Mapping of a Major-Effect Quantitative Trait Locus for Seed Dormancy in Wheat

The excavation and utilization of dormancy loci in breeding are effective endeavors for enhancing the resistance to pre-harvest sprouting (PHS) of wheat varieties. CH1539 is a wheat breeding line with high-level seed dormancy. To clarify the dormant loci carried by CH1539 and obtain linked molecular markers, in this study, a recombinant inbred line (RIL) population derived from the cross of weak dormant SY95-71 and strong dormant CH1539 was genotyped using the Wheat17K single-nucleotide polymorphism (SNP) array, and a high-density genetic map covering 21 chromosomes and consisting of 2437 SNP markers was constructed. Then, the germination percentage (GP) and germination index (GI) of the seeds from each RIL were estimated. Two QTLs for GP on chromosomes 5A and 6B, and four QTLs for GI on chromosomes 5A, 6B, 6D and 7A were identified. Among them, the QTL on chromosomes 6B controlling both GP and GI, temporarily named QGp/Gi.sxau-6B, is a major QTL for seed dormancy with the maximum phenotypic variance explained of 17.66~34.11%. One PCR-based diagnostic marker Ger6B-3 for QGp/Gi.sxau-6B was developed, and the genetic effect of QGp/Gi.sxau-6B on the RIL population and a set of wheat germplasm comprising 97 accessions was successfully confirmed. QGp/Gi.sxau-6B located in the 28.7~30.9 Mbp physical position is different from all the known dormancy loci on chromosomes 6B, and within the interval, there are 30 high-confidence annotated genes. Our results revealed a novel QTL QGp/Gi.sxau-6B whose CH1539 allele had a strong and broad effect on seed dormancy, which will be useful in further PHS-resistant wheat breeding.


