Haiming Han,Xinyuan Ma,Zhen Wang,Kai Qi,Wenjing Yang,Weihua Liu,Jinpeng Zhang,Shenghui Zhou,Yuqing Lu,Xinming Yang,Xiuquan Li,Lihui Li

National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:Agropyron cristatum Wheat Chromosome 5P Translocation Chromosome pairing

ABSTRACT Wide hybridization is a strategy for broadening the genetic basis of wheat.Because an efficient method for inducing wheat–alien chromosome translocations will allow producing useful germplasm,it is desirable to discover new genes that induce chromosomal variation.In this study,chromosome 5P from A.cristatum was shown to induce many types of chromosomal structural variation in a common wheat background,including nonhomoeologous chromosome translocations,as revealed by genomic in situ hybridization,fluorescence in situ hybridization,and DNA marker analysis.Aberrant meiosis was associated with chromosomal structural variation,and aberrant meiotic behavior was observed in wheat–A.cristatum 5P monosomic and disomic addition lines,suggesting that the effect of chromosome 5P was independent of the number of chromosome 5P copies.Chromosome 5P disturbed homologous chromosome pairing at pachytene stage in a common wheat background,resulting in a high frequency of univalent formation and reduced crossing over.Thirteen genes involved in DNA repair or chromatin remodeling,including RAD52-like and MSH6 genes,were differentially expressed(upregulated)in wheat–A.cristatum 5P addition lines according to transcriptome analysis,implicating chromosome 5P in the process of meiotic double-strand break repair.These findings provide a new,efficient tool for inducing wheat–alien chromosome translocations and producing new germplasm.

1.Introduction

The study of breeding practice has shown that a narrow genetic basis is the bottleneck limiting breakthroughs in wheat(Triticum aestivum)improvement.The transfer of desirable genes from wild relatives into wheat is a valuable strategy in wheat breeding.It is necessary to achieve translocations with sufficient compensation of wheat chromosomes and little genetic drag.In the history of wheat breeding,translocation lines carrying alien genes have included T1BL.1R#1S, carrying Lr26/Sr31/Yr9/Pm8, and T1AL.1R#2S,carrying Gb2/Pm17 from Secale cereale;T3DS.3DL-3Ae#1,carrying Sr24/Lr24,and T6AS.6AL-6Ae#1L,carrying Sr26,from Thinopyrum elongatum;and T2B/2G#1,carrying Sr36/Pm6 from Triticum timopheevii[1,2].In addition to T1BL.1R#1S,the T6VS.6VL translocation line,carrying Pm21 from Haynaldia villosa,is increasingly being used in disease resistance breeding in China[3].

Methods or materials that efficiently induce wheat–alien chromosome translocation are needed for creating new wheat breeding materials.In wide hybridization,artificially induced translocations can create translocation lines at a high frequency,and this method is an important part of chromosome engineering.Two common biological methods for inducing wheat–alien chromosome translocations include the gametocidal(Gc)system and the use of the ph1 mutant.When an alien chromosome carrying a Gc gene is introgressed into wheat(to produce monosomic addition lines or substitutions),Gc genes can cause non-Gc gene gametes to abort,resulting in preferential transmission of Gc gene gametes(carrying the alien chromosome)to the next generation[4].Fourteen Gc chromosomes have been identified in the C,S,and M genomes of the Aegilops genus,involving 2,3,and 4 homoeologous groups[5].Gc chromosomes are widely used to induce chromosomal deletions,construct physical chromosome maps,and develop wheat–alien chromosome translocations[6–11].

ph1 mutant is valuable for inducing wheat–alien chromosome translocations.Meiotic recombination plays a central role in evolution,speciation,plant breeding,and crop improvement.In common wheat(with genome formula AABBDD,2n=6x=42),chromosome pairing and recombination are controlled by pairing-homoeologous(Ph)genes[12,13].A major regulator,the Ph1 locus,ensures that recombination is restricted to true homologs rather than homoeologs and that the stability and fertility of the wheat genome are maintained in the process of evolution[14,15].Ph1 recessive or deletion mutants allow a degree of homoeologous synapsis to occur between alien chromosomes and wheat homoeologous chromosomes,resulting in wheat–alien chromosomal variations[15,16].The hexaploid wheat ph1b mutant and the tetraploid wheat ph1c mutant are Ph1 deletion mutants.Hundreds of wild relative chromosome segments have been successfully introgressed into wheat by exploiting Chinese Spring(CS)ph1b[17].The ph1b mutation is favorable for reducing the chromatin surrounding a target alien gene[18,19].

