Identification of effective alleles and haplotypes conferring pre-harvest sprouting resistance in winter wheat cultivars

Background Pre-harvest sprouting (PHS) is a serious limiting factor for wheat (Triticum aestivum L.) grain yield and end-use quality. Identification of reliable molecular markers and PHS-resistant germplasms is vital to improve PHS resistance by molecular marker-assisted selection (MAS), but the effects of allelic variation and haplotypes in genes conferring PHS resistance in winter wheat cultivars are less understood. Results Resistance to PHS was tested in 326 commercial winter wheat cultivars for three consecutive growing seasons from 2018–2020. The effects of alleles and haplotypes of 10 genes associated with PHS resistance were determined for all cultivars and were validated by introgressing the PHS-resistance allele and haplotype into a susceptible wheat cultivar. High level of phenotypic variation in PHS resistance was observed in this set of cultivars and 8 of them were highly resistant to PHS with stable germination index (GI) of less than 25% in each individual year. Allelic effects of nine genes and TaMFT haplotype analysis demonstrated that the haplotype Hap1 with low-GI alleles at five positions had the best PHS resistance. This haplotype has the priority to use in improving PHS resistance because of its high effectiveness and rare present in the current commercial cultivars. Among 14 main allelic combinations (ACs) identified, the AC1 carrying the haplotype Hap1 and the TaSdr-B1a allele had better PHS resistance than the other classes. The introgression of Hap1 and TaSdr-B1a is able to significantly improve the PHS resistance in the susceptible cultivar Lunxuan 13. Conclusions The effectiveness of alleles conferring PHS resistance in winter wheat cultivars was determined and the useful alleles and haplotypes were identified, providing valuable information for parental selection and MAS aiming at improving PHS-resistance in winter wheat. The identification of the PHS-resistant cultivars without known resistance alleles offers an opportunity to explore new PHS-resistant genes. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03710-w.

from PHS, mainly in the Middle and Lower Yangtze River Valleys Winter Wheat Zone (MLWZ), Southwestern Winter Wheat Zone (SWWZ) and Northeastern Spring Wheat Zone (NSWZ) [4]. In recent years, PHS frequently occurred in the Yellow and Huai River Valleys Winter Wheat Zone (YHWZ), resulting in serious reductions in grain yields in 2013, 2015, and 2016 [5]. Thus, resistance to PHS has become an important target trait in many wheat breeding programs.
Wheat grain color is associated with PHS, with better tolerance to PHS for the red-grained wheat cultivars than for the white-grained ones [6]. However, white-grained cultivars had higher flour yield than red-grained wheat cultivars at the same grade of whiteness of flour, which makes them more popular for millers [7,8]. Most Chinese prefer the shine and white color of products, mainly including steamed bread, boiled dumpling and noodle. The production acreages of white-grained wheats increase due to the strong market demand. Among the 75 wheat cultivars with the annual planting area > 667,000 hectares during the last several decades in China, 61 are white grains [9]. Hence, improvement of white-grained wheat with strong resistance to PHS is important for expanding its production in order to meet the market demands.
Resistance to PHS is a complex quantitative trait that is controlled by both genetic factors and external environmental factors [10]. Marker-assisted selection (MAS) on PHS resistance can not only shorten breeding cycles but also enhance selection efficiency. To date, more than 42 quantitative trait loci (QTL) governing PHS resistance are cataloged in wheat, most of which are associated with grain color and seed dormancy [2,11,12]. The transcription factor Tamyb10 is a candidate gene of grain color-related gene R, and three homoeologous genes Tamyb10-A1, Tamyb10-B1, and Tamyb10-D1 were identified [9,[13][14][15]. Gene TaDFR-B affects grain color and PHS resistance by controlling anthocyanin synthesis, and the TaDFR-Bb allele was tightly associated with PHS resistance in TaDFR-B [16].
Resistance to PHS can be assessed under field and controlled environmental conditions [23]. Field evaluation of PHS resistance needs suitable weather conditions including humidity and temperature, hence, it is difficult to obtain consistent phenotypic data in different years or environments. By contrast, controlled environmental evaluation of PHS resistance is relatively easy, and phenotypic data of PHS can be repeated in other environments. Sprouting rate of whole spikes and seed germination test are two of the main methods under controlled environment [30,31]. Calculation of the GI by testing seed germination is the most direct approach to detect seed dormancy, and was widely used to evaluate PHS resistance in previous studies [27][28][29]. However, improvement of PHS resistance based on phenotypic selection is timeconsuming and labor-intensive.
The reliable molecular markers are a prerequisite for MAS, but PHS resistance and effects of allelic variation and haplotypes in known genes affecting PHS resistance in winter wheat cultivars are less studied. The aims of this study were to 1) evaluate PHS resistance in a set of winter wheat cultivars in China; 2) identify the allelic variation and haplotypes of 10 PHS resistance genes and compare the effects of contrasting alleles at each gene, haplotypes of TaMFT, and allelic combinations on PHS resistance; and 3) validate the effects of PHS resistance allele and haplotype under the genetic background of a PHS-susceptible wheat cultivar. 24] and Lunxuan 13 (Shimai 12/Zhoumai 16//Zhoumai 16) are white-grained cultivars but differ in the resistance to PHS.

