Qiuhong Wu ,Yongxing Chen ,b,Jingzhong Xie ,Lingli Dong ,Zhenzhong Wng ,Ping Lu ,Rongge Wng ,Chengguo Yun ,Yn Zhng ,Zhiyong Liu ,b,*

a State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,The Innovative Academy of Seed Design,Chinese Academy of Sciences,Beijing 100101,China

b University of Chinese Academy of Sciences,Beijing 100049,China

c China Agricultural University,Beijing 100193,China

d Hebei Gaoyi Stock Seed Farm,Gaoyi 051330,Hebei,China

ABSTRACT Reduced plant height is one of the most important traits related to lodging resistance and crop yield.The use of reduced height genes has been one of the main features in breeding modern high-yielding wheat varieties with less lodging.A spontaneous dwarf mutant DD399 was identified in a high yielding,gibberellic acid (GA)-insensitive,lodging-resistant variety Nongda 399 (ND399).Significant differences in upper internode lengths between mutant DD399 and wild type ND399 were caused by reduced cell elongation.The plant height of ND399×DD399 F1 hybrids was intermediate between the parents,indicating incomplete dominance or a dose–response effect of a reduced height gene.Plant height showed continuous distribution in the F2 population,and segregation distortion was observed among the 2292 F2:3 progenies.The reduced height mutation was characterized by Illumina 90 K iSelect SNP genotyping and bulked segregant RNA-Seq (BSR-Seq) analysis of the segregating population.A concentrated cluster of polymorphic SNPs associated with the reduced height phenotype was detected in the distal region of chromosome arm 2BL.Co-segregation of reduced height phenotype with the clustered markers revealed a 36 Mb terminal deletion of chromosome 2BL in mutant DD399.

Keywords:BSR-Seq Reduced height Semi-dwarf stature SNP Triticum aestivum

1.Introduction

Common wheat,the most widely grown cereal grain crop,occupies about one-fifth of the total cultivated land in the world and provides more calories and protein in the human diet than any other crop.There is a strong relationship between biomass and plant height in most plant species [1] and a positive correlation between grain yield and plant height [2–6].But the stems of tall plants tend to be too weak to support the grain in high-yielding varieties and lodging and grain loss is a frequent consequence.The Green Revolution in the 1960s was based on the use of reduced height genes in wheat and rice that enabled plants to respond to higher nutrient conditions without excessive lodging [7].The new so-called semi-dwarf varieties not only had stiff straw and were not as prone to stem collapse before harvest but also had a greater proportion of assimilate partitioned into the grain,resulting in further yield increases [8,9].Early in the 20th century,a reduced height wheat variety called ‘Daruma’ was crossed with American high-yielding varieties and produced ‘Norin10′which was used in breeding programs in the United States of America and International Wheat and Maize Improvement Center (CIMMYT) to develop high-yielding reduced height cultivars.TheRht-B1bandRht-D1balleles from‘Norin10′were widely used thereafter in worldwide wheat breeding programs [8,10].

Plant height in bread wheat is generally considered to be a complex trait controlled by major reduced height and minor genes[11,12].To date,25 reduced height genes (Rht1-Rht25) have been cataloged in wheat [13–16].Among them,Rht1(Rht-B1b),Rht2(Rht-D1b),Rht3(Rht-B1c),andRht10(Rht-D1c) have been cloned[17–19] and Ellis et al.[20] developed specific PCR markers to detect the point mutations responsible forRht-B1bandRht-D1b.Rht23likely encodes aQhomeolog with pleiotropic effects on plant height and spike compactness[21].Rht-B1b,Rht-B1c,Rht-D1b,andRht-D1care gibberellic acid 3 (GA3)-insensitive,whereas other genes such asRht4toRht25are GA3-sensitive[13–16].In addition to reducing plant stature,Rht-D1bandRht-B1balso reduce coleoptile length and seedling vigor.However,shorter coleoptile length and decreased seedling vigor were not found in varieties with GA3-sensitive reduced height genes,such asRht8andRht9[22–24].

Although a number of reduced height genes have been characterized only a few of them have been widely applied in wheat improvement [25].Based on GA reaction and pedigree analyses,Jia et al.[26]and Guo et al.[27]showed that the most extensively used reduced height genes in China wereRht-B1b,Rht-D1b,Rht8,andRht9.Rht24was also frequently found in wheat cultivars from China and elsewhere [15].To further enhance wheat yield potential and lodging resistance the search for new reduced height genes continues.