Genes suppressing Ph1 and promoting homoeologous chromosome pairing play similar roles in inducing translocations from wheat relatives and landraces.Chromosome 5U of Ae.umbellulata Zhuk.[20]and chromosomes 3E,4E,and 5E of Lophopyrum elongatum(Host)á.L?ve[21]have been shown to promote homoeologous pairing when introduced into wheat.Chromosome 5 Mgof Ae.geniculata can freely recombine with chromosome 5D of wheat in the presence of Ph1[22].Su1-Ph1 and Su2-Ph1,mapped to Ae.speltoides chromosome arms 3SL and 7SL,have been reported[23,24]to suppress the Ph1 locus.A locus on chromosome 3AL promoted homoeologous pairing in the Kaixian-Luohanmai landrace[25].These genes can also be used for the introgression of alien genes into wheat.

Agropyron Gaertn.,a perennial genus of the tribe Triticeae,contains one P genome and exhibits three ploidy levels:diploid,tetraploid,and hexaploid[26].Agropyron Gaertn.is not only widely cultivated as a forage and sand-stabilizing plant but also a genus of wild relatives of wheat that carries many desirable genes for improving wheat characteristics,including resistance to certain diseases and abiotic stress and the production of multiple spikelets,florets,and fertile tillers[26,27].To transfer these desirable genes to common wheat,wide hybridization of the common wheat cultivar Fukuhokomugi(Fukuho,2n=6x=42,AABBDD)with tetraploid A.cristatum(accession Z559,2n=4x=28,PPPP,)was achieved via embryo rescue[28–30].A series of wheat–A.cristatum derivative lines,including wheat–A.cristatum addition,substitution,deletion,and translocation lines,showing high kernel number per spike,thousand-kernel weight,and fertile tiller number,as well as resistance to leaf rust,stripe rust,and powdery mildew were obtained[31–43].These lines have been used as bridge materials or novel germplasm in wheat improvement.The promotion of homoeologous pairing was observed in hybrids of wheat with A.cristatum.Translocation only among wheat homoeologous chromosomes was found in a wheat–A.cristatum 4P addition line,indicating that a gene with a weaker effect of suppressing Ph1 was present on chromosome 4P[44].Recently[45],some chromosomal variations have been found in the derivatives of a wheat–A.cristatum 5P addition line,suggesting that chromosome 5P can induce chromosomal variation.However,the genetic effect of chromosome 5P in a wheat background awaits further elucidation.

Identification of the novel gene that induces chromosomal structural variations on chromosome 5P would provide a new tool for chromosome engineering.In the present study,chromosomal structural variations induced by A.cristatum chromosome 5P in a common wheat background were revealed by genomic in situ hybridization(GISH),nondenaturing fluorescence in situ hybridization(ND-FISH),and DNA marker analysis.The meiotic behavior of wheat–A.cristatum 5P monosomic and disomic addition lines was investigated to determine whether there was a dose effect of chromosome 5P.Chromosome pairing by tracking the meiotic behavior of chromosome 3B was used to identify the key stage of disturbed homologous chromosome pairing.RNA-seq analysis was used to investigate which process of meiosis influenced by chromosome 5P.

2.Materials and methods

2.1.Plant materials

II-11-1b,a wheat–A.cristatum 5P disomic addition line,was developed by hybridization between T.aestivum cv.Fukuhokomugi(Fukuho)and A.cristatum accession Z559(2n=4x=28,PPPP,from Xinjiang,China).WA5P is a wheat–A.cristatum 5P monosomic addition line from the BC6F1offspring of a cross of II-11-1b to Fukuho as the recurrent parent.There were no chromosomal structural variations in these two addition lines(Fig.S1).A population of BC2F4plants derived from a cross of II-11-1b and Fukuho was used to evaluate the chromosomal constitution in this study.These materials were stored or created in our laboratory.

