Plant materials and growth conditions
The DPW and TPW lines were originally collected from Tulufan, Xinjiang province, China, by Prof. Chi Yen and Junliang Yang (Sichuan Agricultural University, China) in the 1980s. The F1 population of DPW×TPW and the F2 population (401 plants) derived from DPW×TPW were individually developed for trait investigation. Two RIL populations (F7 including 330 lines and F8 including 300 lines) derived from DPW×TPW, and a RIL population (F6 including 194 lines) derived from DPW×Jianyangailanmai (AABB, 2n = 4x = 28, T. turgidum L., Ailanmai), were developed for gene mapping. Two pairs of NILs (D_60/T_58, and D_33/T_35, D and T represent dwarf and tall phenotype, respectively) derived from two heterozygous F7 lines were selected for transcript analyses. Meanwhile, F1 plants and a F2 population (244 plants) derived from the cross of D_60 and T_58 were developed for trait investigation. The haplotype analysis was conducted using 59 tetraploid wheat accessions (Table S1).
DPW, TPW and their F1 plants and F2 population were grown at the Wenjiang experimental field of Sichuan Agricultural University, Chengdu, China, in the 2011–2012 (from October 2011 to June 2012) and 2012–2013 (from October 2012 to June 2013) wheat growing seasons. The F7 and F8 RIL populations of DPW×TPW were grown at two experimental fields (Wenjiang and Chongzhou) of Sichuan Agricultural University (Chengdu, China) in the 2017–2018 (from October 2017 to June 2018) and 2018–2019 (from October 2018 to June 2019) wheat growing seasons, respectively. The F6 RIL population, the F1 plants of D_60×T_58, two pairs of NILs, and 59 tetraploid wheat accessions were grown at the Wenjiang experimental field in the 2018–2019 (from October 2018 to June 2019) wheat growing season. The F2 population of D_60×T_58 was grown at the Wenjiang experimental field in the 2019–2020 (from October 2019 to June 2020) wheat growing season. Each line was planted with 20 plants per row. The rows were 2 m long and the spacing between rows was 30 cm.
Phenotypic measurements and analysis
Plant height, spike length, and stem length were measured at maturity. We selected three individual plants per line and calculated the average value. Data was analysed using SPSS software (version 18.0; SPSS, Chicago, IL, USA) Figures were drawn using SigmaPlot software (version 12.0; Systat, Point Richmond, CA, USA).
Homologous cloning of Rht-B1
According to the genomic sequence of T. aestivum cv. ‘Chinese Spring’ (IWGSC RefSeq v1.0), a pair of Rht-B1-specific primers (forward: 5’-CGATGCCGTC TACAACTACT-3’; reverse: 5’-CAACTCCTAGATCGGGAAACTT-3’) was designed using Beacon designer software (version 7.0; Premier Biosoft International, Palo Alto, CA, USA). These primers were used to amplify the full-length Rht-B1 sequence from DPW and TPW. Each PCR reaction mixture contained 2 μl DNA, 2 μl mixture of forward and reverse primers (4 pmol/μl), 2 μl dNTP (2.5 mM/μl), 1 μl Ex-Taq polymerase (5 U/μl), 2 μl MgCl2 (2.5 mM/μl), 2.5 μl 10× PCR buffer, and 13.5 μl ddH2O. The PCR amplification conditions were 95°C for 5 min, 40 cycles (95°C for 30 s, 58°C for 30 s, and 72°C for 2 min), and final extension at 72°C for 10 min. Each amplified fragment was cloned into the pMD19-T vector for sequencing. Differences in Rht-B1 sequences between DPW and TPW were detected in an alignment analysis using Vector NTI software (version 11.5.1; Invitrogen, Carlsbad, CA, USA).
Exploitation of indel marker of Rht-B1 for mapping
According to the sequence differences in Rht-B1 between DPW and TPW, a pair of Rht-B1-specific primers (Rht-B1 Indel-F: 5’-GGCGGGAGATCGAAGTAC-3’, Rht-B1 Indel-R: 5’-GACACCGTGCACTACAAC-3’) was designed using Beacon designer software.
Exploitation of SSR markers on 4BS for mapping
According to the genomic sequence of 4BS of T. aestivum cv. ‘Chinese Spring’ (IWGSC RefSeq v1.0) (http://plants.ensembl.org/), microsatellites were predicted using the MIcroSAtellite identification tool (https://webblast.ipk-gatersleben.de/misa/) [31-32]. Beacon designer software was used to design SSR markers (Table S2).
Genotyping and genetic mapping
Genomic DNA was extracted from DPW, TPW, Ailanmai and the mapping populations RIL6 (DPW×Ailanmai), RIL7 and RIL8 (DPW×TPW) using a plant genomic DNA kit (TIANGEN BIOTECH, Beijing, China). Each PCR reaction mixture contained 1 μl DNA, 2 μl mixture of forward and reverse primers (4 pmol/μl), 1.5 μl dNTP (2.5 mM/μl), 0.5 μl Taq polymerase (5 U/μl), 1.5 μl MgCl2 (2.5 mM/μl), 2 μl 10× PCR buffer, and 11.5 μl ddH2O. The PCR amplification conditions were 95 °C for 5 min, 35 cycles (95°C for 45 s, 58°C for 45 s, and 72°C for 45s), and final extension at 72°C for 7 min. The PCR products were separated on 8% polyacrylamide gels. The polymorphic bands between the parents were used to genotype individual lines of the mapping populations.
