

[11] Li B, Lu X, Zhao S et al. Genetic mapping and discovery of the candidate gene for black seed coat color in watermelon (Citrullus lanatus). Front Plant Sci. 2020; 10: 1689-9.

[12] Li N, Shang J, Wang J et al. Fine mapping and discovery of candidate genes for seed size in watermelon by genome survey sequencing. Sci Rep. 2018; 8: 17843-3.

[13] Wang K, He J, Zhao Y et al. EAR1 negatively regulates ABA signaling by enhancing 2C protein phosphatase activity. Plant Cell. 2018; 30: 815-34.

[14] Park SY, Fung P, Nishimura N et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 2009; 324: 1068-71.

[15] Zhou X, Stephens M . Genome-wide efficient mixed-model analysis for association studies. Nat Genet. 2012; 44: 821-4.

[16] Lippert C, Listgarten J, Liu Y et al. FaST linear mixed models for genome-wide association studies. Nat Methods. 2011; 8: 833-5.

[17] Sul JH, Eskin E . Mixed models can correct for population structure for genomic regions under selection. Nat Rev Genet. 2013; 14: 300.

[18] Duan P, Xu J, Zheng D et al. Natural variation in the promoter of GSE5 contributes to grain size diversity in rice. Mol Plant. 2017; 10: 685-94.

[19] Miller C, Wells R, McKenzie N et al. Variation in expression of the HECT E3 ligase UPL3 modulates LEC2 levels, seed size, and crop yields in brassica napus. Plant Cell. 2019; 31: 2370-2385.

[20] Ren D, Wang X, Yang M et al. A new regulator of seed size control in Arabidopsis identified by a genome-wide association study. Mol Plant. 2019; 222: 895-906.

[21] Dou J, Zhao S, Lu X et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor Appl Genet. 2018; 131: 947-58.

[22] Li N, Liu Z, Wang Z et al. STERILE APETALA modulates the stability of a repressor protein complex to control organ size in Arabidopsis thaliana. PLoS Genet. 2018; 14: e1007218. http://doi:10.1371/journal.pgen.1007218

[23] Yuan H, Fan S, Huang J et al. 08SG2/OsBAK1 regulates grain size and number, and functions differently in Indica and japonica backgrounds in rice. Rice. 2017; 10: 25.

[24] Xu Q, Yu H, Xia S et al. The C2H2 zinc-finger protein LACKING RUDIMENTARY GLUME 1 regulates spikelet development in rice. Science Bulletin. 2020; 65: 753-64.

[25] Zhuang H, Wang HL, Zhang T et al. NONSTOP GLUMES1 encodes a C2H2 zinc finger protein that regulates spikelet development in rice. Plant Cell. 2020; 32: 392-413.

[26] Yang Y, Qin Y, Xie C et al. The Arabidopsis chaperone J3 regulates the plasma membrane H+-ATPase through interaction with the PKS5 kinase. Plant Cell. 2010; 22: 1313-32.

[27] Huang X, Han B . Natural variations and genome-wide association studies in crop plants. Annu Rev Plant Biol. 2014; 65: 531-51.

[28] Paris HS. Origin and emergence of the sweet dessert watermelon. Ann Bot. 2015;116:133-48.

[29] Olsen KM, Wendel JF . A bountiful harvest: genomic insights into crop domestication phenotypes. Annu Rev Plant Biol. 2013; 64: 47-70.

[30] Wu J, Lawit SJ, Weers B et al. Overexpression of zmm28 increases maize grain yield in the field. Proc Natl Acad Sci U S A. 2019; 116: 23850.

[31] Reichardt S, Budahn H, Lamprecht D et al. The carrot monoterpene synthase gene cluster on chromosome 4 harbours genes encoding flavour-associated sabinene synthases. Hortic Res. 2020; 7: 190.

[32] Xue H, Shi T, Wang F et al. Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method. Hortic Res. 2017; 4: 17053.

[33] Ren Y, McGregor C, Zhang Y et al. An integrated genetic map based on four mapping populations and quantitative trait loci associated with economically important traits in watermelon (Citrullus lanatus). BMC Plant Biol. 2014; 14: 33.

[34] Prothro J, Abdel-haleem H, Bachlava E et al. Main and epistatic quantitative trait loci associated with seed size in watermelon. J Am Soc Hortic Sci. 2012; 137: 452-457.

[35] Guo DL, Zhao HL, Li Q et al. Genome-wide association study of berry-related traits in grape [Vitis vinifera L.] based on genotyping-by-sequencing markers. Hortic Res. 2019; 6: 11.

[36] Zhou X, Hao H, Zhang Y et al. SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-type protein KINASE, is important for abscisic acid responses in arabidopsis through phosphorylation of ABSCISIC ACID-INSENSITIVE5. Plant Physiol. 2015; 168: 659-76.

[37] Price AL, Patterson NJ, Plenge RM et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet. 2006; 38: 904-9.

[38] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25: 402-8.