Identification of the Solid Stem Suppressor Gene Su-TdDof in Synthetic Hexaploid Wheat Syn-SAU-117

Lodging is one of the most important factors affecting the high and stable yield of wheat worldwide. Solid-stemmed wheat has higher stem strength and lodging resistance than hollow-stemmed wheat does. There are many solid-stemmed varieties, landraces, and old varieties of durum wheat. However, the transfer of solid stem genes from durum wheat is suppressed by a suppressor gene located on chromosome 3D in common wheat, and only hollow-stemmed lines have been created. However, synthetic hexaploid wheat can serve as a bridge for transferring solid stem genes from tetraploid wheat to common wheat. In this study, the F1, F2, and F2:3 generations of a cross between solid-stemmed Syn-SAU-119 and semisolid-stemmed Syn-SAU-117 were developed. A single dominant gene, which was tentatively designated Su-TdDof and suppresses stem solidity, was identified in synthetic hexaploid wheat Syn-SAU-117 by using genetic analysis. By using bulked segregant RNA-seq (BSR-seq) analysis, Su-TdDof was mapped to chromosome 7DS and flanked by markers KASP-669 and KASP-1055 within a 4.53 cM genetic interval corresponding to 3.86 Mb and 2.29 Mb physical regions in the Chinese Spring (IWGSC RefSeq v1.1) and Ae. tauschii (AL8/78 v4.0) genomes, respectively, in which three genes related to solid stem development were annotated. Su-TdDof differed from a previously reported solid stem suppressor gene based on its origin and position. Su-TdDof would provide a valuable example for research on the suppression phenomenon. The flanking markers developed in this study might be useful for screening Ae. tauschii accessions with no suppressor gene (Su-TdDof) to develop more synthetic hexaploid wheat lines for the breeding of lodging resistance in wheat and further cloning the suppressor gene Su-TdDof.


Introduction
Wheat is the largest cereal crop in the world, accounting for 220 million hectares with annual global production of 772 million tons [1].The world's population is expected to increase by nearly 2 billion over the next 30 years [2], and there will be greater demand for global wheat production.Therefore, we must strive to increase wheat yield.However, with the continuous improvement of wheat yield and the increase in fertilizer application, lodging has become an important factor affecting the high and stable yield of wheat [3][4][5][6][7].Plant dwarfing can effectively alleviate the lodging damage of wheat [8]; however, severe dwarfism leads to inadequate biomass accumulation and lower yield potential [9].Increasing the mechanical strength of the stem is another breeding strategy [3].The ratio of stem wall thickness to stem diameter and the content of mechanical tissue in solid-stemmed wheat were found to be significantly higher than those in common wheat [10][11][12].Therefore, compared with common wheat, solid-stemmed wheat has higher stem strength and lodging resistance, which play an important role in wheat lodging resistance breeding [11].