Introduction
Plant seeds can evade external adverse factors through dormancy, which is significant for their reproduction [1].Common wheat (Triticum aestivum L., 2n = 6x = 42; AABBDD) is a widely cultivated crop worldwide and the first staple food crop domesticated in history, contributing about one-fifth of the total calories and proteins of the daily dietary intake of global population [2].It originated from hybridization between cultivated tetraploid emmer wheat (Triticum dicoccoides L., 2n = 4x = 28; AABB) and Aegilops tauschii (2n = 2x = 14; DD) approximately 8000 years ago.The cultivation and domestication of common wheat have been directly associated with the spread of agriculture and settled societies [3].In order to obtain the uniformity of seed germination and seedling emergence, the seed dormancy of wheat has gradually weakened in the process of domestication by human ancestors and extensive modern artificial selection, which results in pre-harvest sprouting (PHS) of physiologically mature seeds in spikes of most modern cultivars when the harvest time coincides with untimely rainfall, leading to losses in grain yield and deterioration in grain milling and baking quality [1][2][3].Therefore, enhancing the level of seed dormancy in wheat to a certain extent is a necessary measure to address the negative impact on yield and quality caused by PHS.
Unearthing dormancy loci is fundamental endeavor in enhancing seed dormancy.Numerous genes associated with dormancy have been identified in wheat.The Red-1 (R-1) gene controlling grain color was first observed to be positively associated with seed dormancy due to the fact that red grains usually have stronger dormancy characteristics than white grains [4].R-1 gene encodes the MYB transcription factor and has three subgenomic copies, namely Myb10-A, Myb10-B, and Myb10-D [5].Recent research revealed that Myb10-D confers PHS resistance by enhancing abscisic acid (ABA) biosynthesis to delay germination in wheat [6].Overexpression of wheat DELAY OF GERMINATION-1 (TaDOG1), the homolog of AtDOG1 in Arabidopsis [7], significantly increases seed dormancy levels [8].Wheat MOTHER OF FT AND TFL 1 (TaMFT), the homolog of AtMFT [9], finely regulates seed dormancy by coordinating the ABA and gibberellic acid signaling pathways [10].Moreover, wheat genes MITOGEN-ACTIVATED PROTEIN KINASE KI-NASE 3 (TaMKK3) [11], PLASMA MEMBRANE 19 (TaPM19) [12], and SEED DORMANCY (TaSdr) [13] were identified to play critical roles in the regulation of seed germination.
Moreover, transcriptome sequencing was applied in mining candidate genes for seed dormancy.Comparing the transcriptomes of seeds from two wheat varieties, strong dormant Scotty (PI 469294) and weak dormant Doublecrop (Cltr 17349), 2368 differentially expressed genes (DEGs) were identified at 20 days after anthesis, and several dormancyrelated genes such as TaMFT and wheat FLOWERING LOCUS C (TaFLC) were selected and validated through qRT-PCR [14].Additionally, a total of 13,154 DEGs were identified in four comparison groups between wheat cultivars Woori and Keumgang, with high and low levels of seed dormancy, respectively, and nine DEGs related to the spliceosome and proteasome pathways were verified as candidate genes using qRT-PCR [15].A similar analysis on strong dormant AC Domain and weak dormant RL4452 showed that DEGs including wheat LONELY GUY (TaLOG), cis ZEATIN-O-GLUCOSYLTRANSFERASE (TacZOG), ALDEHYDE OXIDASE (TaAO), UBIQUITIN (TaRUB), and AUXIN RESPONSE FACTOR (TaARF) are involved in seed dormancy during the early stage of seed maturation [16].Transcriptomic comparison of germination seeds and dormant seeds of the wheat variety MingXian169 revealed 3027 DEGs, and qRT-PCR confirmed that that the expressions of ASCORBATE PEROXIDASE (APX), MONODEHYDROASCORBATE REDUCTASE (MD-HAR), β-GLUCOSIDASE (GLU), and β-AMYLASE (AMY) are significantly upregulated in germination seeds [17].
Furthermore, a multitude of dormancy-or PHS-associated quantitative trait loci (QTLs) have been identified on each chromosome of wheat [18,19].Taking chromosome 6B as an example, molecular markers wmc104 [20] and wPt0959 [21] on the short arm of the chromosome were reported to be associated with tolerance to PHS in a recombinant inbred line (RIL) population and a set of 96 winter wheat accessions, respectively.In addition, QPhs.spa-6B [22] with 25% phenotypic variance explained (PVE) for germination index (GI) was identified in a doubled haploid mapping population; QPhs.umb-6B [23] with the PVE value of 3.09~4.33%for speed of germination index (SGI) was mapped in the doubled haploid population; and Qphs.ahau-6B was related to GI, field sprouting, and a period of dormancy in different environments with the R 2 of 6.1~16.7%based on a genome-wide association study for 192 wheat varieties [24].
It is believed that the PHS resistance of wheat is predominantly due to dormancy, and the early interruption of seed dormancy has been considered the major component of PHS [13].A lack of high dormancy level in many wheat cultivars during harvest period results in serious economic losses due to the adverse effects of pre-harvest sprouted wheat grains on end-product quality.Enhancing the tolerance to PHS is a major breeding objective in the world.Therefore, an understanding of the genetic control of seed dormancy and the development of functional markers are very necessary for marker-assisted breeding targeting for PHS tolerance in wheat breeding.CH1539 is a wheat breeding line with strong seed dormancy characteristics bred by our team.In order to clarify its dormant loci and obtain linked molecular markers, we created an RIL population derived from the intercrossing of CH1539 (CH) with the weakly dormant wheat line SY95-71 (SY).Here, this RIL population was used to construct a high-density genetic map using the Wheat17K SNP chip.Then, the germination percentage (GP) and GI of the seeds from each RIL were estimated, and the QTLs for GP and GI were mapped.We detected a major QTL for seed dormancy on chromosome 6B and developed its PCR-based diagnostic marker, which will be useful in further QTL-cloning and the PHS-resistant wheat breeding.

