Fine mapping and characterization of the awn inhibitor B1 locus in common wheat(Triticum aestivum L.)

Jianqing Niu, Shusong Zheng,, Xiaoli Shi, Yaoqi Si, Shuiquan Tian,Yilin He, Hong-Qing Ling,

aState 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

bCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

ABSTRACT Awns play an important role in seed dispersal and photosynthesis of spikes. Three major awn inhibitors (Hd, B1, and B2) are reported in wheat. However, the molecular mechanism underlying awnlessness remained unknown until recently.In this study,we identified two F8 recombinant inbred lines(RILs)that were segregating for awn length.In order to identify the causal gene for awn length in the heterozygous inbred families(HIFs),SNPs were called from RNA sequencing (RNA-Seq) data for HIF-derived progenies with long and short awns.SNPs between long and short awn plants were evenly distributed on chromosomes (chr)other than chromosome 5A.SNPs on chr 5A were clustered in a region distal 688 Mb on the long arm,where inhibitor B1 was located.This suggested that B1 was the causal segregating locus.We precisely mapped B1 to ～1 Mb region using two HIF-derived families.Considering that the lines segregated for long, intermediate and short awn phenotypes we speculated that B1 should have a dosage effect on awn length. Two differentially expressed genes(DEGs)located in the candidate region were regarded as candidate genes for B1,because the molecular expression pattern was consistent with the phenotype.HIFs with long and short awns showed no difference on grain yield and other agronomic traits.

1. Introduction

Awns, bristle-like extensions from the spikelet lemmas are common in gramineous crops, such as wheat (Triticum aestivum), barley (Hordeum vulgare) and oats (Avena sativa).Awns play important roles in seed dispersal and crop production.Barbs on the awn surface can help seed dispersal by attaching to animal fur [1]. Long awns also protect grains from predation [2]. Unlike awns of rice, the awns of wheat consist of three vascular bundles and chlorenchyma that confer the ability of photosynthesis [3]. The pathway for assimilate movement of photosynthate from awns to grains is minimal [4]. Further, the development of awns is later than the flag leaf,which makes it senesce later[4].The total surface area of awns in durum wheat can sometimes equal the ground surface, permitting interception of about 4% of the light [5,6]. The contribution of awns to the carbon exchange rate(CER)is largely dependent on the surface area of awns[7].Awns contribute 40%-80% of spike CER depending on the species [7]. These advantages of awns double the net rate of ear photosynthesis [4]. The water use efficiency (WUE) of awned spikes is 2-3 times higher than awnless spikes during grain filling in barley[8].

Taking all the advantages of awns into account,long awns should be regarded as a useful trait in selection for high yield.Higher WUE and CER, pronouncedly greater photosynthetic potential and thermo-tolerance should lead to equal or higher grain yield for long awned genotypes. However, the issue of whether long awns promote higher grain yield remains controversial. Martin et al. [9] reported that awns increased grain yield by 0-6.2% in winter wheat. Motzo and Giunta[6]concluded that awns increased average grain yield by 10%-16% in comparisons of awned and awnless durum isolines. Removal of awns at ear emergence and after anthesis decreased grain yield by 17.3% and 13.5%, respectively[10].Other studies showed that long awns had negative effects on grain yield [11,12]. Some studies found that awns had little or no influence on the grain yield. For example,Olugbemi et al. [13] found no increase in grain yield by long awns irrespective of genetic background and experimental conditions. Rebetzke et al. [14] found that grain yields of awned and awnletted sister-NILs were equivalent, irrespective of yield potential and genetic background using 45 awned-awnletted NILs in 25 diverse environments in Australia and Mexico. It was also reported that the genes controlling awn length in rice were always associated with pleiotropic effects on other agronomic traits. For example,An-1 and An-2 not only regulated awn development but also grain size, grain number and other grain characteristics[15,16].