To date, two candidate genes, TraesCS3B01G608800 and TRITD3BV1G280530, have been reported for stem solidity in hexaploid wheat and durum wheat, respectively.The differentially expressed gene TraesCS3B01G608800 (KAF7034036.1)showed copy number variations associated with stem solidity in different hexaploid wheat cultivars [13].However, TRITD3BV1G280530 was confirmed as a candidate gene in SSt1 in durum wheat, and the copy numbers of TRITD3BV1G280530 in solid-stemmed and hollow-stemmed durum wheat were different.The protein encoded by TRITD3BV1G280530 is a zinc finger protein, and its physical location is 829.1 Mb on chromosome 3BL of the durum wheat genome v1.0 [14].Liu et al. [15] found that a QTL for pith thickness in wheat was previously discovered on 3BL in a double haploid population of 'Westonia' × 'Kauz'.A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential pith-thickness candidate gene in Australian 'Westonia' wheat [15].
There are many solid-stemmed varieties, landraces, and old varieties in durum wheat (Triticum turgidum L. ssp.durum, AABB, 2n = 4x = 28) [16].Durum wheat has greater stem firmness and more stable genetic characteristics than those of common wheat varieties [17][18][19].Beginning in the 1940s, breeders tried to transfer the solid stem gene from Golden Ball to hexaploid wheat by crossing, but the solid stem trait was suppressed, and only hollow-stemmed lines were created [17,20,21].Then, it was found that the expression of the solid stem gene was suppressed, and the suppressor gene was presumed to be located on chromosome 3D in common wheat [17].Suppression is a common phenomenon in nature.For example, suppression of disease resistance is especially frequent in the expression genes for resistance to fungal plant pathogens causing stem rust, leaf rust, and powdery mildew [22][23][24][25][26][27][28].Inhibition often occurs, especially during the transfer of foreign genes from diploid and tetraploid ancestors to hexaploid wheat [22][23][24]27,29,30].
Synthetic hexaploid wheat was an effective bridge for transferring the solid stem gene from durum wheat to hexaploid wheat.A synthetic hexaploid wheat (P89-77-1 F 4 ) derived from crosses between solid-stemmed Golden Ball durum wheat and Ae.squarrosa L. expressed pith in the culm lumen [31].Then, two solid-stemmed hexaploid spring wheat lines (PI 633,737 and PI 633,738) were developed and released by backcrossing P89-77-1 F 4 with the hollow-stemmed hexaploid wheat cultivar AC Elsa [31,32].In our previous study, the expression of a solid second internode at the base of the stem was stable for two synthetic hexaploid wheat lines (Syn-SAU-117 and Syn-SAU-119), which were developed from a cross of the semidwarf solid-stemmed durum wheat line Ma as the female parent and two different Ae.tauschii accessions (AS92 and AS96) as the male parent [33].The second internode at the base of the stem of Syn-SAU-117 (pedigree: Ma × AS92) was semisolid, while that of Syn-SAU-119 (pedigree: Ma × AS96) was solid in both the greenhouse and field.It was indicated that the D genome of Ae. tauschii AS92 suppressed the expression of the solid stem gene from the 3B chromosome in Syn-SAU-117 [33].The objective of this study was to identify and map the solid stem suppressor gene in Syn-SAU-117 by using bulked segregant RNA-seq (BSR-seq) analysis.

