Development and application of the GenoBaits WheatSNP16K array to accelerate wheat genetic research and breeding

Abstract

Single-nucleotide polymorphisms (SNPs) are widely used as molecular markers for constructing genetic linkage maps in wheat. Compared with available SNP-based genotyping platforms, a genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology has become the favored genotyping technology because it is less demanding and more cost effective, flexible, and user-friendly. In this study, a new GenoBaits WheatSNP16K (GBW16K) GBTS array was designed using datasets generated by the wheat 660K SNP array and resequencing platforms in our previous studies. The GBW16K array contains 14 868 target SNP regions that are evenly distributed across the wheat genome, and 37 669 SNPs in these regions can be identified in a diversity panel consisting of 239 wheat accessions from around the world. Principal component and neighbor-joining analyses using the called SNPs are consistent with the pedigree information and geographic distributions or ecological environments of the accessions. For the GBW16K marker panel, the average genetic diversity among the 239 accessions is 0.270, which is sufficient for linkage map construction and preliminary mapping of targeted genes or quantitative trait loci (QTLs). A genetic linkage map, constructed using the GBW16K array-based genotyping of a recombinant inbred line population derived from a cross of the CIMMYT wheat line Yaco"S" and the Chinese landrace Mingxian169, enables the identification of Yr27, Yr30, and QYr.nwafu-2BL.4 for adult-plant resistance to stripe rust from Yaco"S" and of Yr18 from Mingxian169. QYr.nwafu-2BL.4 is different from any previously reported gene/QTL. Three haplotypes and six candidate genes have been identified for QYr.nwafu-2BL.4 on the basis of haplotype analysis, micro-collinearity, gene annotation, RNA sequencing, and SNP data. This array provides a new tool for wheat genetic analysis and breeding studies and for achieving durable control of wheat stripe rust.

Keywords: genotyping by target sequencing; liquid array; resistance gene; stripe rust; wheat.

Figure 1

Design and statistics of the GenoBaits Wheat16K (GBW16K) array. (A) Design pipeline of the GBW16K array. (B) Number of target SNP regions on each chromosome. Blue line represents the length of individual chromosome. (C) Density of the GBW16K array SNP regions in 1-Mb windows throughout the whole genome. (D) Distribution of SNP regions within genome regions (intergenic, upstream, exonic, downstream, intronic, 3′ UTR, 5′ UTR, ncRNA_exonic, and splicing). (E) Positions of >5-Mb gaps in the genome. Red arrows indicate the locations of gaps.

Figure 2

Validation of the GBW16K array in 239 wheat accessions. (A) Minor allele frequency (MAF) distribution of the coreSNPs from each SNP region. (B) Diversity distribution between any two wheat accessions based on mSNPs. (C) Phylogenetic tree of accessions based on mSNPs. Each branch is color coded to represent accessions from distinct geographic regions. (D) Principal coordinate analysis (PCoA) plots colored by geographic origin. Principal coordinate 1 is plotted along the x axis. Principal coordinate 2 is plotted along the y axis.

Figure 3

Phenotypic and QTL analysis of stripe rust response in the Yaco“S” × MX169 RIL population. (A) Phenotypes of Yaco“S” and MX169 challenged with stripe rust at the adult-plant stage. (B) Phenotypic distributions of mean infection types (IT) and disease severities (DS) for Yaco“S” × MX169 RILs across four environments. Values for the parents Yaco“S” and MX169 are indicated by arrows. (C) Linkage maps and QTL mapping for three chromosome arms. QTLs were identified by inclusive composite interval mapping (ICIM). Confidence intervals of QTLs and markers significantly linked with QTLs are marked in red and blue, respectively. LOD score refers to the logarithm of odds score. YL, JY, and TS represent Yangling, Jiangyou, and Tianshui, and 20 and 21 represent the crop seasons of 2020 and 2021. MIT and MDS represent the mean IT and mean DS of all replicates in each environment.