Grass species often harbor long awns. However, cultivated crops have been selected awnlessness or short awns during domestication to facilitate seed collection,storage and processing,especially in the case of rice.Some genetic factors for awns initiation and elongation have been characterized in rice,such the basic-helix-loop-helix transcription factor An-1[15],cytokinin synthesis enzyme gene An-2[16,17],GRAIN NUMBER,GRAIN LENGTH AND AWN DEVELOPMENT1 (GAD1) [18,19], YABBY transcription factor (DROOPING LEAF [DL]) [20] and auxin response factor OsETTIN2 [20]. Allele Lks2 is a member of the SHORT INTERNODE (SHI) family of transcription factors, mutation which caused a reduction of awn length in barley [21]. A 305 bp duplication in the fourth intron of Knox3 in the chr 7H causes the Hooded phenotype in barley[22].In common wheat,relatively few genes are involved in control of awn length.Three dominant awn length inhibitors located in chr 4AS,5AL and 6BL are named as Hooded (Hd), Tipped1 (B1), and Tipped2 (B2) [23]respectively [24-26]. Nishijima et al. [27] recently identified another awn length locus in the diploid wheat ancestor Aegilops tauschii. The dominant Hd allele causes reduced awn length with the awns hooked and twisted near the base [28]. The B1 allele inhibits awn development at the base and middle of the spikes and reduces awn length to 0.5-2.0 cm at the top.B2 also produces slightly curved awns that never bend as much as Hd.Wheat varieties with genotype hdhdb1b1b2b2 are fully awned,whereas varieties with any two dominant inhibitor alleles are nearly awnless[28].Sears[25]placed B1 on the long arm of 5A using aneuploid lines.Kato et al.[29]located B1 at the distal end region of chr 5AL and distal to the 5A/4A translocation break point using a set of single-chromosome recombination lines(RIL)for chr 5A.Sourdille et al.[30]mapped B1 between markers Xgwm156 and Xcfa2155 by comparing Chinese Spring deletion lines 5AL-10 (awned speltoid phenotype) and 5AL-17 (awnless speltoid phenotype) [28]. With molecular markers and a highdensity genetic map,Yoshioka et al.[28]mapped B1 to the distal end of 5AL beyond Xcfd39 (outside the region flanked by markers Xgwm156 and Xcfa2155)and closely linked with marker Xgwm291.Mackay et al.[31]mapped B1 to a 7.5 cM interval and developed a diagnostic KASP marker using an eight-parent MAGIC population. A genome-wide association study also identified a significant marker-trait association (MTA) on chromosome 5AL[32].These various studies mapped B1 either in a very large region or in different positions.

In this study, we first mapped B1 to the distal end of chr 5AL by RNA-Seq of heterozygous inbred families (HIFs) that segregated for differences in awn length. Furthermore, we finely mapped B1 to a ～1.1 Mb region using 4572 progenies from two HIFs. Differentially expressed gene (DEG) analysis identified two DEGs that exhibited a consistent expression pattern between awn phenotype and the candidate region.There was a high possibility that one of these DEGs was B1.The results presented a sound foundation for cloning B1.

2. Materials and methods

2.1.Plant materials and field trials

The parents of the F8RIL population were the semi-dwarf,long awned cultivar ‘Lankao 86’ (LK86) and short awned Chinese landrace‘Ermangmai’(EMM).Both parents differed in other agronomic traits and yield components (Fig. S1).Segregating RILs L71 and L132 contained 18 and 16 individuals, respectively (Fig. 1). Each individual within the HIFs showed no detectable difference other than their awn length(Fig. 2a, c). The progenies from the plants that segregated for awn length(9 of 18 plants for L71;6 of 16 plants for L132)were used to construct a fine mapping population, a total of 4572 HIF-derived plants. Young spikes(～1 cm in length) with long,intermediate and short awns from one HIF were subjected to RNA sequencing. The progenies of homozygous long awned and short awned plants were used to evaluate the grain yield and several agronomic traits in a field trail.Seeds for the field trials were sown in standard experimental plots (1.4 m width and 10 m long)with the seeding rate at 300 seedlings m−2in a randomized complete block design with three replicates during 2018-2019 at Zhaoxian in Hebei province. Agronomic practices regarding fertilizer and pest control were in accordance with local practices.