Field trials
The whole set of wheat cultivars were planted at the experimental station of the Chinese Academy of Agricultural Sciences (CAAS) in Xinxiang (35°31′N, 113°85′E), Henan province, during the 2017-2018, 2018-2019, and 2019-2020 wheat cropping seasons. All cultivars were arranged in one-row plots of 2-m length and 0.25-m width with 40 seeds. The soil of field is a typical clay loam. Fertilization, irrigation, and other field managements were carried out as described previously [32]. The meteorological data of daily average temperature (°C), relative humidity (%) and precipitation (mm) of the three cropping seasons is showed in Fig. S1.

Germination index assay
Resistance of the wheat entries to PHS was assessed using the seed GI method for three years from 2018-2020. The GIs of 206 F 2 plants and derived F 3 lines from the Lunxuan 13 × Bainong 3217 population were assessed in 2019 (F 2 ) and 2020 (F 3 ), respectively. The F 2 plants were individually harvested and separately evaluated of GI. About 30 spikes were harvested from each cultivar and F 3 line at the physiological maturity stage (about 35 d after flowering), air dried at ambient temperature (~ 25 °C) for 3 d, hand-threshed, and stored in a refrigerator at -20 °C. Fifty seeds were sterilized with 5% NaClO, evenly embedded on two layers of filter paper in Petri dishes (15 cm in diameter), and incubated in a growth cabinet at 25 °C for 7 d with 50 mL of sterile water. Germinated seeds were counted daily and removed. Germination index was calculated according to the method described by Zhang et al. [27]. This experiment was carried out thrice.

Data analysis
Analysis of variance (ANOVA) for the GI values of 326 winter wheat cultivars over three years was performed using the PROC GLM program in the Statistical Product and Service Solutions (SPSS) software 22.0 (International Business Machines Corporation, Armonk, New York, USA) [34]. Phenotypic comparison between whitegrained and red-grained wheats, and differences of PHS resistance between contrasting alleles of each gene were determined by the t-test in SPSS software 22.0. Multiple comparisons (PROC GLM) for the GI values of wheat cultivars from different wheat zones or provinces and phenotypic differences among haplotypes or allelic combinations (ACs) were performed using Tukey-Kramer at P < 0.05 in SPSS software 22. The broad-sense heritability (h 2 ) and correlation coefficients between years were estimated according to the method described by Li et al. [35].

Phenotypic evaluation on PHS resistance
The mean squares of genotypes, years and genotype × year interaction were significant as shown by ANOVA (P < 0.01) ( Table S3). The broad-sense heritability (h 2 ) of GI was 0.96. There were significant differences of GI in different years, and the mean GI values of the 326 cultivars were 48.9%, 59.2%, and 34.5% in 2018, 2019 and 2020, respectively. The wide range of phenotypic variation in GI was observed in each of the three years (Fig.  S2a). A total of 43, 10 and 86 cultivars showed the GI values lower than 25.0% in 2018, 2019 and 2020, respectively (Fig. S2a, Table S4), and 8 cultivars had stable PHS resistance across the three years.
The difference in GI was significant among the four wheat zones, in which MLWZ had the lowest GI value in each year (Fig. S2b). The cultivars from Jiangsu and Shaanxi provinces showed better PHS resistance than those from the other provinces (Fig. S2c). Grain color was associated with PHS resistance (Fig. S2d), and the red-grained cultivars had lower mean GI than the whitegrained cultivars (P < 0.05). The GI values for the 326 cultivars measured in different years were significantly correlated with a range of correlation coefficients from 0.69 to 0.79 (Fig. S3).

