Phenotype of plants harbouring Rht-dp
The average heights of DPW and TPW were 91.52 ± 2.97 cm and 189.88 ± 1.72 cm, respectively. No significant difference of plant heights between F1 plants (179.12 ± 3.65 cm) and TPW was observed (Fig S1). The plant heights of F2 plants ranged from 65 to 185 cm. According to the frequency distribution of plant height, F2 plants were separated into two groups of dwarf and tall phenotypes at 110 cm (Fig. 1A). The dwarf and tall phenotype groups included 107 and 294 plants, respectively, consistent with the expected Mendelian segregation ratio of 1:3 (t = 0.606, P = 3.841, α = 0.05). These results validate that Rht-dp should be a major recessive gene. However, the separated threshold of plant height with 110 cm was significant larger than the plant height of DPW with 91.52 ± 2.97 cm, which implied that the effect of Rht-dp on reducing plant height might be partially covered by one or more non-allelic loci.
To fine-map Rht-dp, two RIL populations including 330 F7 and 300 F8 plants were constructed. The plant heights of F7 and F8 plants ranged from 65 to 165 cm (Fig. 1B) and from 65 to 170 cm (Fig. 1C), respectively. For F7 population, the average heights of dwarf and tall phenotypes were 84.07 ± 1.97 cm and 133.75 ± 2.01 cm, respectively. Compared with the tall phenotype, the lines harbouring Rht-dp showed a reduction in plant height of up to 37.14%. The reduced plant height was because of the shortened first internode (by 14.83%), second internode (by 7.15%), and basal internode (by 1.46%), but the length of the spike was not affected (Fig. 1D). These results indicate that Rht-dp reduces plant height mainly by restricting elongation of the first and second internodes at the booting stage.
To validate the candidate region of Rht-dp in a different genetic background, an F6 RIL population including 194 lines derived from DPW × Ailanmai was constructed. The average height of Ailanmai was 100.98 ± 0.37 cm. Ailanmai has a recessive dwarfing gene Rht22, which has an additive effect with Rht-dp. The RIL population was grouped into dwarf and tall phenotypes with heights ranging from 20 to 60 cm and from 120 to 160 cm, respectively (Fig. 1E).
Differences in sequence of Rht-B1 between DPW and TPW
To test the hypothesis that the candidate gene of Rht-dp is Rht-B1b or one of its alleles, the sequences of Rht-B1 were cloned from DPW and TPW. Sequence analysis showed that Rht-B1 of DPW is Rht-B1b, with a single nucleotide change from C to T at the nucleotide position 190 when compared with Rht-B1a (Fig. 2A) that results in a premature termination codon at amino acid position 64 (Fig. 2B). Although Rht-B1 of TPW did not have this single nucleotide change from C to T at nucleotide position 190, it had a three-nucleotide deletion at nucleotide position 386–388 when compared with Rht-B1a (Fig. 2A), resulting in a serine (S) deletion at amino acid position 129 (Fig. 2B). These results imply that the candidate gene of Rht-dp might be Rht-B1b. An Rht-B1 Indel marker was developed from the three-nucleotide deletion of Rht-B1 in TPW for further analysis.
Mapping of Rht-dp
To confirm that the candidate gene of Rht-dp is Rht-B1b, the Rht-B1Indel marker was first used to determine whether Rht-B1 was tightly linked with Rht-dp. Genetic mapping analyses confirmed that the Rht-B1Indel marker completely co-segregated with Rht-dp in three RIL populations (Fig. 3).
To further confirm that Rht-B1b is located in the candidate region of Rht-dp, 190 pairs of SSR markers were exploited according to the genome reference of 4BS (Table S2). Fifteen pairs of SSR markers exhibited polymorphism between DPW and TPW, and were linked with Rht-dp in the F7 RIL population. Of them, two SSR markers, Xgpw2994.1 and Xgpw3128.1, were tightly linked with Rht-dp with a genetic distance of 0.6 cM (Fig. 3A). Xgpw2994.1 and Xgpw3128.1 were confirmed as tightly linked markers flanking Rht-dp in the F6 (Fig. 3B) and F8 (Fig. 3C) RIL populations.
Based on the gene annotation of wheat 4BS from 29.94 to 31.29 Mbp, flanked by Xgpw2994.1 and Xgpw3128.1, there were five potential genes: TraesCS4B01G042700 (encodes a teosinte branched 1 protein), TraesCS4B01G042800 (encodes an uncharacterized protein), TraesCS4B01G042900 (a RING finger protein), TraesCS4B01G043000 (EamA-like transporter family), and TraesCS4B01G043100 (Rht-B1 encodes a DELLA protein) (Fig. 3D). Except of Rht-B1, sequence difference of other four genes between DPW and TPW was not found. These results implied that the candidate gene of Rht-dp should be Rht-B1b.