Assessment of Seed Dormancy for SY and CH
The two breeding lines SY and CH have large differences in seed dormancy (Figure 1).SY had a weak seed dormancy with a GP of 52.42~70.21%and a GI of 28.30~34.40,whereas CH had a strong seed dormancy with a GP of 0~2.17% and a GI of 0~0.62.The best linear unbiased estimation (BLUE) values of both GP and GI in SY were significantly higher than those in CH (p < 0.001) (Table 1).
with strong seed dormancy characteristics bred by our team.In order to c dormant loci and obtain linked molecular markers, we created an RIL population from the intercrossing of CH1539 (CH) with the weakly dormant wheat line SY95 Here, this RIL population was used to construct a high-density genetic map u Wheat17K SNP chip.Then, the germination percentage (GP) and GI of the seeds fr RIL were estimated, and the QTLs for GP and GI were mapped.We detected a ma for seed dormancy on chromosome 6B and developed its PCR-based diagnostic which will be useful in further QTL-cloning and the PHS-resistant wheat breedin

Assessment of Seed Dormancy for SY and CH
The two breeding lines SY and CH have large differences in seed dormancy 1).SY had a weak seed dormancy with a GP of 52.42~70.21%and a GI of 28.3 whereas CH had a strong seed dormancy with a GP of 0~2.17% and a GI of 0~0 best linear unbiased estimation (BLUE) values of both GP and GI in SY were sign higher than those in CH (p < 0.001) (Table 1).

Phenotypic Variance of GP and GI in the SY × CH RILs
GP and GI showed segregations in the RIL population derived from the cro and CH, which ranged from 0 to 100% and from 0 to 98.00, with the coefficient of v values of 0.75~0.88 and 0.83~0.97,respectively (Table 1).According to the phenoty of three repeat tests and the derived BLUE datasets, GP and GI in the RILs exhib tinuous distribution (not normal distribution) and transgressive segregation, w lines having stronger germination than SY, indicating that seed dormancy was a trait controlled by multi-genes (Figure 2).

Phenotypic Variance of GP and GI in the SY × CH RILs
GP and GI showed segregations in the RIL population derived from the cross of SY and CH, which ranged from 0 to 100% and from 0 to 98.00, with the coefficient of variation values of 0.75~0.88 and 0.83~0.97,respectively (Table 1).According to the phenotypic data of three repeat tests and the derived BLUE datasets, GP and GI in the RILs exhibited continuous distribution (not normal distribution) and transgressive segregation, with some lines having stronger germination than SY, indicating that seed dormancy was a complex trait controlled by multi-genes (Figure 2).

Genetic Mapping of GP and GI
A total of 3328 SNPs from the 17K SNP chip showed allelic variation between SY and CH, and 2437 of which were mapped in the SY × CH RIL population after removing the redundant or excessive missing data.These SNP markers were assembled into 21 chromosomes to form a genetic map for the RILs with a marker density of 1.93 cM per marker.Based on the linkage map and the phenotypic BLUE data, two genomic regions on chromosomes 5A and 6B were found to have significant effects on the GP trait, with a PVE of 5.78% and 17.66%, respectively; and four genomic regions on chromosomes 5A, 6B, 6D and 7A were found to have significant effects on the GI trait, with a PVE of 6.41%, 34.11%, 5.06% and 9.02%, respectively (Table 2).The QTLs on chromosomes 5A and 6B controlled both GP and GI, and their additive effects came from weak dormant SY and strong dormant CH, respectively.Among them, the QTL on chromosomes 6B, temporarily named QGp/Gi.sxau-6B,has the maximum PVE values and negative additive effects, which indicates that QGp/Gi.sxau-6B is a major QTL for aggravating seed dormancy (Table 2).

Verification of QGp/Gi.sxau-6B
To verify the mapping results, PCR-based markers for QGp/Gi.sxau-6Bwere developed.QGp/Gi.sxau-6B was mapped to a 0.1 cM interval flanked by markers 995614 (chr.6B:28,712,133) and 1091526 (chr.6B:30,888,979), corresponding to a physical range of 2.18 Mbp (Figure 3a,b).We randomly designed 25 pairs of primers within the genome segment that ranged from 1 Mbp upstream of 995614 to 1 Mbp downstream of 1091526, to amplify parental DNA and then sequence the products.Fortunately, sequence differences of insertion/deletion (InDel) were found on both sides and in the middle of QGp/Gi.sxau-6B.For the InDel2 on the outer site of SNP marker 995614, there was a 14 bp deletion in SY but a 14 bp insertion in CH; for the InDel5 on the outer site of SNP marker 1091526, there