Gene mapping in common wheat is time-consuming and laborious due to the enormous genome size (～17 Gb),allohexaploidy(2n=42,genomes AABBDD),and abundance of repetitive DNA.Next generation sequencing technology (NGS) provides abundant sequence data for development of single nucleotide polymorphism(SNP) markers linked to traits of interest via SNP arrays [28] and BSR-Seq [29].For example,by high-throughput 90 K SNP array genotyping of pooled extremes Wu et al.[30] identified major effect adult plant stripe rust resistance QTLQyr.nwafu-6BLin hexaploid wheat.Using Illumina 90 K iSelect SNP chip and bulked segregate analysis (BSA) Wu et al.[31] performed a large-scale detection of powdery mildew resistance genes in 36 wheat lines.Trick et al.[32] mapped the high grain protein content geneGPCB1to a 0.4 cM interval in polyploid wheat by BSR-Seq analysis.By combining BSR-Seq and comparative genomics analyses the stripe rust resistance geneYrZH22was mapped to a 5.92 cM genetic interval spanning a 4.0 Mb physical genomic region on wheat chromosome 4BL [33].

In the present study,we report the identification of a GA3-insensitive spontaneous reduced height mutant DD399 from the high yielding,lodging-resistant wheat cultivar Nongda 399.The reduced height mutation was mapped to a terminally deleted fragment of chromosome 2BL using Illumina 90 K SNP assay and BSRSeq analysis.

2.Materials and methods

2.1.Materials

The DD399 mutant was identified during ear-to-row seed purification cultivar Nongda 399 (ND399,pedigree Torino/2*2552//9516/3/5*Shi 4185).A ND399 × DD399 cross was made to produce F1,F2,and derivative F2:3materials for mapping the reduced height gene.Chromosomal arm assignment and bin mapping of markers linked to the reduced height gene were conducted using Chinese Spring (CS) and CS deletion line 2BL6-0.80-1.00 (kindly provided by Drs.W.J.Raupp and B.S.Gill,Wheat Genetics Resource Center,Kansas State University,USA).

2.2.Field experiment and phenotype measurement

ND399 and DD399 were grown at Beijing,China (BJ,N40.08°,E116.10°) and Gaoyi Stock Seed Farm at Gaoyi in Hebei province(GY,N37.38°,E114.36°) during the 2010–2011 and 2013–2014 growing seasons for evaluation of phenotypic traits.The F1was evaluated at BJ in 2011–2012 and an F2population and F2:3families were evaluated at GY in 2012–2013 and 2013–2014,respectively.CS and CS deletion line 2BL6-0.89-1.00 were tested in 2014 at BJ.Plots were represented by 3 m rows sown with 40 seeds and a row spacing of 25 cm.Field management was the same as commonly practiced in wheat production.

Plant heights(from the surface of the soil to the tip of the main spike) of ND399,DD399,and F1,F2individuals,F2:3rows,CS and del-2BL6-0.89-1.00 were recorded.Five plants were sampled for ND399,DD399,and the F1and three plants were recorded for CS and del-2BL6-0.89-1.00.Plant height of the main spike was recorded for each F2individual.Uniformity in plant height of F2:3families was used to verify the homozygosity versus heterozygosity of each F2individual.One representative plant was measured for each homozygous family,whereas the shortest and tallest plants were measured in segregating families.Thousand grain weight (TGW),main spike length (SL),and individual internode lengths of ND399,DD399,the F1,CS,and del-2BL6-0.89-1.00 were also determined.TGW was recorded using an electronic balance to determine the average weight of three independent 100-grain samples.Effective tiller number(TN)and grain number of the main spike (GN) of ND399,DD399,and the F1were also measured.CS(tall) and Aibian 1 (Rht-D1c) were used as GA3-sensitive and GA3-insensitive controls.

2.3.Gibberellic acid 3 (GA3) response test

ND399,DD399,CS(sensitive control),and Aibian 1 (insensitive control)were tested for response to foliar application of GA3at the seedling stage using the method reported by Wu et al.[19].

2.4.Microscopic analysis and cell dimensions

The central elongating section of main tiller peduncles of DD399 and ND399 were sampled at flowering for determination of cell dimensions.The peduncles were fixed in formalin-acetic alcohol (FAA) and vacuum-infiltrated overnight.These cut tissues were embedded in paraffin and transverse and longitudinal sections were stained with safranin and fast green,and then observed using an Olympus stereo fluorescence microscope (SEX16,Olympus,Japan).