2.2.Meiotic and mitotic preparations

When the plants reached the flag leaf stage,spikes were sampled.In each dissected floret,one of the three synchronized anthers was squashed in 45% acetic acid in water to identify the meiotic stage.If appropriate stages were present,the two remaining anthers were either fixed in ethanol:acetic acid(3:1)for 24 h and stored in 70% ethanol at 4 °C for the cytological analysis of meiocytes or harvested for RNA-seq experiments.Slide preparations were examined under a BX51 Olympus phase-contrast microscope(Olympus Corporation,Tokyo,Japan)and then placed over liquid nitrogen prior to removal of the cover glass.

Seeds were germinated on moistened filter paper in Petri dishes.Actively growing roots were removed from seedlings and subjected to nitrous oxide treatment for 2 h,fixed in 90% acetic acid for 8 min,and stored in 70% v/v ethanol.Chromosome preparation was performed following Kato et al.[46]and Han et al.[47].Cytological observations were performed under the phase-contrast microscope.

2.3.GISH and FISH

GISH was performed with A.cristatum genomic DNA as probe and Fukuho genomic DNA as blocker.Multicolor GISH(mcGISH)was performed following Han et al.[48].Total genomic DNA of T.urartu was labeled with fluorescein-12-dUTP(PerkinElmer,Boston,MA,USA),and total genomic DNA of Ae.tauschii was labeled with Texas Red-5-dCTP(PerkinElmer)via the nick-translation method.Total genomic DNA of Ae.speltoides was used for blocking.NDFISH with oligo-pTa535 and oligo-pSc119.2 or oligo-3BL as probes was performed according to Tang et al.[49]and Tang et al.[50].The oligonucleotide sequences were 5′-or 3′-end labeled with 6-carboxyfluorescein or 6-carboxytetramethylrhodamine.All images were acquired with an Olympus AX80 fluorescence microscope(Olympus Corp.)and processed with Adobe Photoshop CS 3.0(Adobe,San Jose,CA,USA).

2.4.DNA marker analysis

A.cristatum-specific sequence-tagged site(STS)markers were developed from expressed sequence tag(EST)sequences identified in A.cristatum transcriptome sequences[51].These STS markers were used to characterize wheat–A.cristatum 5P derivative lines.To detect the presence of the Ph1 locus on chromosome 5B in the wheat–A.cristatum 5P addition line,two specific markers for the Ph1 locus,Xwgc2111 and Xwgc2049[52],were used.PCR amplification was performed as described by Luan et al.[53].The amplified products were separated by 8%polyacrylamide or 1.5%agarose gel electrophoresis.

2.5.RNA-seq library preparation and sequencing

Anthers at the pachytene stage were collected from common wheat Fukuho and WA5P plants.Three biological replicates were prepared for each genotype,so that a total of six samples were obtained.Six libraries were constructed following the protocol of the Gene Expression Sample Prep Kit(Illumina,San Diego,CA,USA).The libraries were sequenced by Allwegene Tech.Corporation(Beijing,China)using the Illumina HiSeq 2500 platform with a paired-end read length of 150 bp.

2.6.Alignment of RNA-seq reads and expression analysis

The raw sequence reads were cleaned by removing RNA adapters and trimming reads with＞50% low-quality bases(Q≤20)using Trimmomatic(version 0.36)[54].The clean reads of the six samples were de novo assembled using the Trinity package(version 2.4.0)with default parameters[55].Unigenes were subjected to BLASTx searches and annotation against the NCBI nr and nt(https://www.ncbi.nlm.nih.gov/),Pfam[56],Swiss-Prot(https://www.uniprot.org/),eggNOG,KEGG,and GO databases[57].The fragments per kilobase of transcript per million mapped reads(FPKM)values of the unigenes were calculated using RSEM(version 1.3.0)[58].Genes showing a mean expression level＞0.3 FPKM were retained for differential expression analysis.Significantly differentially expressed transcripts were identified using DESeq2 software[59]according to a|log2(fold change)|＞1 and adjusted P-value≤0.05.GO and KEGG enrichment analyses of differentially expressed genes were performed with GOseq(version 3.8)[60]and KOBAS(version 2.0)[61].BLASTn was used to search against the IWGSC RefSeqv1.0 genome[62]and the A.cristatum transcriptome database[63]to verify locations of the unigenes.