Genetic Mapping of GP and GI
A total of 3328 SNPs from the 17K SNP chip showed allelic variation between SY and CH, and 2437 of which were mapped in the SY × CH RIL population after removing the redundant or excessive missing data.These SNP markers were assembled into 21 chromosomes to form a genetic map for the RILs with a marker density of 1.93 cM per marker.Based on the linkage map and the phenotypic BLUE data, two genomic regions on chromosomes 5A and 6B were found to have significant effects on the GP trait, with a PVE of 5.78% and 17.66%, respectively; and four genomic regions on chromosomes 5A, 6B, 6D and 7A were found to have significant effects on the GI trait, with a PVE of 6.41%, 34.11%, 5.06% and 9.02%, respectively (Table 2).The QTLs on chromosomes 5A and 6B controlled both GP and GI, and their additive effects came from weak dormant SY and strong dormant CH, respectively.Among them, the QTL on chromosomes 6B, temporarily named QGp/Gi.sxau-6B,has the maximum PVE values and negative additive effects, which indicates that QGp/Gi.sxau-6B is a major QTL for aggravating seed dormancy (Table 2).

Verification of QGp/Gi.sxau-6B
To verify the mapping results, PCR-based markers for QGp/Gi.sxau-6Bwere developed.QGp/Gi.sxau-6B was mapped to a 0.1 cM interval flanked by markers 995614 (chr.6B:28,712,133) and 1091526 (chr.6B:30,888,979), corresponding to a physical range of 2.18 Mbp (Figure 3a,b).We randomly designed 25 pairs of primers within the genome segment that ranged from 1 Mbp upstream of 995614 to 1 Mbp downstream of 1091526, to amplify parental DNA and then sequence the products.Fortunately, sequence differences of insertion/deletion (InDel) were found on both sides and in the middle of QGp/Gi.sxau-6B.For the InDel2 on the outer site of SNP marker 995614, there was a 14 bp deletion in SY but a 14 bp insertion in CH; for the InDel5 on the outer site of SNP marker 1091526, there was a 10 bp deletion in SY but a 10 bp insertion in CH; and for the InDel3 located inside the QGp/Gi.sxau-6B,there was a 29 bp deletion in SY but a 29 bp insertion in CH (Figure 3b).Based on these InDels, three PCR markers Ger6B-2, Ger6B-3 and Ger6B-5 were developed and showed the expected parental polymorphisms by polyacrylamide gel electrophoresis (Figure 3c). the QGp/Gi.sxau-6B,there was a 29 bp deletion in SY but a 29 bp insertion in CH (Figure 3b).Based on these InDels, three PCR markers Ger6B-2, Ger6B-3 and Ger6B-5 were developed and showed the expected parental polymorphisms by polyacrylamide gel electrophoresis (Figure 3c).Verification results in the RIL population showed that the SY allele of Ger6B-2, Ger6B-3 and Ger6B-5 all corresponded to the higher GP and GI values, while the CH allele of the three markers corresponded to the lower values, confirming the effect of QGp/Gi.sxau-6B.The phenotypic differences between the two alleles of Ger6B-3 (p < 0.0001) within the QTL were more significant than that of markers Ger6B-2 and Ger6B-5 (p < 0.01) on both sides (Figure 4).Hence, Ger6B-3 was used as the diagnostic marker for allelic variation in QGp/Gi.sxau-6B.Verification results in the RIL population showed that the SY allele of Ger6B-2, Ger6B-3 and Ger6B-5 all corresponded to the higher GP and GI values, while the CH allele of the three markers corresponded to the lower values, confirming the effect of QGp/Gi.sxau-6B.The phenotypic differences between the two alleles of Ger6B-3 (p < 0.0001) within the QTL were more significant than that of markers Ger6B-2 and Ger6B-5 (p < 0.01) on both sides (Figure 4).Hence, Ger6B-3 was used as the diagnostic marker for allelic variation in QGp/Gi.sxau-6B.the QGp/Gi.sxau-6B,there was a 29 bp deletion in SY but a 29 bp insertion in CH (Figure 3b).Based on these InDels, three PCR markers Ger6B-2, Ger6B-3 and Ger6B-5 were developed and showed the expected parental polymorphisms by polyacrylamide gel electrophoresis (Figure 3c).Verification results in the RIL population showed that the SY allele of Ger6B-2, Ger6B-3 and Ger6B-5 all corresponded to the higher GP and GI values, while the CH allele of the three markers corresponded to the lower values, confirming the effect of QGp/Gi.sxau-6B.The phenotypic differences between the two alleles of Ger6B-3 (p < 0.0001) within the QTL were more significant than that of markers Ger6B-2 and Ger6B-5 (p < 0.01) on both sides (Figure 4).Hence, Ger6B-3 was used as the diagnostic marker for allelic variation in QGp/Gi.sxau-6B.