Differential Expression of the TdDof Gene in Different Materials
The gene expression levels of TdDof were determined in two synthetic wheat cultivars, Syn-SAU-117 and Syn-SAU-119, and two durum wheat cultivars, Ma (solid) and Cocorit (hollow), as solid-and hollow-stemmed contrasts during early internode elongation (Zadoks stage 32 and Zadoks stage 34).The expression level of TdDof was higher in Syn-SAU-119 than in Syn-SAU-117 in both periods (Figure 1a,b).

Differential Expression of the TdDof Gene in Different Materials
The gene expression levels of TdDof were determined in two synthetic wheat cultivars, Syn-SAU-117 and Syn-SAU-119, and two durum wheat cultivars, Ma (solid) and Cocorit (hollow), as solid-and hollow-stemmed contrasts during early internode elongation (Zadoks stage 32 and Zadoks stage 34).The expression level of TdDof was higher in Syn-SAU-119 than in Syn-SAU-117 in both periods (Figure 1a,b).

BSR-Seq Analysis of the RNA of Bulks with Contrasting Stem Solidity
The RNA samples of the solid bulk and the semisolid bulk were subjected to RNAseq analysis, which generated 56,746,340 and 82,341,750 raw reads, respectively.After quality control, 56,740,782 and 82,334,750 high-quality reads from the solid bulk and semisolid bulk, respectively, were uniquely mapped to the Chinese Spring genome (IWGSC RefSeq v1.1).A total of 8581 SNPs (p < 1 × 10 −8 and AFD > 0.6) were identified from these reads by using the GATK v4.0 software (Figure 4).One hundred and twenty-three SNPs were located in an 8 Mb genomic interval (4-11 Mb) on the short arm of chromosome 7D in the Chinese Spring genome (IWGSC RefSeq v1.1), and these were regarded as candidate SNPs linked to Su-TdDof (Figure 5).

BSR-Seq Analysis of the RNA of Bulks with Contrasting Stem Solidity
The RNA samples of the solid bulk and the semisolid bulk were subjected to RNA-seq analysis, which generated 56,746,340 and 82,341,750 raw reads, respectively.After quality control, 56,740,782 and 82,334,750 high-quality reads from the solid bulk and semisolid bulk, respectively, were uniquely mapped to the Chinese Spring genome (IWGSC RefSeq v1.1).A total of 8581 SNPs (p < 1 × 10 −8 and AFD > 0.6) were identified from these reads by using the GATK v4.0 software (Figure 4).One hundred and twenty-three SNPs were located in an 8 Mb genomic interval (4-11 Mb) on the short arm of chromosome 7D in the Chinese Spring genome (IWGSC RefSeq v1.1), and these were regarded as candidate SNPs linked to Su-TdDof (Figure 5).