Allelic effects and haplotype analysis
Cultivars carrying the low-GI allele showed better PHS resistance than those carrying the high-GI allele in each of the 9 genes identified ( Table 2). Among them, cultivars with the low-GI alleles TaSdr-B1a and Tamyb10-D1b had significantly lower GI values than those with the contrasting high-GI alleles in TaSdr-B1 and Tamyb10-D1 in the three years, respectively (P < 0.05). The allele Tamyb10-D1b had the largest phenotypic effect on PHS resistance at the single gene level. Compared to Tamyb10-D1a, the allele Tamyb10-D1b decreased GIs by 10.4%, 14.9% and 10.6 in 2018, 2019 and 2020, respectively.
Haplotypes Hap1-Hap8 with at least one PHS resistance allele at five positions exhibited lower GIs than the Hap9 with five PHS susceptibility alleles (Fig. 2). Among them, Hap1 showed stable and better PHS resistance than the other haplotypes in each individual year. Compared to haplotype Hap9, Hap1 had significantly lower GIs, and averagely decreased by 32.1%, 28.5%, and 23.3% in 2018, 2019, and 2020, respectively (P < 0.05).

Effects of allelic combinations
Among the 10 genes associated with PHS resistance analyzed, 3 genes had significant difference in GIs between the contrasting alleles or among haplotypes. Hence, these genes were used to analyze effects of the ACs. A total of TaMFT-A1b/a TaPHS1-646G/A + + - TaPHS1-666A/T + + -+ + + ---14 major ACs were detected in the 326 cultivars (Fig. 3).

Validation of effects of Hap1 and TaSdr-B1a
Based on the haplotypic analysis of TaMFT S4a) and F 3 (Fig. S4b) lines. More than 90% F 2 plants and F 3 lines showed significantly lower GIs than the PHS-susceptible parent Lunxuan 13. Thirteen F 2 plants and five F 3 lines were not significantly different in GI values from the PHS-resistant parent Bainong 3217.
Significant phenotypic difference in GIs was found between the progenies with different TaMFT haplotypes and TaSdr-B1 alleles in the Lunxuan 13 × Bainong 3217 population (Fig. 4). The Hap1 progenies reduced the GI values by 13.5% and 14.4% compared to those with the haplotype Hap4 in the F 2 and F 3 populations (Fig. 4a), respectively (P < 0.05). Consistently, the progenies with the low-GI allele TaSdr-B1a showed significantly lower GI values than those with the high-GI allele TaSdr-B1b (P < 0.05) (Fig. 4b). Compared to the PHS-susceptible parent Lunxuan 13, the progenies with the Hap1 and TaSdr-B1a reduced the GI values by 29.9% and 27.1% in the F 2 population, respectively, and 40.8% and 40.0% in the F 3 population, respectively (P < 0.05). The AC TaMFT-Hap1/TaSdr-B1a had the smallest GIs among the four ACs in the F 2 (Fig. 5a) and F 3 (Fig. 5b) populations. In comparison with Lunxuan 13, the progenies with the TaMFT-Hap1/TaSdr-B1a genotype reduced the GI values by 32.7% and 44.3% in the F 2 and F 3 populations, respectively (P < 0.05).

Performance of PHS resistance in winter wheat cultivars
ANOVA and broad-sense heritability analysis suggested that the genetic variation of PHS resistance was mainly  controlled by genotype, but year and genotype × year interaction also affected PHS resistance. The consistent finding was reported in the previous study [23]. Temperature is a major external determinant on seed dormancy during seed development in wheat [36]. High temperatures during seed development reduces the level of seed dormancy [19]. Late flowering delayed sampling and continuous high temperature ( Figure S1) occurred at late maturity stage in 2019, which might result in reducing the depth of seed dormancy and making all wheat cultivars with higher GIs than other years. In all 10 genes identified, the Low-GI alleles/ haplotype had better PHS resistance than the corresponding high-GI alleles/haplotype in each individual year ( Table 2), suggesting that these Low-GI alleles/ haplotype had stable effects on PHS resistance in different environment, and they can be used to improve wheat PHS in different breeding programs. A wide variation in the GI values was observed in the winter wheat cultivars examined, but most of them were susceptible to PHS (Fig. S2a, Table S4). This is attributed to the fact that modern wheat cultivars have been domesticated by human in order to improve their adaptation and productivity, so they have relative uniform and rapid germination ability [37]. The PHSresistant cultivars identified will be useful as donors for PHS improvement.
The red-grained cultivars showed usually more resistant to PHS than the white-grained ones [6,15]. All the red-grained cultivars examined proved to carry at least one resistance allele from the Tamyb10-A1, Tamyb10-B1, and Tamyb-D1 genes. Furthermore, these cultivars mainly adapted to the MLWZ and SWWZ where PHS occurs frequently during harvesting seasons due to wet weather conditions [4]. This might make wheat cultivars more tolerant to PHS in those wheat areas by natural and artificial selections.