Distribution of QGp/Gi.sxau-6B Alleles in Wheat
Ger6B-3 was used to amplify a set of wheat germplasm containing 97 varieties.The results showed that 39 varieties carried the SY allele of QGp/Gi.sxau-6B and 58 varieties carried the CH allele of QGp/Gi.sxau-6B.The GP and GI values corresponding to the SY allele were significantly higher than those corresponding to the CH allele, with p < 0.01 and p < 0.05, respectively, indicating that QGp/Gi.sxau-6Bhad a wide effect on the dormancy of different wheat germplasms (Figure 5).Further analysis revealed that the distribution frequency of the strong-dormancy QGp/Gi.sxau-6BCH allele was high in landraces (78.95%), but decreased in cultivars (47.46%) (Figure 5c), suggesting that artificial selection in wheat breeding significantly affected seed dormancy ability and was one of the reasons for the decrease in resistance to PHS.

Distribution of QGp/Gi.sxau-6B Alleles in Wheat
Ger6B-3 was used to amplify a set of wheat germplasm containing 97 varieties.The results showed that 39 varieties carried the SY allele of QGp/Gi.sxau-6B and 58 varieties carried the CH allele of QGp/Gi.sxau-6B.The GP and GI values corresponding to the SY allele were significantly higher than those corresponding to the CH allele, with p < 0.01 and p < 0.05, respectively, indicating that QGp/Gi.sxau-6Bhad a wide effect on the dormancy of different wheat germplasms (Figure 5).Further analysis revealed that the distribution frequency of the strong-dormancy QGp/Gi.sxau-6BCH allele was high in landraces (78.95%), but decreased in cultivars (47.46%) (Figure 5c), suggesting that artificial selection in wheat breeding significantly affected seed dormancy ability and was one of the reasons for the decrease in resistance to PHS.

CH1539 Can Be Used to Improve the Resistance of Cultivars to PHS
Modern wheat cultivars generally have poor resistance to PHS, as the breeding process artificially weakens the dormancy ability of their seeds to obtain quick and consistent seedling emergence after sowing.An improvement in seeds' dormancy of different varieties can enhance their resistance to PHS.CH1539 is a strong dormant breeding line.In this study, the GP of CH1539 seeds was only 0~2.17% after 7 days of germination under suitable conditions of moisture and temperature, indicating that CH1539 will show effective resistance to PHS in the field when encountering continuous rainfall before harvest.CH1539 carries a major-dormancy QTL, QGp/Gi.sxau-6B, with a PVE of 17.66~34.11%.The distribution frequency of QGp/Gi.sxau-6BCH allele in cultivars is significantly lower than that in landraces, suggesting that the dormant effect of this locus has been attenuated during the breeding process and needs to be strengthened in the future.We developed the PCR-based diagnostic marker Ger6B-3 for molecular-assisted selection of QGp/Gi.sxau-6B.In addition, CH1539 also carries the leaf rust resistance gene LrCH1539 with the co-separated InDel-marker sxau-2BS210 [25], which can be used for aggregate breeding of diseaseand PHS resistance.