Molecular Mapping of Su-TdDof
Fifty-one out of the 123 clustered SNPs on 7DS were chosen to develop KASP markers.Four of them were successfully converted into KASP markers (KASP-533, KASP-669, KASP-1055, KASP-1166) (Table 2) and scored reliably on the parents, as well as the solid and semisolid bulks.All tested markers exhibited identical haplotypes between Syn-SAU-117 and its male parent AS92 but were distinct from those of Syn-SAU-119 and its male parent AS96 (Table 3).Subsequently, these KASP markers were used to genotype 134 F 2 plants derived from the cross between solid-stemmed Syn-SAU-119 and semisolid-stemmed Syn-SAU-117 plants.Linkage analysis indicated that KASP-669 was potentially mapped 1.88 cM distally, and KASP-1055 was placed 2.65 cM proximally to Su-TdDof (Figure 6).

Molecular Mapping of Su-TdDof
Fifty-one out of the 123 clustered SNPs on 7DS were chosen to develop KASP markers.Four of them were successfully converted into KASP markers (KASP-533, KASP-669,

Molecular Mapping of Su-TdDof
Fifty-one out of the 123 clustered SNPs on 7DS were chosen to develop KASP markers.Four of them were successfully converted into KASP markers (KASP-533, KASP-669,

Gene Analysis of the Genomic Region of Su-TdDof
The sequences of the closely linked markers KASP-669 and KASP-1055 were blasted against the Chinese Spring genome and the Ae.tauschii genome to obtain their physical positions.Su-TdDof was physically mapped to a 3.86 Mb region between the 6.69 Mb and 10.55 Mb regions of the Chinese Spring 7DS chromosome (IWGSC RefSeq v1.1) and be-

Gene Analysis of the Genomic Region of Su-TdDof
The sequences of the closely linked markers KASP-669 and KASP-1055 were blasted against the Chinese Spring genome and the Ae.tauschii genome to obtain their physical positions.Su-TdDof was physically mapped to a 3.86 Mb region between the 6.69 Mb and 10.55 Mb regions of the Chinese Spring 7DS chromosome (IWGSC RefSeq v1.1) and between the 6.58 Mb to 8.87 Mb regions (2.29 Mb) in the Ae.tauschii AL8/78 7DS chromosome (Ae.tauschii AL8/78 v4.0) (Figure 6).There were 180 and 125 predicted genes in the target physical regions in Chinese Spring and Ae.tauschii AL8/78, respectively (IWGSC RefSeq v1.1; Ae. tauschii AL8/78 v4.0; Supplementary Tables S1 and S2).In the Chinese Spring genome, ten genes may be associated with the growth and development of plant stems, including six zinc-finger-protein-related genes (TraesCS7D02G015800, TraesCS7D02G019600LC, TraesCS7D02G022000LC, TraesCS7D02G023300LC, TraesCS7D02G023400LC, TraesCS7D02G-024900LC), two biofunction inhibitor genes (TraesCS7D02G020000, TraesCS7D02G020100), one pectin lyase-like superfamily protein gene (TraesCS7D02G016300), and one homeoboxlike protein BEL1 gene (TraesCS7D02G019800).Five genes, including two zinc-finger-proteinrelated genes (AET7Gv20034800, AET7Gv20042400), one 36.4kDa proline-rich protein gene (AET7Gv20040700), one transcription factor gene (AET7Gv20036400), and one homeoboxlike protein BEL1 gene, were found in the Ae.tauschii genome, which had a good collinear relationship with those of Chinese Spring (Figure S1).Transcriptome analysis revealed a total of 43,362 differentially expressed genes, including 341 upregulated genes, 227 downregulated genes, and 42,794 nondifferentially expressed genes (Figure 7).There were 12 and 7 significantly differentially expressed genes in the target physical regions in the Chinese Spring genome and Ae.tauschii genome (Supplementary Tables S3 and S4, Figure S1).Among them, the annotations of TraesCS7D02G016300, AET7Gv20040000, and AET7Gv20040700 were probably associated with the growth and development of plant pith [14,[35][36][37][38][39][40][41].TraesCS7D02G016300 was a gene encoding pectin lyase superfamily proteins (PG) that acted on pectin and lignin in the cell wall and promoted cell wall degradation and shedding, thus promoting cell apoptosis (Supplementary Table S3).The homologous genes of TraesCS7D02G016300 were two polygalacturonase genes (AET7Gv20034200 and AET7Gv20034900) in the Ae.tauschii genome (Ae.tauschii AL8/78 v4.0), and their gene annotations were not correlated with the inhibition of solid stems (Supplementary Tables S3 and S4).AET7Gv20040700 and AET7Gv20040000 in the Ae.tauschii genome (Ae.tauschii AL8/78 v4.0) had a good collinear relationship with those of Chinese Spring (Figure S1).AET7Gv20040000 was annotated as a homeobox-like protein BEL1 gene (Supplementary Table S4).The homologous gene of AET7Gv20040000 in Chinese Spring was TraesCS7D02G019800, and its functional annotation was a homeobox-like protein BEL1 gene (Supplementary Table S1), which was the same as AET7Gv20040000 in the Ae.tauschii AL8/78 genome.AET7Gv20040700 was annotated as a 36.4 kDa proline-rich protein gene (Supplementary Table S4).The homologous gene of AET7Gv20040700 in Chinese Spring was TraesCS7D02G020100, and its functional annotation was that of a bifunctional (protease/α-amylase) inhibitor/plant lipid transfer protein/seed storage helical domain (Supplementary Table S1).
protein BEL1 gene (Supplementary Table S1), which was the same as AET7Gv20040000 in the Ae.tauschii AL8/78 genome.AET7Gv20040700 was annotated as a 36.4 kDa prolinerich protein gene (Supplementary Table S4).The homologous gene of AET7Gv20040700 in Chinese Spring was TraesCS7D02G020100, and its functional annotation was that of a bifunctional (protease/α-amylase) inhibitor/plant lipid transfer protein/seed storage helical domain (Supplementary Table S1).