Fig.1-Flow chart for development of heterozygous inbred families(HIFs)derived from the cross between Lankao 86(LK86)and Ermangmai(EMM).

Fig.2- Phenotype of plants within heterozygous inbred families(HIFs).Whole plants and spikes of plants in HIF1 (L71)(a, b)and HIF2(L132)(c,d).(e,f)Mean awn lengths for plants in HIF1 and HIF2.The significance of differences was tested by Students t-tests.Error bars indicate standard deviations.

2.2. Phenotype evaluations

2.3. SSR marker development

Two hundred and twenty simple sequence repeat (SSR)markers located distal to 688 Mb of 5AL were developed to fine-map B1. The reference sequence of Chinese Spring(http://www.wheatgenome.org/) after 688 Mb was used to design SSR markers on the Batchprimer3 v1.0 website (https://wheat.pw.usda.gov/demos/BatchPrimer3/). Polymerase chain reactions (PCR) were conducted using a touchdown program as follows: 94 °C for 5 min; 8 cycles of 94 °C for 30 s, decreasing from 65 °C to 57 °C by 0.8 °C per cycle for 25 s, 72 °C for 30 s;24 cycles of 94 °C for 30 s, 57 °C for 25 s, 72 °C for 30 s; 72 °C for 5 min; and 24 °C for 3 min. PCR products were separated by 12% non-denaturing polyacrylamide gel electrophoresis(PAGE) or in 5% agarose [33]. Polymorphic markers were listed in Table S1.

2.4. RNA sequencing analysis

Young spikes of HIFs with long, intermediate and short awns were used for RNA sequencing. Two biological replicates were conducted for each sample. Total RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturers instructions. RNA-seq was conducted by the BGISEQ-500 platform of the Beijing Genomics Institute (BGI). About 15.54 Gb of 150 bp paired-end raw data were generated for each sample. The quality of reads was evaluated by SOAPnuke(v1.4.0) with parameters: l = 5, q = 0.5, n = 0.1 [34].Trimmomatic (v0.36) was used to remove the low quality reads with parameters: illuminaclip:2:30:10 leading:3 trailing:3 slidingwindow:4:15 minlen:50 [35]. The remaining clean reads were aligned to the wheat reference genome sequence IWGSC RefSeq v1.0 (http://www.wheatgenome.org/)by Bowtie2 (v2.2.5) with parameters: -q --phred64 --sensitive--dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200 [36]. The HaplotypeCaller module of Genome Analysis Toolkit (GATK) was used for SNP calling [37]. Only repeatable SNPs across the two replicates were applied in identifying SNPs between long awn and short awn individuals. The distribution of polymorphic SNPs on chromosomes was plotted in R. The software RSEM (v1.2.8) with default parameters [38] was used to quantify the reads mapped to respective high-confidence genes annotated from IWGSC_v1.1_HC_gene in each sample. Bioconductor package“DESeq2” was used to calculate the differential expression[39].Genes with an absolute value of log2(fold change)≥1 and adjusted P-value ≤0.001 were considered to be DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes(KEGG) analyses were implemented using a TopGO package[40]. GO terms with Q_value ≤ 0.05 were regarded as significantly enriched.

2.5.Fine mapping B1

A mapping population of 4572 individuals was derived from HIFs L71 and L132. Polymorphisms of the 220 SSR markers located distal to 688 Mb on 5AL were checked on LK86 and EMM by PAGE. We genotyped a subset of 192 random individuals with the polymorphic markers to identify flanking markers that could delimit the candidate region of B1. The remaining 4380 individuals were screened with the two flanking makers to identify recombinant plants. The recombinant plants were genotyped using all polymorphic markers.