QGp/Gi.sxau-6B Is a Novel Dormancy Locus
QGp/Gi.sxau-6B was located in the chr.6B:28.7~30.9Mbp physical position flanked by SNP markers 995614 and 1091526.Currently, a total of five loci related to dormancy or PHS have been reported on chromosome 6B (Figure 6).Among them, QPhs.spa-6B was mapped in a Xgwm508-Xgdm113 interval corresponding to the physical position range from chr.6B:47.8Mbp to 77.1 Mbp [22], and it should be in the same locus as a PHS-associated single marker wPt-0959 [21], but different from the position of QGp/Gi.sxau-6B.Moreover, the other PHS-linked single marker Xwmc104 was located in the chr.6B:149.2

CH1539 Can Be Used to Improve the Resistance of Cultivars to PHS
Modern wheat cultivars generally have poor resistance to PHS, as the breeding process artificially weakens the dormancy ability of their seeds to obtain quick and consistent seedling emergence after sowing.An improvement in seeds' dormancy of different varieties can enhance their resistance to PHS.CH1539 is a strong dormant breeding line.In this study, the GP of CH1539 seeds was only 0~2.17% after 7 days of germination under suitable conditions of moisture and temperature, indicating that CH1539 will show effective resistance to PHS in the field when encountering continuous rainfall before harvest.CH1539 carries a major-dormancy QTL, QGp/Gi.sxau-6B, with a PVE of 17.66~34.11%.The distribution frequency of QGp/Gi.sxau-6BCH allele in cultivars is significantly lower than that in landraces, suggesting that the dormant effect of this locus has been attenuated during the breeding process and needs to be strengthened in the future.We developed the PCR-based diagnostic marker Ger6B-3 for molecular-assisted selection of QGp/Gi.sxau-6B.In addition, CH1539 also carries the leaf rust resistance gene LrCH1539 with the co-separated InDel-marker sxau-2BS210 [25], which can be used for aggregate breeding of disease-and PHS resistance.

Prediction of Causing Gene for QGp/Gi.sxau-6B
There are thirty high-confidence genes in the QGp/Gi.sxau-6Binterval according to the annotation of coding sequences (RefSeq v1.1) in the Chinese Spring genome database, five of which were highly expressed in germinated seeds but lowly expressed in dormant seeds based on published data [17].Among these differentially expressed genes (DEGs), two genes TraesCS6B02G049100 and TraesCS6B02G049300 encoded histone H2B, two genes TraesCS6B02G049400 and TraesCS6B02G049500 encoded histone H2A, and the remain gene TraesCS6B02G050700 encoded carboxypeptidase (CP) (Figure 6).
It has been established that histone H2A and H2B play important roles in the regulation of seed dormancy [26][27][28].The epigenetic factor POWERDRESS (PWR) can interact with ABA-INSENSITIVE 3 (ABI3) and HISTONE DEACETYLASE 9 (HDA9) to reduce histone acetylation level and increase H2A deposition at the SOMNUS (SOM) locus, a positive regulator of seed dormancy, thus repressing the expression level of SOM and promoting seed germination process [29].Moreover, histone H2B monoubiquitylation regulated by HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 can increase DOG1 expression and then regulate the seed dormancy level [30][31][32].Therefore, the H2A and H2B genes exhibit significant transcriptional changes during the epigenetic regulation of seed dormancy in wheat.
Additionally, the expression level of the CP gene TraesCS6B02G050700 was significantly upregulated in germinated seeds.Carboxypeptidase is a key hydrolytic enzyme during germination in cereal grains [33,34].In wheat, CP genes were involved in the acidification of endosperm starchy [35], mobilization of endosperm storage proteins [36], and other biochemical reactions during germination processes [37].More importantly, TraesCS6B02G050700 was highly expressed specifically in grains (Figure S1), indicating that it may be the causing gene for QGp/Gi.sxau-6B.Further experiments are needed to confirm this speculation.