Discussion
To date, lodging is still a problem in wheat-growing regions worldwide, despite scientists having made great efforts to solve it for many years.The selection of excellent germplasms with alternative semidwarf genes or good stem mechanical strength may be an effective way to solve this problem [42].Solid-stemmed wheat has strong lodging resistance due to its higher stalk strength [11,43].The pith is composed of undifferentiated parenchymatous cells in the solid stems, which accounted for 11% of the total stem dry weight 10-14 days after anthesis and contributed 13% of ethanol-soluble carbohydrates in the entire stem [44].Water-soluble carbohydrates (WSCs), including sugars, such as fructans, sucrose, glucose, and fructose, are stored in the stems [45,46], and they could be remobilized and transported to the developing grains [47], thus playing an important role in buffering grain yield [48,49]. Stem solidness was reported to be negatively correlated with yield in a few older reports [50,51].However, later studies showed no such association of the variation of solid stem traits with a decrease in wheat yield [52].Moreover, the development of high-yielding solid-stemmed cultivars is not related to the degree of stem solidness [53][54][55][56][57].
Durum wheat has many solid-stemmed varieties, landraces, and old varieties [16].However, attempts to transfer solid stem genes to hexaploid wheat via direct crossing have been unsuccessful because the expression of solid stem genes is suppressed by the suppressor gene on chromosome 3D in common wheat [10,20,21].In this study, a new solid stem suppressor gene, Su-TdDof, was identified in the synthetic hexaploid wheat Syn-SAU-117 and mapped on chromosome arm 7DS; it was flanked by the markers KASP-669 and KASP-1055 within a 4.53 cM genetic interval corresponding to the 3.86 Mb physical region in the Chinese Spring genome (IWGSC RefSeq v1.1).In addition, the expression of the solid stem gene TdDof in Syn-SAU-117 was lower than that in Syn-SAU-119, thus confirming the existence of the solid stem suppressor gene Su-TdDof.
In common wheat, the existence of many suppressor genes affects the normal expression of some important genes and the utilization of excellent foreign genes [28,43,[58][59][60][61][62].To date, many disease resistance genes and corresponding suppressor genes have been found in the D genome of common wheat and the D genome donor Ae. tauschii [43,61,63,64], such as the leaf rust suppressor gene Su-Lr23 on chromosome 2DS [62] and the stem rust suppressor gene SuSr-D1 on chromosome 7DL of the hexaploid wheat cultivar 'Canthatch' (CTH) [65][66][67][68].A recent study showed that the gene SuSr-D1 encoded Med15, a subunit of the Mediator complex that suppressed the expression of stem rust resistance [28].In the present study, Su-TdDof was from Ae. tauschii, which was different from the suppressor gene presumed to be located on chromosome 3D in common wheat found by Larson et al. [10].
Su-TdDof was physically mapped to the region between 6.58 Mb and 8.87 Mb (2.29 Mb) on the Ae.tauschii AL8/78 7DS chromosome (Ae.tauschii AL8/78 v4.0) (Figure 6).Based on the gene functional annotation and screening of differentially expressed genes in the transcriptome, there were two protein-coding genes, AET7G20040000 and AET7Gv20040700, in the target physical regions in the Ae.tauschii genome (Supplementary Tables S3 and S4, Figure S1).AET7Gv20040000 was annotated as a homeobox-like protein BEL1 gene, and the homologous gene of AET7Gv20040000 in Chinese Spring was TraesCS7D02G019800.Its functional annotation was the same as that of AET7Gv20040000.The genes of the BEL1 protein family play an important role in the growth and development of plant stems, leaves, flowers, and other organs [36,38].For example, in Arabidopsis thaliana, the specific interaction between the BEL1 protein-like family genes BLH6 and KNAT7 inhibits the transcription factor REVOLUTA (REV), affecting growth and development in the stem of Arabidopsis inflorescences and, thereby, regulating secondary cell wall development [37].AET7Gv20040700 was annotated as a 36.4 kDa proline-rich protein gene, and the homologous gene of AET7Gv20040700 in Chinese Spring was TraesCS7D02G020100.Its functional annotation was that of a bifunctional (protease/α-amylase) inhibitor/plant lipid transfer protein/seed storage helical domain.Studies have shown that the formation of pith in the stem is related to starch [14,35,39].AET7Gv20040700 may inhibit the hydrolysis of starch and affect the formation of pith.These two genes, AET7Gv20040000 and AET7Gv20040700, will be cloned and sequenced in future studies to further develop markers for verification.
During the introduction of foreign genes into common wheat, with the increase in ploidy, the expression of superior genes decreased or was completely inhibited because of the existence of suppressor genes [43,58].Therefore, exploring new suppressor genes, screening accessions without suppressor genes, or carrying out artificial mutation of suppressor genes can enable breeders to break through this restriction and provide beneficial help for the introduction of foreign genes into common wheat [69].The flanking markers KASP-669 and KASP-1055 developed in this study could be used as molecular markers to screen recombinant heterozygous plants, construct secondary F 2 populations and develop markers, and further narrow the location interval to finely map and clone Su-TdDof.The flanking markers KASP-669 and KASP-1055 were also used to screen Ae. tauschii accessions with no suppressor gene (Su-TdDof) to develop more synthetic hexaploid wheat lines with solid stems for the breeding of lodging resistance.Solid-stemmed synthetic hexaploid wheat can be used as a bridge to cross with elite wheat cultivars [70].Combined with molecular-marker-assisted selection, the transfer of solid stem genes from tetraploid wheat into common wheat cultivars and the breeding of new wheat cultivars with solid stems will provide new materials for the breeding of lodging resistance in wheat.