2.5.Statistical analysis

Comparisons of tested traits was performed with GraphPad Prism 8.0.1 (GraphPad Software,San Diego,CA,USA).Chisquared (χ2) tests for goodness-of-fit were used to compare observed and theoretically expected segregation ratio.

2.6.Illumina iSelect 90 K SNP array for detection of genetic differences between DD399 and ND399

High-density SNP genotyping arrays containing 81,587 SNPs[28] were used to detect genetic differences between DD399 and ND399.SNP genotyping was conducted at the Genome Center of the University of California at Davis,USA,according to the manufacturer’s protocols(Illumina,San Diego,CA,USA).SNP allele clustering and genotype calling was performed with GenomeStudio v2011.1 software as described in Cavanagh et al.[34].

2.7.BSR-Seq

BSR-Seq analysis as described in Wang et al.[33] was used to identify candidate SNP variations.Leaves of 60 extremely tall and 60 extremely short F2plants were pooled to prepare contrasting RNA bulks.RNA libraries were prepared from 3 μg of RNA and sequenced on a Hiseq 2000 platform with 100 bp paired end reads(Illumina).Both RNA extraction and RNA-Seq were conducted by Beijing Novogene Bioinformatics Technology Co.Ltd.The implementation of SNP calling and filtration and the identification of SNPs associated with the target trait were performed as described in Wang et al.[33].

2.8.SSR genotyping

Wheat simple sequence repeat (SSR) markers [35] (GWM,WMC,BARC,CFD,and GDM series;http://wheat.pw.usda.gov) on homoeologous group 2 chromosomes were screened for polymorphisms between ND399 and DD399.The polymorphic markers were used to genotype the F2population.PCR were performed on total volumes of 10 μL using an ABI9700 analyzer and the products were separated in 8% non-denaturing polyacrylamide gels as described by Li et al.[36] and visualized by silver staining.

2.9.Polymorphic SSR and STS markers development

The sequences of polymorphic SNPs detected by 90 K SNP array and BSR-Seq were used as queries in BLAST searches of CS chromosome sorting survey sequences(https://urgi.versailles.inra.fr/blast/blast.php) and whole genome assembly (IWGSC WGS v1,NRGene DeNovoMAGIC),and the homologous contigs were used to screen SSR motifs via BatchPrimer3 [37] or as templates to design sequence-tagged site (STS) markers (http://bioinfo.ut.ee/primer3-0.4.0/) with the following parameters:amplification product size of 200–600 bp,primer length of 18–24 bp,Tmof 55–65 °C,and GC content of 40%–60%.

3.Results

3.1.Phenotypic characterization of the semi-dwarf mutant DD399

The plant height of DD399 in cropping seasons 2011–2012 and 2013–2014 at BJ and GY varied from 54 to 60 cm and 55–61 cm,and the plant height of ND399 ranged from 71 to 72 cm and 70.2–77 cm,respectively (Fig.1A,B).The average plant height of DD399 was 15 cm shorter than ND399.There were significant differences in TGW,peduncle length and internode lengths between DD399 and ND399 at both sites,except for internode 4 at BJ(Fig.1A,B).

3.2.Gibberellic acid 3 (GA3) response

Seeding and coleoptile length responses of ND399,DD399,CS,and Aibian 1 to exogenous GA3treatment were evaluated on 10-day old seedlings treated with 50 mg L-1GA3solution after germination.The average seeding and coleoptile lengths of CS to such treatment were significantly higher than the non-treated control while no significant change was observed on DD399 and ND399(Table 1).

Table 1 Effects of GA3 on seedling and coleoptile lengths of 10-day-old wheat plants.

3.3.Cell dimensions

The transverse and longitudinal sections of fully elongated peduncles in ND399 and DD399 at flowering were observed.The number of vascular bundles of ND399 was around 30 compared to about 26 for DD399.The arrangement of vascular bundles in ND399 appeared to be loose (Fig.2A and B) throughout the transverse section.The longitudinal sections of ND399 and DD399 were also compared to search for cytological evidence of plant height reduction.Peduncle tissues of DD399 were composed of shorter cells than those of ND399 (Fig.2C and D),indicating the likely cause of reduced plant height.