The Rht-B1 Indel marker and 15 polymorphic SSR markers were first used for genetic mapping of Rht-dp in the F7 RIL population. Then, Rht-B1Indel and its four flanking SSR markers (Xgpw2994.1, Xgpw3128.1, Xgpw3427.1, and Xgpw4800.1) were further used to confirm the candidate region in the F8 RIL and F6 RIL populations. The F7 RIL population was hybridized on the wheat 55K SNP array by CapitalBio Technology (Beijing, China) (unpublished data).
Linkage analysis was performed using the JoinMap software (version 4.0; Kyazma BV, Wageningen, Netherlands) with a logarithm of odds (LOD) threshold of 3.0. The Kosambi mapping function was used to convert the recombination frequencies into genetic distances (cM) .
Haplotype analysis of Rht-B1 in 59 tetraploid wheat accessions
Genomic DNA was extracted from each tetraploid wheat accession using a plant genomic DNA kit (TIANGEN BIOTECH, Beijing, China), and PCR amplification was performed as described in the section “Homologous cloning of Rht-B1”. The amino acid sequence was deduced using ExPASy software (http://web.expasy.org/ translate/). All sequences were aligned using Vector NTI software (Invitrogen). A phylogenetic tree was constructed using the neighbour-joining algorithm in MEGA5 (https://www.megasoftware.net/).
Expression analysis of Rht-B1b
Tissues at the three growth stages (jointing, booting, and grain filling stages) were collected, including roots, basal stems, leaf sheaths, leaf blades, young leaves, lower leaf blades, first and second internodes, flag leafs, and spikes. The collected tissues were snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Total RNA was extracted using a Plant RNA Kit (Omega Bio-Tek, American). cDNA was synthesized using the M-MLV First Strand cDNA Synthesis kit (Invitrogen).
Quantitative real-time PCR (qPCR) was performed on the CFX-96 system as described by Wang et al. using a pair of Rht-B1b-specific primers (forward: 5′-GGCGGGAGATCGAAGTAC-3′; reverse: 5′-GACACCGTGCACTACAAC-3′) . To normalize gene expression levels, the Actin gene was used as the reference gene . Relative expression levels were calculated according to the 2ΔΔCt method using the CFX Manager (version 3.1; Bio-Rad, Hercules, CA, USA).
Transcript analysis of two pairs of NILs
At the booting stage, the first internode was collected individually from two pairs of NILs, and then snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction.
RNA extraction, library preparation and sequencing
Total RNA was isolated as described above, and RNA degradation and contamination were monitored on 1% agarose gels. A NanoPhotometer® spectrophotometer (Implen GmbH, Munich, Germany) RNA purity was used to check RNA purity. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and divided into short fragments using NEBNext First Strand Synthesis Reaction Buffer (5×) (New England Biolabs, Ipswich, MA, USA). The cDNA was synthesized using the fragments as templates and then purified and resolved with EB buffer for the end-repair step and addition of a single adenine (A) nucleotide. To select cDNA fragments 250~300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, CA, USA), and suitable fragments were chosen for a PCR amplification. The PCR products were purified (AMPure XP system) and the library quality was assessed using the Agilent Bioanalyzer 2100 system. The prepared libraries were sequenced on the Illumina Hiseq platform.
RNA-seq data analysis
Raw data (raw reads) of in fastq format were first processed using in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly-N, and low-quality reads from the raw data. All the downstream analyses were conducted using clean, high-quality data.
The Chinese Spring (IWGSC RefSeq v1.0) reference genome and gene model annotation files were downloaded from the genome website (https://urgi.versailles. inra.fr/download/iwgsc/IWGSC_RefSeq_Assemblies/v1.0). The D genome sequences were excluded from the reference before mapping the processed reads of the tetraploid lines (A and B genomes). An index of the Chinese Spring reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count the number of reads mapped to each gene. The mean fragments per kilobase of transcript per million mapped reads (FPKM) value for each gene was calculated based on the length of the gene and the number of reads mapped to it .
Differential expression analysis
Read counts were adjusted by the edgeR program package through one scaling normalized factor. Analysis of differential gene expression between two pairs of NILs (D33/T35 and D60/T58) was performed using the DEGSeq R package. The P values were adjusted using the Benjamini and Hochberg method. A corrected P-value of 0.005 and log2 (fold change) of 1 were set as the thresholds for significantly different gene expression.
QPCR for validation
Two differentially expressed genes Auxin-repressed protein (ARP) and L-ascorbate oxidase homolog (ASCO) from RNA-Seq were verified by qPCR, and their gene-specific primers sequences were APR (forward: 5′-ATTAAGCAGTCGCCG TCGAT-3′; reverse: 5′-TCGCTGTAAAGCCAG TCGTA -3′) and ASCO (forward: 5′-AATGGCAATAGGTTCACAGTAGA-3′; reverse: 5′-CTTCACGAGGAACGAGT AGG-3′), respectively.