Plant Materials
Two synthetic hexaploid wheat lines (Syn-SAU-117 and Syn-SAU-119), two different durum wheats (Ma and Cocorit), and two different Ae.tauschii (2n = 2x = 14, DD) accessions (AS92 and AS96) were used in this study.Syn-SAU-117 and Syn-SAU-119 were generated via natural chromosome doubling of Ma × AS92 F 1 and Ma × AS96 F 1 , respectively.Syn-SAU-117 and Syn-SAU-119 were identified by using FISH with the oligonucleotide probes Oligo-pSc119.2-1and Oligo-pTa535-1 [33].Plants with 42 chromosomes were used in this study.The durum wheats Ma (solid-stem) and Cocorit (hollow-stem) were supplied by George Fedak at the Ottawa Research and Development Center for Agriculture and Agri-Food (Ottawa, ON, Canada).The lines with the AS code were stored in our institute.All materials used in this study were kept at the Triticeae Research Institute of Sichuan Agricultural University.

Population Construction and Phenotypic Investigation
Two synthetic hexaploid wheat lines were sown in the greenhouse in July 2020, and Syn-SAU-117/Syn-SAU-119 F 1 plants were subsequently generated.These F 1 seeds were sown in a greenhouse in March 2021.Syn-SAU-117/Syn-SAU-119 F 2 seeds were sown in the greenhouse in July 2021.Syn-SAU-117/Syn-SAU-119 F 2:3 plants were sown in the field in November 2021.Each plant was 10 cm apart within rows, 30 cm apart between rows, and 1.5 m in length.The stems were sampled according to the method of Kong et al. [11].More than ten stems from the main tiller were randomly selected after flowering and were cross-sectionally cut at the center of each internode.The level of stem solidity was rated as 1-5 (1 for hollow and 5 for solid) following Pauw et al. [34].

Observation of the Anatomical Structures of Stems
The internodes were numbered consecutively from the base to the top of the stem.At the jointing stage, the main tiller was selected.The center of the second internode of the wheat stem base was cut into 1 cm pieces and then soaked in FAA fixative for more than 24 h [43].The samples were sent to Wuhan CVI Biotechnology Co., Ltd.(Wuhan, China) (https://www.servicebio.cn/accessed on 10 July 2022) for the preparation of paraffin sections.CaseViewer 2.3 (https://www.3dhistech.com/solutions/caseviewer/accessed on 20 July 2022, Budapest, Hungary) was used to view the results of the paraffin section analysis.

Solid Stem Gene Expression Analysis
A quantitative reverse transcription polymerase chain reaction (qRT-PCR) system (Bio-Rad, Shanghai, China) was used to analyze the gene expression of TdDof [13].The D1 probe primers (D1_F: GTTCCTGCACGCCATGGAC; D1_R: TCCCCCATCGTCGCCATTA) were specifically designed to distinguish differences in expression levels between different plants, and the housekeeping gene GAPDH was used as a reference for gene expression analysis.The main stems of three plants were sampled at Zadoks Stage 32 and Zadoks Stage 34 when the first two and four nodes were present on the stem.Approximately 0.5 cm of the stem was sampled, measuring from the bottom of the lowermost node toward the uppermost node.Total RNA extraction was performed by using the Tiangen DP441 RNA prep Pure RNA Extraction Kit according to the manufacturer's protocol.The quality of RNA was assessed by using polyacrylamide gel electrophoresis, and RNA reverse transcription was performed by using the Fermentas K1622 RT Reverse Transcription Kit (Thermo Scientific, Waltham, MA, USA).

Bulked Segregant RNA-Seq (BSR-Seq)
Solid and semisolid RNA pools for RNA-Seq were constructed by using the F 2 generations with different stem solidity in the greenhouse.Equal amounts of RNA from 20 homozygous solid-stemmed and 20 semisolid-stemmed generations were pooled to conduct bulked segregant analysis [71].The RNA samples were sequenced on the platform of Chengdu Tiancheng Future Technology Co., Ltd.(https://www.tcuni.com/,accessed on 27 December 2021).Sequence quality control was performed by using the fastp software v0.19.5 [72].RNA reads of the solid stem and semisolid stem bulks were aligned to the reference genome sequence of Chinese Spring v1.1 [13] and Ae.tauschii AL8/78 v4.0 by using the STARv2.5.1b software [73].The unique and confident alignments were applied to call SNP variants by using the GATK v3.6 software [74].The SNP variants with p-values from Fisher's exact test (FET) of <1 × 10 −8 and an allele frequency difference (AFD) of >0.6 were considered to be associated with solid stem suppression and further used as templates for developing SNP markers [71].

Kompetitive Allele-Specific PCR (KASP) Assays
The solidity-related SNPs and the 500 bp flanking sequences were used to design the KASP primers and test polymorphisms on the parental lines and the solid and semisolid stem DNA bulks.Polymorphic markers that could be reliably scored were genotyped on the F 2 population of Syn-SAU-117 × Syn-SAU-119.For each KASP assay, a 10 µL reaction volume containing 5 µL of 2 KASP master mix (Biosearch Technologies, Shanghai, China), 1.4 µL of primer mix (mixture of 0.168 µM each forward A1 and A2 primers and 0.42 µM of reverse primer), 100 ng of genomic DNA, and 2.6 µL of ddH 2 O was prepared.The CFX96Touch™ real-time PCR detection system (Bio-Rad, USA) was used for amplification under the following conditions: 15 min at 94 • C, 10 touchdown cycles of 20 s at 94 • C, 60 s at 65-57 • C (decreasing by 0.8 • C per cycle), and 32 cycles of 20 s at 94 • C, followed by 60 s at 57 • C.