Fig.1.Comparison of agronomic traits in ND300 and DD399.(A)and(B)Traits measures for ND399 and DD399 in 2014 at Beijing(A)and Gaoyi(B),respectively.(C)Heights of ND399,DD399,and the F1.(D)Comparison of trait measures for Chinese Spring and Chinese Spring deletion line 2BL6-0.89–1.00 in 2014 at Beijing.P-values are from oneway ANOVA.**and*,significantly different at P<0.01 and P<0.05,respectively.TN,tiller number;GN,grain number of the main spike;SL,spike length of the main spike;PH,plant height;TGW,thousand grain weight.

Fig.2.Transverse and longitudinal culm sections from ND399 and DD399 at flowering.Anatomical structures in ND399(A) and DD399(B).Bar,200 μm.Microstructure of parenchyma cells in the peduncles of ND399 (C) and DD399 (D).Bar,100 μm.

3.4.Genetic analysis and mapping of the dwarf mutation

The height of the ND399×DD399 F1(62–65 cm)was intermediate between the parents(Fig.1C).The heights of the 2292 F2population were continuously distributed or unequally bimodal(Fig.3A).The F2:3families segregated 354 homozygous short(45–59 cm):1045 segregating:893 homozygous tall (60–80 cm)(=271.31,P2df<0.001).The distribution was clearly highly skewed in favor of tall plants (Fig.3B).

Illumina 90 K iSelect SNP genotyping was applied to detect genetic differences between DD399 and ND399.Of the 81,587 potential SNPs arrayed in the chip,only 538(0.66%)were polymorphic providing supportive evidence that DD399 was a spontaneous mutant of ND399.Three hundred and seventy one of the 538 SNPs could be mapped to 19 wheat chromosomes excluding 4D and 5D(Fig.4A);279 (75%) were located on homeologous group 2,with the majority (187/279=67%) located on chromosome 2B(Fig.4A).These results indicated that a reduced height gene,tentatively designatedRhtDD399,was located on chromosome 2B.

RNA-Seq analysis in 100 bp paired-end mode generated 74,744,011 and 79,234,419 clean reads for the tall and short bulks,respectively.With filtration criteria of allele frequency difference(AFD) >0.8 and Fisher Exact TestP-value <1e-10 313 SNPs were identified as putatively associated with plant height.Among them,286(91.37%)were located on chromosome 2BL(Fig.4B),indicating thatRhtDD399was located on 2BL.This result was consistent with the 90 K SNP chip results.

Only two(Xwmc317andXwmc361)of the 160 SSR markers distributed on homoeologous group 2 chromosomes were polymorphic between ND399 and DD399 (Fig.5A and B).Twenty two molecular markers linked withRht8on chromosome 2D[38]were also tested but yielded none polymorphism between ND399 and DD399.Genotyping ofXwmc317andXwmc361on 90 extremely tall and 90 extremely short F2plants revealed that the target DNA bands were present only in the tall plants as in ND399,and no products were produced by the short plants.In addition,the CS deletion line del-2BL6-0.89-1.00 was selected to determine the physical bin location ofXwmc317andXwmc361.Target bands of these two markers were also detected in CS but not in deletion line CS del-2BL6-0.89-1.00,indicating thatRhtDD399was located in distal bin 2BL6-0.89-1.00.

Fig.3.Distribution of plant height in the segregating F2 population(n=2292)(A)and homozygous F2:3 families(n=1247)(B).

3.5.Polymorphic marker development

The flanking sequences ofRhtDD399-associated SNPs identified from the Illumina 90 K SNP chip assay and BSR-Seq analysis were used to blast the CS contigs and whole genome assembly to find homologous contigs for use in PCR primer design.Eight new SSR and seven STS markers polymorphic between ND399 and DD399 were developed(Table 2;Fig.5C–E).All were dominant with bands amplified only in ND399 and not in the mutant.Genotyping of these markers on the 90-F2plant extreme phenotype groups also showed amplification by the tall plants as with ND399,but not the short plants,consistent with the results for SSR markersXwmc317andXwmc361.In addition,all these markers were located in the CS distal bin 2BL-0.89-1.00.

Fig.5.PCR amplification of polymorphic markers.(A–C)Amplifications for markers Xwmc317, Xwmc361,and Xwggc1605 in non-denaturing polyacrylamide gel electrophoresis(PAGE),respectively.1,ND399;2,DD399;3,Chinese Spring;4,Chinese Spring del2BL6-0.89–1.00;5–14,tall F2 plants;15–24,short F2 plants.(D and E)Amplification of STS markers Xdd8 and Xfh17.1,ND399;2,DD399;3–10,tall F2 plants;11–18,short F2 plants.Arrows indicate polymorphic DNA bands.M,DNA Ladder 2000.