Prediction of Causing Gene for QGp/Gi.sxau-6B
There are thirty high-confidence genes in the QGp/Gi.sxau-6Binterval according to the annotation of coding sequences (RefSeq v1.1) in the Chinese Spring genome database, five of which were highly expressed in germinated seeds but lowly expressed in dormant seeds based on published data [17].Among these differentially expressed genes (DEGs), two genes TraesCS6B02G049100 and TraesCS6B02G049300 encoded histone H2B, two genes TraesCS6B02G049400 and TraesCS6B02G049500 encoded histone H2A, and the remain gene TraesCS6B02G050700 encoded carboxypeptidase (CP) (Figure 6).
It has been established that histone H2A and H2B play important roles in the regulation of seed dormancy [26][27][28].The epigenetic factor POWERDRESS (PWR) can interact with ABA-INSENSITIVE 3 (ABI3) and HISTONE DEACETYLASE 9 (HDA9) to reduce histone acetylation level and increase H2A deposition at the SOMNUS (SOM) locus, a positive regulator of seed dormancy, thus repressing the expression level of SOM and promoting seed germination process [29].Moreover, histone H2B monoubiquitylation regulated by HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 can increase DOG1 expression and then regulate the seed dormancy level [30][31][32].Therefore, the H2A and H2B genes exhibit significant transcriptional changes during the epigenetic regulation of seed dormancy in wheat.
Additionally, the expression level of the CP gene TraesCS6B02G050700 was significantly upregulated in germinated seeds.Carboxypeptidase is a key hydrolytic enzyme during germination in cereal grains [33,34].In wheat, CP genes were involved in the acidification of endosperm starchy [35], mobilization of endosperm storage proteins [36], and other biochemical reactions during germination processes [37].More importantly, TraesCS6B02G050700 was highly expressed specifically in grains (Figure S1), indicating that it may be the causing gene for QGp/Gi.sxau-6B.Further experiments are needed to confirm this speculation.

Analysis of QTL Alleles in Germplasms
GP and GI were tested for the 97 wheat accessions according to the method described in Section 4.2.Then, allelic variation in target QTL in germplasm was identified using the diagnostic marker to evaluate the correlation between seed dormancy and the target QTL alleles.The distribution frequency of each allele in landraces and cultivars was also counted.In addition, the fragments per kilobase of exon model per million mapped fragments (FPKM) values of high-confidence annotated genes within target QTL in germinated seeds (GS) and dormant seeds (DS) of wheat variety Mingxian169 were downloaded from published data [17], and then DEGs between GS and DS were identified on the BMKCloud platform (www.biocloud.net(accessed on 18 January 2024)) with a threshold of |log2FC| ≥ 1 and FDR ≤ 0.01 (Table S2) and selected for visualization on heat map.

Statistical Analysis
The Origin v3.1 software (OriginLab, Northampton, MA, USA) was used to perform the statistical analysis by one-way analysis of variance (ANOVA), and p < 0.05 was considered a statistically significant difference, while p < 0.01 was considered an extremely statistically significant difference.

Conclusions
This study clarified the dormant loci carried by CH1539 and obtained close-linked molecular markers.Two QTLs for GP on chromosomes 5A and 6B and four QTLs for GI on chromosomes 5A, 6B, 6D and 7A were identified.Among them, QGp/Gi.sxau-6Bcontrolling both GP and GI was a major QTL for seed dormancy with the maximum PVE of 17.66~34.11%.One PCR-based diagnostic marker Ger6B-3 for QGp/Gi.sxau-6Bwas developed, and the genetic effect of QGp/Gi.sxau-6B on the RIL population and wheat germplasm was successfully confirmed.

Figure 6 .
Figure 6.The physical position of QGp/Gi.sxau-6Bcompared with the previously reported dormancy-or PHS-associated loci on chromosome 6B.The boxes or bars indicate intervals harboring QTLs flanked by markers, and the rhombic dots indicate single markers.Differentially expressed genes between germinated seeds (GS) and dormant seeds (DS) in QGp/Gi.sxau-6Bregion are selected from published data [17] and visualized by heatmaps.

Figure 6 .
Figure 6.The physical position of QGp/Gi.sxau-6Bcompared with the previously reported dormancyor PHS-associated loci on chromosome 6B.The boxes or bars indicate intervals harboring QTLs flanked by markers, and the rhombic dots indicate single markers.Differentially expressed genes between germinated seeds (GS) and dormant seeds (DS) in QGp/Gi.sxau-6Bregion are selected from published data [17] and visualized by heatmaps.

Table 1 .
Assessment of seed dormancy for SY × CH RIL population and the parents.

Table 1 .
Assessment of seed dormancy for SY × CH RIL population and the parents.

Table 2 .
QTL mapping for GP and GI in SY × CH RIL population.

Table 2 .
QTL mapping for GP and GI in SY × CH RIL population.