Figure 4

Phenotypic responses of YM106R/YM106S NILs to stripe rust and fine mapping of QYr.nwafu-2BS/YrAc. (A) Upper: stripe rust responses of YM106R and YM106S at different developmental stages (two- to five-leaf stage). Observe the increasing resistance of YM106R. Lower: stripe rust responses of YM106R and YM106S in the field. (B) Genetic differences (purple) between YM106R and YM106S based on the GBW16K array. (C) Distribution of polymorphic SNPs across each chromosome. The SNPs were identified from bulked segregant analysis of stripe rust response in the YM106 F₆ population. (D) High-resolution genetic and physical map of YrAc. The region in red marks the confidence interval of YrAc; markers significantly linked to QTLs are marked in red and blue. n indicates the number of lines used in mapping; R, S, and H denote homozygous resistant, homozygous susceptible, and heterozygous, respectively. R1–R21 represent different recombinant types. Numbers in parentheses represent the number of each recombinant type. (E) Polymorphisms in the CDS of the candidate gene TraesCS2B01G182800.1 among wheat cultivars Kariega (Yr27), Yaco“S,” Zhoumai 22 (Lr13/Ne2), and MX169. Red indicates nucleotide sequence consistency; beige indicates nucleotide sequence inconsistency.

Figure 5

Physical location, haplotype analysis, and candidate genes of QYr.nwafu-2BL.4 in chromosome arm 2BL. (A) QYr.nwafu-2BL.4 and previously mapped Pst resistance genes/QTLs are positioned on IWGSC RefSeq v.1.0. QYr.nwafu-2BL.4 and reported genes/QTLs are marked in red and blue, respectively. Detailed information on genes/QTLs is provided in Supplemental Table 12. References: ^[1]Draz et al. (2021); ^[2]Zegeye et al. (2014); ^[3]Zhou et al. (2015); ^[4]Jighly et al. (2015); ^[5]Marchal et al. (2018); ^[6]Singh et al. (2013); ^[7]Cheng and Chen (2010); ^[8]Kumar et al. (2013); ^[9]Xu et al. (2013); ^[10]Sui et al. (2009); ^[11]Ren et al. (2012); ^[12]Chhetri et al. (2023); ^[13]Ramburan et al. (2004); ^[14]Cheng et al. (2022); ^[15]Rosewarne et al. (2008); ^[16]Mallard et al. (2005); ^[17]Vazquez et al. (2015); ^[18]Buerstmayr et al. (2014); ^[19]Paillard et al. (2012); ^[20]Liu et al. (2015); ^[21]Ren et al. (2015); ^[22]Farzand et al. (2021); ^[23]Guo et al. (2008); ^[24]Powell et al. (2013); ^[25]Boukhatem et al. (2002); ^[26]Zeng et al. (2019a); ^[27]Christiansen et al. (2006); ^[28]Hou et al. (2015); ^[29]Zeng et al. (2019b). (B) Phylogenetic tree of the candidate QYr.nwafu-2BL.4 region using 660K array data. Hap_1 (Yaco“S”), Hap_2 (MX169), and Hap_3 are found mainly in CIMMYT and ICARDA derivatives, landraces, and modern Chinese cultivars. Numbers of wheat accessions and their frequencies are listed alongside the corresponding haplotypes. Details of results based on 660K wheat SNP markers are available in Supplemental Table 13. (C) Stripe rust responses of different haplotype groups in the field. ns, no significant difference. ∗∗∗p < 0.001 (Student’s t-test). (D) Expression levels of high-confidence genes within the interval of QYr.nwafu-2BL.4. Black dots highlight nine genes with SNP mutations in the coding sequence between Yaco“S” and MX169. W, inoculated with water; SR, inoculated with stripe rust. (E) Structures and positions of amino acid variations in the six candidate genes. Arrows indicate the locations of amino acid variations. Detailed information on the high-confidence genes is provided in Supplemental Table 14.