Characterization of Rht-dp in F1 plants and F2 population derived from the cross of a pair of NIL
Although the current data supported that the candidate gene of Rht-dp should be Rht-B1b, there was a discrepancy that Rht-dp is a recessive dwarfing gene resulted from the genetic analysis of F1 and F2 of DPW × TPW, but Rht-B1b is a semi-dominant dwarfing gene [6]. Additionally, the threshold of plant height separated dwarf and tall plants in F2 population was larger than the plant height of DPW. The information promoted us to do a hypothesis that the effect of Rht-dp on reducing plant height was probably influenced by one or more non-allelic loci derived from TPW. To test this hypothesis, a QTL analysis was performed on F7 RIL population using the Wheat 55K SNP Array. Except of a major-locus on 4BS (Rht-dp) derived from DPW caused dwarfism, a micro-locus on 5A derived from TPW heightened plant was detected (unpublished data). To further confirm the information of Rht-dp, we measured the plant height of F1 plants and F2 population derived from the cross of a pair of NIL (D_60 and T_58). The average heights of D_60 and T_58 were 93.52 ± 1.83 cm and 159.67 ± 2.72 cm, respectively; the average plant height of F1 was 123.23 ± 2.55 cm. Compared with T_58, F1 plants harbouring Rht-dp showed a reduction on plant height up to 22.82%. The plant heights of F2 plants ranged from 65 to 155 cm. According to the frequency distribution of plant height, F2 plants were separated into two groups of dwarf and tall phenotypes at 95 cm (Fig. 4A). The dwarf and tall phenotype groups included 62 and 182 lines, respectively, consistent with the expected Mendelian segregation ratio of 1:3 (t = 0.021, P = 3.841, α = 0.05). Meanwhile, the separated threshold of plant height with 95 cm was similar to the plant height of D_60 with 93.52 ± 1.83 cm. In addition, the Rht-B1 Indel marker completely co-segregated with Rht-dp in this F2 populations (Fig. 4B). Thus, these results indicated that the dwarfing gene of Rht-dp is a single semi-dominant gene, and confirmed that the candidate gene is Rht-B1b.
Expression patterns of Rht-B1b in DPW
To confirm that Rht-B1b reduces plant height via its effects on elongation of the first and second internodes at the booting stage, the transcriptional patterns of Rht-B1b were investigated in different DPW tissues at the jointing, booting, and grain-filling stages. Rht-B1b was mainly expressed in the first and second internodes at the booting stage, and at dramatically higher levels in those tissues than in other tissues at the jointing, booting, and grain-filling stages (Fig. 5).
Allelic variations of Rht-B1 in tetraploid wheat accessions
Since Rht-B1b is the candidate gene of Rht-dp in DPW, the haplotypes of Rht-B1b in 59 tetraploid wheat accessions were analysed. Among them, five accessions were dwarf phenotypes including two T. turgidum (AS313 and AS2239), two T. polonicum [AS304 (DPW) and IC12196], and one T. durum (ZH2237). The 59 sequences cloned from the 59 tetraploid wheat accessions were grouped into eight types. Rht-B1b was only obtained from DPW and IC12196; and Rht-B1t and Rht-B1u were only obtained from T. turgidum. subsp. dicoccon (PI191781) and T. turgidum. subsp. Turanicum (PI184543), respectively. Of them, five novel types (named Rht-B1q–B1u, respectively) were identified by comparison with Rht-B1a (Fig. 6B). Rht-B1q contained an S deletion at position 129 (S129); Rht-B1r carried a mutation at position 30 (A30S) and an S deletion at position 129 (S129); Rht-B1s contained a mutation at position 363 (P363S). Rht-B1t had two mutations at positions 15 (G15R) and 363 (P363S). Rht-B1u also had two mutations at positions 136 (Y136D) and 363 (P363S) (Fig. 6B).
Among these variations, Rht-B1q had the highest frequency (43.9%). The frequencies of Rht-B1a, Rht-B1b, Rht-B1h, Rht-B1r, Rht-B1s, Rht-B1t, and Rht-B1u were 15.3%, 3.4%, 13.6%, 13.6%, 6.8%, 1.7%, and 1.7%, respectively.