3. Results

3.1.Phenotypic and genetic analysis of HIFs

For simplification, the two HIFs segregating for awn length locus were designated as HIF1 (L71) and HIF2 (L132). In the progenies of HIFs,awned plants exhibited long awns throughout the spike (Fig. 2b, d-f). However, plants of short awns displayed awns only at the top of the spike and were nearawnless at the spike base (Fig. 2b, d-f). Some plants showed medium awn length at the top of the spike and slightly longer length at the central and basal parts (Fig. 2b, d-f). A genetic linkage analysis was performed using 381 HIFs-derived progenies. Among them, 99 individual plants showed long awns and 282 plants showed short or intermediate awns.The results indicated that long awns were controlled by a single recessive gene(χ21:3=0.197,P ＜0.05).

To evaluate whether this awn controlling locus has effects on agronomic traits in our HIFs,grain yield,plant height,spike length, spikelet number, tiller number, thousand-grain weight, grain number per spike, seed length and seed width were investigated in HIFs displaying significant difference in awn length. There were no significant differences between the two groups(Fig.3).

3.2.Primary mapping B1 by RNA-Seq

In order to map the region underlying awn length, young spikes(～1 cm in length)of progenies of HIF2 with long,medium and short awns were performed RNA sequencing with two biological replicates. For simplification, we named these samples HIF2_LK, HIF2_ZH, and HIF2_EM with long, medium and short awn,respectively.More than 100 M of clean reads(150 PE) were obtained for each sample (Table S2). All clean reads were aligned to the Chinese Spring reference genome sequence(IWGSC RefSeq v1.0) and more than 180,000 high quality SNPs were identified by GATK with default parameters [37]. Finally,there were 17,356 SNPs between HIF2_LK and HIF2_EM. Many SNPs were evenly distributed along all chromosomes (Fig. 4a;Fig. S2a, b). However, 66.02% (1158/1754) were clustered at the distal end of chromosome 5AL (distal to 688 Mb) where the B1 locus was located(Fig.4b).These results suggested that B1 was the determinant of awn length in these HIFs materials,and that B1 was distal to the 688 Mb point.

Fig.3- Comparisons of grain yield and agronomic traits for homozygous long awned and short awned plants from the two HIFs.(a)Grain yield.(b)Plant height.(c)Spike length.(d)Spikelet number.(e) Thousand-grain weight.(f)Grain number per spike.(g)Seed length.(h)Seed width.(i)Tiller number.Long,HIFs-derived long awn materials;Short,HIFs-derived short awn materials.Students t-tests.Error bars indicate standard deviations.

3.3.Fine mapping of B1

Among the 18 and 16 individuals in HIF1 and HIF2 the progenies of 9 (9/18) and 6 (6/16) individuals, respectively,segregated for awn length. In order to fine map B1 all the seeds from the 15 segregating plants were planted, and the 4572 progenies were used as a fine mapping population. For this, 220 pairs of SSR primers were developed for the chr 5AL region distal to 688 Mb. Ten SSR markers were polymorphic between LK86 and EMM.Following genotyping a subset of 192 random individuals from the mapping population using these markers the B1 locus was delimited to the region flanked by NQ-60 and 5A-3.1 (Fig. 4). These markers were then used to screen all individual plants. We identified seven key recombinant plants that enabled B1 to be mapped between NQ-60 and NQ-143 (Fig. 4). Markers NQ-5, NQ-156, and NQ-37 cosegregated with the B1 allele. The physical distance between these two flank markers was ～1.1 Mb according to the IWGSC RefSeq v1.0 of Chinese Spring[41],and there were 29 predicted high confidence genes in the region[41].

Fig.4-Mapping of the B1.(a)Distribution of SNPs in the A sub-genome.(b)Distribution of SNPs at the distal end of chr 5AL.(c)Fine mapping of B1.Genotype and phenotype of seven recombinant plants.Black and white blocks indicate genomic fragments from EMM and LK86,respectively.Grey blocks indicate heterozygous regions.

