Wheat is a major worldwide food crop, and China is the largest wheat producer and consumer in the world. In 2022, Chinese wheat harvest area was 22,911.2 thousand hectares, and the total yield reached 135.76 million tons. In Henan, wheat harvest area and yield accounts for 24.8% and 28.1% in China, respectively, being the largest main wheat producing area in China. Its genetic improvement of wheat varieties has played an important role in its continuous improvement of wheat production capacity.

Pre-harvest Sprouting (PHS) refers to the phenomenon of seeds germinating and sprouting on the spike under rainy or humid conditions before wheat harvest. It is a worldwide natural disaster, and has been reported in China (Zhou et al., 2017), Japan (Kashiwakura et al., 2016), the United States (Nonogaki et al., 2014), Canada (Cabral et al., 2014), Europe (Rakoczy-Trojanowska et al., 2017), South Africa (Sydenham and Barnard, 2018), and Australia (Barrero et al., 2010). In China, the frequent and severe PHS spike hazards happened in the middle and lower reaches of Changjiang River winter wheat zone, southwest winter wheat zone, and northeast spring wheat zone (Jin, 1996; Zhang et al., 2010). In these zones, PHS resistance depends on dormant genes linked to red seed coat. In northern China, such as Henan, however, white-grained wheat varieties are used in production, and with the overall popularization of wheat mechanization harvest, that wheat should be harvested after being fully mature and dehydrated in the field. The varieties with PHS susceptibility have an increased probability of spike sprouting due to rainfall during the mature harvest period. Therefore, it is of great urgency to breed resistance varieties via PHS resistance quantitative trait nucleotides (QTNs) or genes of white-grained wheat.

Wheat PHS resistance is a complex quantitative trait controlled by multiple genes (Imtiaz et al., 2008). Thus, it is very important and necessary to identify these resistance loci and develop their molecular markers in crop breeding. In previous linkage analysis, a series of QTLs for PHS resistance has been located on all the 21 wheat chromosomes (Mohan et al., 2009; Cabral et al., 2014; Cao et al., 2016; Fakthongphan et al., 2016), in which repeatedly and stably QTLs were found on chromosome 3 (Kato et al., 2001; Osa et al., 2003; Kulwal et al., 2004; Mori et al., 2005; Liu and Bai, 2010). Currently, red-grained wheat varieties generally exhibit higher PHS resistance, because the PHS resistance genes on chromosomes 3A, 3B and 3D are thought to be closely linked to red seed coat, which is controlled by R dominant allele (Himi et al., 2011). Recently, genome-wide association studies (GWAS) have been used to identify QTLs and their candidate genes for wheat grain weight and plant height (Zanke et al., 2014; Chen et al., 2016; Wang et al., 2017), especially, Zhu et al. (2019) identified some QTLs and developed their molecular markers on wheat chromosomes 1AL, 3BS, and 6BL for PHS resistance, and Lin et al. (2017) identified two candidate genes for PHS resistance in 80 wheat varieties. However, the studies on wheat PHS resistance are relatively limited.

Chinese wheat local varieties showed higher PHS resistance than improved varieties (Wang et al., 2011; Liu et al., 2014), which provided valuable genetic resources for mining the loci of PHS resistance. In this study, 629 wheat varieties were measured for PHS resistance in 2020-2021 and 2021-2022, including 373 wheat local varieties over 70 years ago and 256 wheat improved varieties over the last 70 years in Henan Province, China. To mine some valuable QTNs for PHS resistance, these phenotypes were used to associate with SNP markers in the above 629 wheat varieties using several multi-locus GWAS methods. The results were validated by RNA-seq datasets between PHS resistance and susceptibility varieties, one confirmed QTN was used to develop Kompetitive Allele-Specific PCR (KASP) marker, and the KASP marker was further confirmed to be associated with PHS resistance. Thus, this study provides a valuable locus and white-grained wheat PHS resistance materials, which is available in main producing zones.

Genome-wide association studies for wheat PHS resistance in 629 local and improved varieties (lines) in Henan Province, China provide new insights into the genetic foundation of the important trait and variety breeding. In previous studies, most of them focused on the PHS resistance in southwest and southern wheat zones in China, for example, Zhou et al. (2017) found that the landraces in Chinese wheat zones with high precipitation showed strong PHS resistance in 717 Chinese wheat landraces, but there were few studies on PHS resistance in northern wheat zones with less rain. In this study, 373 local varieties before 1950 and 256 improved varieties after 1950 in Henan Province were included. It was found that genes for wheat PHS resistance were gradually lost in the process of selection and breeding for yield, quality, and other important breeding traits. The main reason is that most of the loci or genes for wheat PHS resistance are found to be linked with red seed coat, while most improved varieties are white grained, resulting in the generally reduced PHS resistance of modern improved varieties. In this study, 38 white grain resistant varieties were observed, and this study provides a material basis for breeders to select white-grained resistant varieties for PHS.