3.6.Deletion of a distal 2BL chromosome fragment in DD399

Based on the physical positions of the polymorphic markers on the CS 2BL reference sequence,four markersXwggc1596,Xwggc5434,Xwmc317,andXwggc5801were selected to genotype all 2292 F2plants.Target DNA bands were successfully amplified in the 1045 heterozygous (segregating F2:3families) and 893 homozygous tall plants (homozygous F2:3families),but were absent in the 354 homozygous short lines(homozygous F2:3families).All 17 dominant markers co-segregated with the tallness allele ofRhtDD399even though they were dispersed across a 36 Mb(763 to 799 Mb)range in CS chromosome 2BL(Fig.6;Table 2).

Table 2 Polymorphic markers linked with RhtDD399.

Comparison of the plant heights of CS and CS del-2BL6-0.89–1.00 also indicated a 24 cm difference (Fig.1D).Significant differences were also observed in peduncle length,the 1st–3rd internode lengths and TGW (Fig.1D).The consistent effects of CS del-2BL6-0.89-1.00 and DD399 on plant height and related traits,suggested a distal deletion of the chromosome 2BL in DD399.

4.Discussion

4.1.Relationship between RhtDD399 and known Rht genes

Two types ofRhtgenes have been described in wheat.Some,such asRht1(Rht-B1b),Rht2(Rht-D1b),Rht3(Rht-B1c),andRht10(Rht-D1c),were identified in natural populations and others,such asRht4,Rht5,Rht12,andRht23,were obtained by artificial mutagenesis [13].Pleiotropism has been found for manyRhtgenes.For example,Rht4,Rht5,Rht6,Rht7,Rht23,Rht24,andRht25showed negative impacts on agronomic traits whereasRht24showed variable effects in different genetic backgrounds[14–16].DD399 was a spontaneous mutant of cultivar ND399 with a 15 cm reduction in plant height.There was no significant difference in tiller number,panicle grain number and panicle length between ND399 and DD399,however,there was a significant decrease in 1000-grain weight relative to ND399.

Among the known reduced height genesRht4obtained following radiation was located on chromosome 2BL[39].LikeRhtDD399,Rht4was reported to be linked with SSR markerXwmc317in a doubled haploid population,accounting for up to a 15.4 cm height difference relative to its wild type [40].Given that radiation predominantly causes deletions,it is likely that mutant allelesRhtDD399andRht4involve the same locus.Tests of allelism,genetic fine-mapping,and cloning ofRht4are required to clarify this situation.

Fig.6.Genetic (left) and physical (right) maps of RhtDD399. The physical positions of molecular markers at the far right of the physical map are according to the Chinese Spring reference sequence (http://www.wheatgenome.org/).

4.2.Deletion of the chromosome segment led to reduced plant height and distorted segregation

Deletions of large chromosomal fragments of wheat chromosomes have significant impacts on agronomic traits.Ma et al.[41] found that some CS ditelosomic lines showed variations in response to Fusarium head blight (FHB) caused byFusariumspecies,suggesting that the missing chromosome arms carried genes affecting FHB response.A similar phenomenon was also observed by Garvin et al.[42]whereby loss of a large chromosome 3DL fragment in cultivar Apogee led to enhanced FHB resistance.Sun et al.[43] mappedRht12,a dominant GA-responsive reduced height gene in a gamma ray-induced wheat mutant that corresponded to a 11.21 Mb physical region at the terminal end of CS chromosome 5AL.They further found the reduced height was associated with deletion of a 10.73 Mb fragment of the distal region of 5AL.In the present study,both the Illumina iSelect 90 K SNP chip and BSR-Seq analyses revealed the absence of 17 molecular markers in theRhtDD399mutant relative to the wild type.Based on the complete association of all 17 markers with the tall phenotype and their physical locations it was evident that the mutant had a 36 Mb deletion that led to reduced height.Reduced plant height and TGW of CS deletion line del-2BL6-0.89–1.00 provided further evidence that a gene(s) involved in plant height was located in the distal region of chromosome 2BL.