3.4. Transcriptomic analysis and candidate genes prediction underlying B1

A transcriptomic analysis was conducted to identify phenotypically linked DEGs.We identified 1353 DEGs between HIF2_LK and HIF2_EM (|Fold change| ≥ 2, Adjusted Pvalue ＜0.001). Among them, 1028 genes were upregulated and 325 genes were downregulated. Gene ontology (GO)functional annotation of all DEGs revealed statistically significant enrichment for biological processes related to‘cellular process’; for example: GO:0042743 (hydrogen peroxide metabolic process) and GO:0072593 (reactive oxygen species metabolic process) (Table S3). Regarding cellular components, terms associated with cells (GO:0031226) and extracellular regions (GO:0005576) were enriched. Molecular function was mainly enriched for terms of DNA binding(GO:0003677) and sulfate transmembrane transporter activity(GO:0015116). All 1353 DEGs were classified into 125 KEGG pathways among which carbon metabolism(ko01200),pyrimidine metabolism(ko00240),ether lipid metabolism(ko00565),homologous recombination (ko03440), mismatch repair(ko03430), mRNA surveillance pathway (ko03015) and RNA transport (ko03013) represented the most significant pathways(Table S4).

Fig.5- Venn diagram of DEGs and expression levels of candidate genes.(a) Venn diagram of DEGs in HIF2_LK vs.HIF2_EM,HIF2_LK vs.HIF2_ZH,and HIF2_EM vs.HIF2_ZH.(b-d) Expression level of TraesCS5A01G541900, TraesCS5A01G542000, and TraesCS5A01G542800 in three genotypes.

Considering the different phenotypes of HIF2_LK,HIF2_ZH,and HIF2_EM we speculated that B1 might have a dosage effect on awn length and hence might be differentially expressed whereby homozygous B1B1 would have highest expression (in HIF2_EM), heterozygous B1b1 would have intermediate expression (in HIF2_ZH), and homozygous b1b1 would have the lowest expression (in HIF2_LK). In order to determine whether the DEGs fitted this expression pattern a DEG analysis was undertaken by comparing expression levels among the three types. There were 1353, 1075, and 219 DEGs in HIF2_LK vs.HIF2_EM,HIF2_LK vs.HIF2_ZH,and HIF2_EM vs.HIF2_ZH,respectively(Fig.5a).A total of 20 DEGs were shared among HIF2_LK vs. HIF2_EM, HIF2_LK vs. HIF2_ZH, and HIF2_EM vs.HIF2_ZH.Furthermore,8 of the DEGs were located on chr 5A and 3 were located in the candidate region between markers NQ-60 and NQ-143. Among these three DEGs, the expression of TraesCS5A01G541900 was the highest in HIF2_LK, second in HIF2_ZH, and the lowest in HIF2_EM (Fig.5b). The expressions of TraesCS5A01G542000 and TraesCS5A01G542800 were opposite (Lowest in HIF2_LK, medium in HIF2_ZH and highest in HIF2_EM) (Fig. 5c, d). Spatial and temporal expression of these three DEGs was investigated through Wheat Expression Browser (http://www.wheatexpression.com/).TraesCS5A01G541900 was mainly expressed in the spike, stigma and ovary during anthesis (Fig. S3a); the expression of TraesCS5A01G542000 did not have tissue and temporal preference(Fig.S3b)making it an unlikely candidate for B1; and TraesCS5A01G542800 predominately expressed in the spike and pistillody stamen during reproductive stage(Fig.S3c). TraesCS5A01G541900 and TraesCS5A01G542000 contain multiple domains, such as nucellin_like, TAXi_N and PLN03146 (Fig. S4a). The proteins that carry these domains mainly function as inhibitors of xylanases[42].The predicted protein TraesCS5A01G542800 is a member of the C2H2-type zinc finger family of transcription factors that share a conserved zf_C2H2 domain.The C2H2-type zinc finger family is involved in a variety of processes, including floral organogenesis, leaf initiation and gametogenesis. Considering we postulated TraesCS5A01G541900 and TraesCS5A01G542800 to be candidate genes for B1.