2.7.Quantitative reverse transcription PCR(qRT-PCR)analysis

Total RNA(2 μg)from each sample was employed to generate complementary DNA(cDNA)templates using M-MLV reverse transcriptase(TaKaRa,Tokyo,Japan)according to the manufacturer’s instructions.qRT-PCR was performed using SYBR Green Real-time PCR Master Mix(TaKaRa)on a StepOnePlus Detection System(Applied Biosystems,Inc.,Foster City,CA,USA).The PCR conditions were as follows:initial step at 95°C for 3 min,followed by 40 cycles of 95°C for 15 s,60°C for 15 s and 72°C for 30 s.For each sample,the PCR analysis was independently repeated in triplicate,and quantification of gene expression followed the relative quantification method(2-ΔCT method)with β-actin as an endogenous control.Specific primer pairs for qRT-PCR are listed in Table S1.

3.Results

3.1.Agropyron cristatum chromosome 5P induced nonhomoeologous chromosome translocation

To clarify the role of chromosome 5P in inducing chromosomal structural variation,the chromosome constitutions of 121 BC2F4plants from the hybridization of wheat–A.cristatum 5P disomic addition line II-11-1b and Fukuho were identified.Four types of chromosomal structural variation were identified using GISH:5P chromosome deletion,5P chromosome translocation,translocation between wheat chromosomes and the 5P chromosome,and wheat chromosome deletion.To further determine the breakpoints of the 5P deletions and translocations,these lines were evaluated using 56 DNA markers specific to chromosome 5P(Fig.1a).Based on the results of GISH and marker analysis,18 5P deletion lines could be categorized into 12 types.The breakpoints most commonly occurred on the long arm of chromosome 5P,followed by the centromere,with only one breakpoint on the short arm(Fig.1b).Among the five identified wheat chromosome deletions,the breakpoints occurred mainly at the centromere and on the long arm of the chromosome(Fig.S2).The single 5P chromosome translocation was determined to be T5PS.5PS based on the specific markers located only on the 5PS chromosome and the characteristics of heterochromatin at the terminus of 5PS(Fig.1b).To identify the wheat chromosomes involved in the wheat–A.cristatum 5P translocated chromosomes and potential translocations between wheat chromosomes,ND-FISH was performed using oligopTa535 and oligo-pSc119.2 as probes.The FISH map of the Fukuho chromosomes was used as a reference(Fig.S3),and 10 translocations between wheat chromosomes and A.cristatum 5P were identified and categorized into six types.Four types(seven plants)were shown to be nonhomoeologous translocations,including T5PS.5PL-2A,T5PS-1BS.1BL,T5P-7BS.7BL,and T5P-2AS.2AL(Fig.2),and the specific translocations in two cases were not determined.Only one translocation was a homoeologous translocation(T5PS.5PL-5B)(Fig.2a,d).Two nonhomoeologous translocations between wheat chromosomes(T3A.4B and TA.D)were identified(Fig.S4).

Fig.1.Physical map and chromosomal structural variations of the A.cristatum 5P chromosome.(a)Physical map divided into 14 bins with 56 STS markers.Left panel,chromosomal bins;Right panel,locations of STS markers.(b)Deletions and translocations of chromosome 5P were identified by GISH,and breakpoints were determined using STS markers.‘‘+”and‘‘-”indicate respectively the presence and absence of chromosome 5P-specific markers.