Data Analysis
Chi-square (χ 2 ) tests were used to determine the goodness of fit for the observed segregation and expected ratios of the F 2 and F 2:3 populations.Linkage analysis was performed by using MAPMAKER/EXP v3.0b [75].The Kosambi function was used to convert recombination values into genetic distances [76].A logarithmic odds (LOD) ratio of 3.0 and a maximum distance of 50.0 cM were set as thresholds for the declaration of linkage.The genetic linkage map was drawn by using the Mapdraw v2.1 software [77].

Candidate Gene Analysis
The sequences of the KASP-669 and KASP-1055 markers linked to Su-TdDof were used for BLAST against the genomes of Chinese Spring v1.1 [13] and Ae.tauschii AL8/78 v4.0 [78].Gene annotations between the flanking markers of the two genomes were retrieved from the Ensembl Plants (http://plants.ensembl.org/index.html,accessed on 12 November 2022) and Swiss-Prot (http://www.gpm-aw.com/html/swi-ss-prot.html,accessed on 12 November 2022) databases.Furthermore, the differentially expressed genes within the interval were screened and analyzed based on the results of RNA-seq with the screening criteria of FDR < 0.05 and |LogFC| > 1. Collinearity analysis was performed on the differentially expressed genes related to the function of solid stems among parents and mixed pools.

Figure 1 .
Figure 1.The expression differences of the TdDof gene in Ma, Syn-SAU-119, Syn-SAU-117, and Cocorit at Zadoks Stage 32 and Zadoks Stage 34: (a) expression differences in the TdDof gene at the Zadoks Stage 32; (b) expression differences in the TdDof gene at the Zadoks Stage 34.

Figure 1 .
Figure 1.The expression differences of the TdDof gene in Ma, Syn-SAU-119, Syn-SAU-117, and Cocorit at Zadoks Stage 32 and Zadoks Stage 34: (a) expression differences in the TdDof gene at the Zadoks Stage 32; (b) expression differences in the TdDof gene at the Zadoks Stage 34.

Figure 5 .
Figure 5.The enrichment of SNPs within a window size of 1 Mb on wheat chromosomes.The green arrow indicates the high-density SNP enrichment that occurred on chromosome 7DS.

Figure 5 .
Figure 5.The enrichment of SNPs within a window size of 1 Mb on wheat chromosomes.The green arrow indicates the high-density SNP enrichment that occurred on chromosome 7DS.

Figure 5 .
Figure 5.The enrichment of SNPs within a window size of 1 Mb on wheat chromosomes.The green arrow indicates the high-density SNP enrichment that occurred on chromosome 7DS.

Figure 6 .
Figure 6.Genetic linkage map of the Su-TdDof gene on chromosome 7DS showing the physical location of Su-TdDof: (a) linkage map of Su-TdDof; (b) the physical interval (blue part) where the four KASP markers linked to Su-TdDof were anchored in Chinese Spring, with orange dots represent centromeres, do ed lines indicating the physical positions of each marker; (c) physical intervals anchored by markers linked to Su-TdDof in Aegilops tauschii.

Figure 6 .
Figure 6.Genetic linkage map of the Su-TdDof gene on chromosome 7DS showing the physical location of Su-TdDof : (a) linkage map of Su-TdDof ; (b) the physical interval (blue part) where the four KASP markers linked to Su-TdDof were anchored in Chinese Spring, with orange dots represent centromeres, dotted lines indicating the physical positions of each marker; (c) physical intervals anchored by markers linked to Su-TdDof in Aegilops tauschii.

Figure 7 .
Figure 7. Volcano map of differentially expressed genes.Figure 7. Volcano map of differentially expressed genes.

Figure 7 .
Figure 7. Volcano map of differentially expressed genes.Figure 7. Volcano map of differentially expressed genes.

Table 2 .
Primer sequences of KASP markers used for the genetic mapping of Su-TdDof.
a AA, CC, GG, and TT represent the haplotype results of SNP genotyping.Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 7 of 15