Zhou et al. (2017) found three major PHS resistant QTLs on chromosomes 3A, 3D, and 5D, in which the marker AX-95124645 was located on Chr 3D. In this study, AX-95124645 locus was identified by several multi-locus methods in two environments to be associated with PHS resistance, especially, its R² value was 38%. However, this study provides three new results compared with previous studies. First, resistant and susceptible PHS varieties were used to conduct RNA-seq analysis, and 9 DEGs were found in the 2.192 Mb upstream and downstream intervals of AX-95124645, and two candidate genes were predicted. Then, the KASP marker QSS.TAF9-3D-TT/CC was developed based on the AX-95124645 locus. The results showed that QSS.TAF9-3D-TT/CC haplotypes with only one T/C base allele variation could completely distinguish all the PHS resistant and susceptible varieties. Finally, all the QSS.TAF9-3D-TT haplotypes were found in 11 white-grained varieties to be resistant for PHS.

It should be point out that the KASP marker QSS.TAF9-3D developed in this study is valuable. First, the KASP marker was used to select 11 white-grained resistant varieties with excellent haplotype QSS.TAF9-3D-TT, indicating its possibility of marker-assisted selection in white-grained varieties for PHS resistance. But among 629 varieties, the numbers of white-grained varieties resistant to spike germination in the two years was 38. Because Wheat PHS resistance controlled by multiple genes (Imtiaz et al., 2008), so we consider the spike sprouting resistance of the remaining 27 white grain varieties was caused by other genes or QTLs. Second, this marker uses high-throughput KASP genotyping technology. In particular, KASP is based on conventional PCR and fluorescence detection, which can meet the requirements of low, medium, and high throughput genotyping on the basis of ordinary laboratory operation (Semagn et al., 2014), indicating that it is flexible, cheap, high-throughput, automated, and accurate. As we known, KASP, as an alternative to TaqMan, is similar in principle to TaqMan (also based on terminal fluorescence reading), but it differs from TaqMan technology in the following ways. It uses a universal probe, which can be used with a variety of different gene-specific primers, without the need for probe synthesis for each specific site, which greatly reduces the reagent cost of the experiment (Majeed et al., 2018). In conclusion, QSS.TAF9-3D-TT/CC markers can be used for higher throughput and more accurate screening of PHS resistance varieties, especially in white-grained varieties, which provides a strong theoretical basis for molecular mark-assisted breeding.

Myb10 is an important regulatory gene in the pathway of pigment synthesis. The earliest MYB-type transcription factor identified was maize Colorless 1 (Paz-Ares et al., 1987). In wheat, Tamyb10 gene is believed to be related to seed dormancy, because it may affect the sensitivity of wheat embryo to ABA. Lang et al. (2021) found that myb10-D gene, as a candidate gene for PHS-3D, not only regulates the synthesis of flavonoid compounds, but also increases the ABA concentration in developing seeds, thus inhibiting the wheat PHS. In this study, the candidate gene TraesCS3D01G468400 was found to be consistent with Tamyb10-D in the annotation information of 61 genes in the QSS.TAF9-3D region. Although Himi et al. (2011) designed the Tamyb10-D marker to screen PHS resistance materials, Tamyb10-D is an important regulatory gene involved in the pigment synthesis of wheat seed coat so that its corresponding molecular marker is mainly used to screen the PHS resistance of red-grained wheat varieties, indicating its difficulty in the application of white-grained varieties. We identified two differentially expressed genes TraesCS3D01G466100 and TraesCS3D01G468500 in the QSS.TAF9-3D region using RNA-seq. TraesCS3D01G466100 GO annotation shows that it encodes C3HC4-RING fifinger E3 ubiquitin ligase. Yang et al. (2016) identified AtAIRP4 in Arabidopsis, which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4-RING finger domain in its C-terminal side, which has in vitro E3 ligase activity. A large number of studies have shown that the dormancy period of wheat seeds is negatively correlated with the degree of PHS (Flintham et al., 2000; Biddulph et al., 2008; Shu et al., 2016), and ABA plays a crucial role in promoting seed dormancy and inhibiting seed germination (Martínez-Andújar et al., 2011). Thus, it is possible for TraesCS3D01G466100 to affect PHS resistance by regulating seed ABA levels. TraesCS3D01G468500 gene encodes the initiation transcription factor TAF9. Yang (2015) cloned a gene CpTAF9 in the woody ornamental plant Chimonanthus melanoides. Salt stress, high temperature or ABA application promoted the expression of CpTAF9 gene in leaves. ABA is an important hormone regulating seed dormanness. TraesCS3D01G468500 gene may affect wheat spike germination by indirectly regulating seed ABA content. We selected these two genes as new PHS resistance candidate genes.

We firstly identified 38 white-grained varieties with PHS resistance in 629 wheat varieties (lines) from Henan Province, China, stably identified a major QTN AX-95124645 on chromosome 3D, and developed its KASP marker QSS.TAF9-3D-TT/CC. This marker haplotype can effectively detect the PHS resistance materials, especially, all the white-grained varieties with QSS.TAF9-3D-TT haplotype are resistant to spike sprouting, which can be used for molecular mark-assisted breeding of spike sprouting resistance in white-grained varieties. This study provides material and methodological basis for breeding wheat PHS resistance in the future.

The sequencing data have been successfully submitted to the GEO database and applied for public disclosure. The GEO accession number is GSE222342, and the BioProject accession number is PRJNA919175.

W-GX conceived and managed the research and revised the manuscript. CK and C-JP analyzed datasets. CK measured the phenotypes of the traits. LH and H-BD provided the research materials and Instruments. CK wrote the draft. All authors contributed to the article and approved the submitted version.