Dwarfism-related DEGs induced by DELLA mutant Rht-B1b
To understand the molecular networks of Rht-B1b, the DEGs induced by the DELLA mutation Rht-B1b in the first internode of two pairs of NILs were investigated. A total of 41 DEGs was obtained, 30 of which were successfully functionally annotated (Table S3). Twenty-eight DEGs were further classed into five sub-groups; hormone-related signalling transduction genes, transcription factor genes, cell wall structure-related genes, reactive oxygen-related genes, and nitrogen regulation-related genes (Table 1). Among the hormone-related signal transduction genes, two brassinolide (BR) signal-related genes serine carboxypeptidase II-3 (SCP) and cytochrome P450 710A1 (CYP450) were down-regulated; and genes encoding salicylic acid (SA)-binding protein 2 and auxin-repressed protein (ARP) were up-regulated in the dwarf phenotype. The only down-regulated transcription factor gene was MybAS2. Fifteen DEGs were grouped into cell wall structure-related genes (seven pectin-related genes and eight xylan acetylation-related genes). In the dwarf phenotype, five pectin-related genes [encoding a pectate lyase 15 (PEL15), three subtilisin-like protease (SBT1.7), and an alpha-galactosidase (α-Gal)] involved in pectin modification were down-regulated; while all eight xylan acetylation-related genes, including three GDSL esterase/lipase genes, two ESKIMO genes, IRX15-L, ALTERED XYLOGLUCAN 4-like (AXY-L), and an uncharacterized acetyltransferase gene were up-regulated. For the reactive oxygen-related genes, plant cysteine oxidase 2 (PCO2) and L-ascorbate oxidase homolog (ASCO) were down-regulated; and genes encoding germin-like protein 5 − 1 (GLP) and blue copper protein (BCP) were up-regulated in the dwarf phenotype. For nitrogen assimilation-related genes, two phosphoenolpyruvate carboxylase kinase 2 (PPCK2) genes and early nodulin (ENOD) were down-regulated; and asparagine synthetase (APS) was up-regulated in the dwarf phenotype.
Table 1
Dwarfism-related DEGs induced by DELLA mutant Rht-B1b
Gene ID | Description | Fold change of transcript |
D_60/T_58 | D_33/T_35 |
Hormone-related signaling transduction genes |
TraesCS2B01G157100 | Serine carboxypeptidase II-3 | -32 | -20 |
TraesCS3B01G167400 | Cytochrome P450 710A1 | -25 | -18 |
TraesCS2B01G471800 | Salicylic acid-binding protein 2 | 28 | 39 |
TraesCS4B01G070300 | Auxin-repressed 125 kDa protein | 12 | 15 |
Transcription factor |
TraesCS1B01G055200 | Myb-related protein MYBAS2 | -13 | -26 |
Cell wall structure-related genes Pectin-related genes |
TraesCS2A01G016500 | Pectate lyase 15 | -17 | -29 |
TraesCS4A01G237500 | Subtilisin-like protease SBT17 | -20 | -22 |
TraesCS4B01G077600 | Subtilisin-like protease SBT17 | -29 | -17 |
TraesCS6A01G339400 | Subtilisin-like protease SBT17 | -14 | -12 |
TraesCS6B01G332900 | Alpha-galactosidase | -16 | -11 |
TraesCS1B01G249000 | (1–3,1–4)-beta-D-glucanase | 21 | 28 |
TraesCS2A01G341400 | Sugar transport protein 5 | 11 | 14 |
Xylan acetylation-related genes |
TraesCS3A01G258100 | GDSL esterase/lipase | 15 | 11 |
TraesCS3B01G290800 | GDSL esterase/lipase | 13 | 10 |
TraesCS7B01G250700 | GDSL esterase/lipase | 13 | 23 |
TraesCS4A01G110000 | ESKIMO 1 | 14 | 10 |
TraesCS4B01G194100 | ESKIMO 1 | 17 | 10 |
TraesCS6A01G131900 | IRX15-like | 13 | 11 |
TraesCS7A01G191700 | ALTERED XYLOGLUCAN 4-like | 17 | 11 |
TraesCSU01G204900 | Uncharacterized acetyltransferase | 28 | 16 |
Reactive oxygen-related genes |
TraesCS5A01G025200 | Plant cysteine oxidase 2 | -11 | -15 |
TraesCS7A01G459400 | L-ascorbate oxidase homolog | -34 | -51 |
TraesCS3A01G165500 | Germin-like protein 5 − 1 | 16 | 22 |
TraesCS6A01G315800 | Blue copper protein | 13 | 12 |
Nitrogen regulation-related genes |
TraesCS6A01G375800 | Phosphoenolpyruvate carboxylase kinase 2 | -12 | -20 |
TraesCS6B01G413500 | Phosphoenolpyruvate carboxylase kinase 2 | -12 | -15 |
TraesCS7A01G091800 | Early nodulin-93 | -40 | -12 |
TraesCS3B01G385400 | Asparagine synthetase | 11 | 12 |