研究区土壤重金属内梅罗综合指数平均值为1.02，超过了警戒线，属于轻度污染等级，土壤安全、警戒线、轻度、中度和重度污染样点的比例分别为50.72%、23.56%、17.98%、4.00%和3.74%，污染样点的比例为25.72%。对比单项和综合污染指数空间分布图可知，As和Cd的单项指数污染评价划分区域与综合污染评价结果高度一致，中度、重度污染区域包含在As和Cd的中度、重度污染区域内，警戒线区域包含As和Cd的轻度污染区域。As和Cd是造成局部区域污染指数等级较高的主要影响元素。

4. Discussion

Awns, long slender extensions of the lemma of gramineous crops, are photosynthetically active and contribute to grain filling by supplying the carbohydrate to developing grains[6,43]. Many genes have been identified to underly awn formation and elongation in rice, including An-1 and An-2[15-18]. In barley, a SHORT INTERNODES (SHI) transcription factor (Lks2) was found to regulate awn elongation [21].However, little is known about the genes involved in awn length in wheat.In this study,we firstly mapped the B1 locus to a small region of chromosome 5AL (～22 Mb) by RNA sequencing of a set of HIFs. We then fine mapped to a～1.1 Mb region using the progenies of HIFs. Compared to biparental and multi-parental gene mapping there are advantages in gene mapping by RNA-seq of HIF(s). First, we can easily identify a heterozygous fragment where the candidate genes located by RNA-seq HIFs can be recognized by phenotypic differences. Second, we can sequence multiple HIFs pairs (if necessary) to delimit the candidate gene to a narrow region.Third,SNPs and Indels associated with phenotype are valuable for marker development from RNA-seq.Finally,DEGs from RNA-seq provide valuable information for identifying a causal gene.In this study,we used this method to locate B1 in the region between markers NQ-60 and NQ-143. Mackay et al.[31] identified a 7.5 cM region containing B1 by GWAS, and developed a dignostic KASP marker converted from peak marker. The dignostic KASP marker was located between the co-segregated markers NQ-5 and NQ-37 in our study.However,that region was much larger than the region delimited in this study.The closest marker Xgwm291 identified by Yoshioka et al. [28] was actually between co-segregating markers NQ-156 and NQ-5 in our study. The significant DarT-Seq markers(B14805/B14806) associated with awn type [32] were outside our candidate region and marker B14816 was anchored between marker NQ-37 and NQ-143. Sourdille et al. [30]mapped B1 between SSR loci Xgwm156 and Xcfa2155. We found that B1 was located at the distal end of chromosome 5AL, consistent with previous reports [28]. Briefly, we precisely mapped B1 in a narrow region using HIFs-derived progenies and RNA-seq HIFs.

Awns can greatly increase surface area for light interception,promote WUE and transfer assimilate to filling grain [6,8].However, whether awns can increase grain yield and awn length in association with pleiotropic effects on other agronomic traits are still controversial issues.Genes underlying awn length are often associated with pleiotropic effects on other agronomic traits and grain yield such as tiller number,spikelet number and plant height [6,14,44,45]. We investigated nine traits (grain yield, plant height, spike length, spikelet number,tiller number, thousand-grain weight,grain number per spike,seed length and seed width) in two HIFs and found no significant difference in these traits between long and short awn materials.This means B1 does not have pleiotropic effects on grain yield and other agronomic traits when NILs are evaluated under regular growing conditions. The discrepancy between the current and previous studies may be due to test environments and materials[46-48].Awns may affect yield and other agronomic traits under drought conditions [4,7,45].Previously, researchers found three major dominant loci involved in awn suppression,viz.B1(Tipped1),B2(Tipped2)and Hd(Hooded)designated as awn inhibitors.In barley,the absence of awns is a recessive trait.This implies unidentified factors are involved in initiation and development of awns in wheat and that the current genetics of wheat versus barley and rice involves different genes.Interestingly,the intermediate phenotype was observed in our HIFs,which was not observed in B2,Hd locus and other cloned genes in rice.This phenomenon implied that B1 might have dosage effects on awn length or have interactions with other unknown loci.Phenotypic variation can be associated with the relative dosage of alleles, which can be related the amount of encoded protein and mRNA abundance[49].A well-known example is plants of monosomic chr 5A that have speltoid spikes. Plants that are nullisomic, monosomic,disomic, trisomic, and tetrasomic for chr 5A exhibit speltoid,semi-speltoid, square headed, subcompact and compact spike architecture caused by dosage dependent expression of allele Q[25,50,51]. Another example is VRN-A1, a MADS box transcription factor,whose expression level was closely associated with frost tolerance and flowering time[52].Because B1 is right next to 5AQ and VRN-A1 on chromosome 5AL,it is easily to think that the expression level of B1 was closely related with awn length.