Comparison of PHS resistance of resistance allele, haplotype and allelic combination
Cultivars with the low-GI allele showed higher PHS resistance than those with the contrasting high-GI allele for each gene examined, which is in agreement with the previous studies [16,[18][19][20][21][22][26][27][28][29]. However, the level of PHS resistance between the contrasting alleles at certain genes varied in different genetic backgrounds [38]. For example, a main-effect locus TaMKK3-A associated with PHS resistance was identified in different populations and explained 30-38% phenotypic variations [10]. The difference of GI between alleles TaMKK3-Aa and TaMKK3-Ab was 15.5% in the Tutoumai A/NW97S186// NW97S186 BC 2 population [10], but the corresponding value was only 2.3%, 1.8% and 1.7% in 2018, 2019, and 2020, respectively (Table 2) in this study. This might be attributed to the impacts of genetic backgrounds.
It is noteworthy that the resistance allele TaPHS1-222C always present in the TaMFT haplotype Hap1, but absent in the other haplotypes. Wang et al. [39] also found that the TaPHS1-222C allele was consistently present with TaPHS1-646G and TaPHS1-666A alleles in haplotype GCA. Furthermore, there was no phenotypic difference in GI between the allele TaPHS1-222C and haplotype Hap1 in this study (data not shown). This suggests that the TaPHS1-222C marker is effective to select Hap1 genotypes in the process of MAS.
Among the 14 ACs, AC1 carrying Hap1 and TaSdr-B1a showed smaller GI than the other ACs (Fig. 3), and the pyramiding effect was further verified in the Lunxuan 13 × Bainong 3217 population (Fig. 5). Even though the Hap1 and Tamyb10-D1b alleles had the largest phenotypic effects on GIs at a single locus level, the effects of pyramiding two alleles are not clear because no cultivar carries both alleles in this study.
The known PHS resistance genes were not detected in the low-GI cultivars such as Yangmai 20 (11.6%), Lunan 11 (13.8%), and Luomai 4 (15.5%), demonstrating that these cultivars may carry new genes associated with the low GI. It warrants genetic analysis to dissect the QTL for their low GI performance.

Distribution of the low-GI alleles and allelic combinations
The haplotype Hap1 with the largest effect on PHS resistance was detected only in 11 cultivars (Table S4), suggesting that it is a rare haplotype in modern wheat cultivars. The low frequencies of the Hap 1 were also reported in Chinese accessions (2.79%) [39] and landraces (2.0%) from the Fertile Crescent and surrounding areas [40], and wheat accessions from the USA (24.4%) [40]. Hence, it is necessary to introgress the Hap1 into PHS-susceptible cultivars by molecular marker of TaPHS1-222C allele due to tightly association between the TaPHS1-222C allele with the other low-GI alleles [39]. Another allele Tamyb10-D1b also showed low frequency (8.6%), and mainly present in the red-grained cultivars (Fig. 2). In China, white-grained cultivars have been preferentially selected than red-grained cultivars by wheat breeders [6], which might result in low frequency of this allele.

Validation of effects of low-GI allele and haplotype on resistance to PHS
Lunxuan 13 is an elite high-yielding wheat cultivar, but susceptible to PHS [41]. We tried to introgress the low-GI Hap1 and allele TaSdr-B1a from the PHS-resistant parent Bainong 3217 to Lunxuan 13. The PHS resistance of the progenies were significantly enhanced. This indicates that they can efficiently improve PHS resistance of a susceptible cultivar. Pyramiding of Hap1 and TaSdr-B1a showed lower GIs than those carrying single haplotype or allele, suggesting that they have additive effects. Similar result was reported by analyzing the combining effects of TaPHS1 and TaMKK3-A [42]. Even if Hap1 and TaSdr-B1a showed additive effects, there was significant difference in mean GI between the pyramiding progenies and PHS resistance parent Bainong 3217, suggesting that Bainong 3217 might carry other unknown PHS-resistant loci.

Conclusions
The comparison of effects between contrasting alleles on GI in single gene combining haplotype analysis showed that the haplotype Hap1 of TaMFT gene had the best PHS resistance. This haplotype can be preferentially used to enhance PHS resistance due to its high effectiveness and low distribution frequency. Combining haplotype Hap1 and the TaSdr-B1a allele in AC1 exhibited additive effects on GIs in winter wheat cultivars and validated in genetic population. This study will facilitate the parental selection and MAS for wheat PHS resistance, and provide important materials for identifying new PHS-resistant genes.