Distorted segregation,that is,deviation from expected Mendelian segregation ratios has been attributed to competition between gametes or abortion of gametes and/or zygotes in animals[44].Distorted segregation caused by differential transmission of gametes[45]in wheat aneuploid stocks[46]was the basis of gene location prior to molecular markers.Distorted inheritance of stem rust resistance geneSr11was observed in the cross of Timstein and CS(ratio about 2.4:1),whereas a normal(ratio about 3:1)segregation was found in the crosses of Timstein with other varieties[47,48].A pollen-killing gene linked toSr11was proposed to be the reason of the differential transmission of gametes [45,47,48].Similar situation was also observed in favor of these alleles (ratio about 4:1) that derived fromT.timopheeviiin the case ofSr36/Pm6transmission [49].Peng et al.[50] and Li et al.[36] reported that gametes carrying certain alleles from Langdon durum had stronger vigor and higher competition ability than those alleles from wild emmer.Miao et al.[51] reported that genetic factor(s)in the centromere region caused distorted segregation of linked genetic markers on wheat chromosome 1B.In our study,it was evident that flower numbers of homozygous short plants in a segregating population were due to lower transmission of gametes carrying the deleted 2B chromosome.Such effects occur through competition of normal and deficient male gametes,whereas female transmission is usually normal.

4.3.Comparison of SNP chip and BSR-Seq in mapping RhtDD399

Both SNP-based pool genotyping and BSR-Seq have been used to map wheat genes.Both methods allow rapid identification of SNPs associated with traits of interest.For example,SNP arrays were used for identification of stripe rust resistance genesYr26[52],Qyr.nwafu-6BL[30],QYrcen.nwafu-7BL[53],QYr.nwafu-3BS.2andQYr.nwafu-7BL[54],leaf rust resistance QTLQLr.hebau-1BL,QLr.bebau-2AS,andQLr.hebau-2DS[55],and powdery mildew resistance genes in 36 wheat lines [31].BSR-Seq provided an alternative approach for mapping stripe rust resistance genesYrZh22[33] andYr17[56],early leaf senescence geneels1[57],and powdery mildew resistance genesPm4b[58],Pm61[59],andPmQ[60].In this study,Illumina 90 K SNP and BSRSeq assays were both applied for mappingRhtDD399and both associated reduced height with the terminal region of chromosome 2BL.The very low frequency of SNPs (538/81,587=0.66%) between ND399 and DD399 confirmed that DD399 was a spontaneous reduced height mutant in cultivar ND399.About one-half of the polymorphic SNPs (279/538=51.86%) were located on homoeologous group 2 chromosomes and 187(187/279=67%) of those were located at the distal end of 2BL(Fig.4A).BSR-Seq analysis identified 313 SNPs associated withRhtDD399between the two contrasting RNA bulks;97.76% were located on homoeologous group 2 chromosomes and 86.26% of those were in the distal end of 2BL (Fig.4B).It seems that BSR-Seq was more efficient than the Illumina 90 K SNP assay in identifying SNPs associated with the target trait.

Validation of the SNP markers in the segregating population revealed PCR amplification only in ND399 but not in DD399 and all of these dominant markers co-segregated with reduced height.The results indicated that these SNPs associated with the chromosome segment deletion identified by Illumina SNP assay and BSRSeq analysis were not SNPs from the 2BL allelic DNA sequences or transcripts of ND399 and DD399 (null alleles),but from the DNA sequences or transcripts of ND399 2BL and ND399 or DD399 2AL,2DL homoeolgs,and other homologs.High sequence identities among homeologs and homologs are major causes of false SNP calling in polyploid wheat.The unusually high frequencies of SNPs identified for chromosomes 2A and 2D compared to other background chromosomes (Fig.4) likely reflected sequence polymorphisms between homoeologous chromosomes detected at reduced stringency in the absence of a homologous 2BL sequence.

5.Conclusions

We determined that reduced height in spontaneous dwarf mutant DD399 was caused by deletion of a 36 Mb terminal segment of chromosome 2BL.The predicted locus controlling plant height in the deleted region might be the same asRht4.

CRediT authorship contribution statement

Qiuhong Wu,Yongxing Chen,and Zhiyong Liuconceived and designed the experiments.Jingzhong Xie and Lingli Dongperformed bioinformatics analysis.Qiuhong Wu,Yongxing Chen,Zhenzhong Wang,Ping Lu,and Yan Zhangperformed experiment and analyzed the data.Rongge Wang,Chengguo Yuan,and Zhiyong Liudeveloped the DD399 mutant.Qiuhong Wu and Zhiyong Liuwrote the paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2016YFD0100302) and the Science and Technology Service Network Initiative of Chinese Academy of Sciences (KFJ-STS-ZDTP-024).