Three DEGs shared between HIF2_LK vs.HIF2_EM,HIF2_EM vs. HIF2_ZH, and HIF2_LK vs. HIF2_ZH were identified in the candidate region(～1.1 Mb).TraesCS5A01G542000 was excluded as a candidate for B1 because its expression pattern was not consistent with the awn phenotype.Sequence analysis of the other two candidate genes showed that there were two SNPs in TraesCS5A01G542800 between LK86 and EMM.The SNP(G to C, 148 bp downstream of the ATG start codon) resulted in a change from alanine to proline. The second SNP (C to T at 322 bp downstream of ATG) caused premature termination with the protein lacking an ethylene response factorassociated amphiphilic repression (EAR2) domain (Fig. S5a).We could not amplify the full length of TraesCS5A01G541900 after designing many pairs of primers. The protein of TraesCS5A01G542800 contained a C2H2 zinc finger domain and two EAR motif-like domains(Fig.S5a).The C2H2 family of zinc finger proteins are involved in many biological processes.EAR motifs are typically present in many transcriptional plant repressors [53]. Phylogenetic analysis showed that TraesCS5A01G542800 grouped with the cellular proliferation repressor KUN and various abscisic acid signaling negative regulators (e.g. ZFP4 and ZFP7) in Arabidopsis (Fig. S5b). The TraesCS5A01G541900 protein contained multiple domains and grouped with ACPB1, a member of the aspartyl protease family in Arabidopsis(Fig.S4).

During preparation and review of this paper three research groups reported that TraesCS5A01G542800 was the causal gene of B1 [54-56]. However, they did not report that awn length differences between B1 heterozygotes and homozygotes could be associated with variation in gene expression.Our conclusion was that TraesCS5A01G542800 was a likely candidate for B1.We recognized that the C to T change at 322 bp downstream from the start codon was the likely causal variation because of loss the EAR2 domain. This variation resulted in premature termination of the protein and longer awns. The three published papers found no variation in the CDS of TraesCS5A01G542800.Apparently the C to T change detected in our study represents a different allele that influences awn length.

5. Conclusions

We firstly mapped B1 to the distal end of chr 5AL by RNA-Seq of HIFs that exhibited difference in awn length. Furthermore,we fine mapped B1 to a ～1.1 Mb region using 4572 HIF-derived progenies. DEGs analysis suggested two DEGs(TraesCS5A01G541900 and TraesCS5A01G542800) in the candidate region that associated expression pattern and phenotype. There was a high probability that one of these DEGs was B1. We preferred TraesCS5A01G542800 because of the differences in the CDS of this gene between long and short awn materials. According to the results of three recently published papers, we identified a unique variation in the CDS region of TraesCS5A01G542800 that influences awn length.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.12.005.

Author contributions

HL and SZ conceived the project.SZ and JN developed the RIL and mapping populations.JN conducted the experiments and analyzed the data. ST, YS, and MN assisted in field works. XS and YH helped in data processing. JN wrote the manuscript.HL and SZ revised the manuscript.

Acknowledgments

This study was supported by the National Key Research and Development Program of China (2016YFD0101802) and Exploring Candidate Genes of heat root length by integrative genomics(PCCE-KF-2018-02).