3.2.The effect of chromosome 5P was independent of the number of 5P chromosomes

Fig.2.GISH and ND-FISH identification of wheat–A.cristatum 5P translocated chromosomes.(a–c,g–i)GISH patterns of wheat–A.cristatum 5P translocated chromosomes obtained using the A.cristatum P genome(red)as the probe;(d–f,j–l)FISH patterns of wheat–A.cristatum 5P translocated chromosomes obtained using oligo-pTa535(red)and oligo-pSc119.2(green)as probes.Scale bars,10 μm.

Chromosomal structural variations could be caused by aberrant meiosis.To determine whether the 5P chromosome could induce chromosomal variation in both monosomic and disomic states,wheat–A.cristatum 5P disomic and monosomic addition lines in which there were no structural variations were used to investigate the meiotic pachytene stage,metaphase I,and anaphase I in pollen mother cells.In comparison with Fukuho,abnormal chromosomal behavior occurred in the metaphase I and anaphase I of the wheat–A.cristatum 5P monosomic addition line and was also observed in 5P disomic addition lines.Many univalents(involving both the wheat chromosomes and chromosome 5P)and chromosomal adhesions were observed in metaphase I(Fig.3).Multivalents formed by wheat chromosome pairing were also recorded(Fig.S5a,b).In anaphase I,chromosomal bridges,various numbers of lagging chromosomes,asynchronous chromosomal dissociation,and homologous chromosomes migrating toward the same pole were observed(Fig.3).Chromosome fragments were also observed in both stages(Fig.S5c,d).This observation suggested that the phenomenon of abnormal meiosis was not stochastic,but was associated with chromosome 5P.The similar chromosomal abnormalities induced by the 5P chromosome were not affected by the number of 5P chromosomes.In addition,translocations were observed between wheat chromosomes and chromosome 5P(Fig.S5e,f),suggesting that translocation induced by the 5P chromosome might have occurred in prophase.

3.3.Chromosome 5P disturbed homologous chromosome pairing

The chromosomal constitutions during meiotic metaphase I were further compared between the wheat–A.cristatum 5P monosomic addition line WA5P and Fukuho.WA5P was obtained from BC6,in which structural variations of wheat chromosomes were not present.Chromosome 5P caused increases in the numbers of univalents(2.39,n=30)and rod bivalents(5.82,n=30)in the wheat background.Correspondingly,the number of crossovers decreased(34.68,n=30).The wheat chromosomes that formed univalents in metaphase I came from three subgenomes,as revealed by mcGISH(Fig.S6),suggesting an absence of genomic bias.Thus,chromosome 5P influenced homologous chromosome pairing.Observation of anaphase I during meiosis revealed abnormal segregation in 73%(22 of 30)of the cells.

To investigate the behavior of wheat homologous chromosomes during meiosis I in the wheat–A.cristatum 5P addition line,a specific oligo-3BL probe targeting chromosome 3B was used to track the behavior of this chromosome.In the pachytene and metaphase I stages,two adjacent green signals,representing the closely paired homologous chromosomes,were observed in common wheat Fukuho(Fig.4a,b).However,13%(4 of 30)of the cells showed two separate oligo-3BL signals in these two stages in WA5P(Fig.4g,h),showing that the chromosome 3B pair had separated.Chromosome 3B homologs that were not separated at anaphase I were also observed(Fig.4c,i).The other cells showed normal chromosome behavior in WA5P(Fig.4d,e,f).These results indicated that homologous pairing was severely disturbed by chromosome 5P in the pachytene stage.

3.4.Chromosome 5P affected double-strand break(DSB)repair

Based on the abnormal meiotic stage,anthers from common wheat Fukuho and WA5P were sampled in the pachytene stage.The six transcriptome libraries generated 40,660,022 and 37,783,776 sequence reads on average in Fukuho and WA5P,respectively.After removal of low-quality reads,the mean total number of clean RNA-seq reads retained for subsequent analysis was 39,873,510 in Fukuho and 37,018,166 in WA5P.

Fig.3.Chromosome spreads of pollen mother cells(PMC)from common wheat Fukuho,a wheat–A.cristatum 5P disomic addition line,and a 5P monosomic addition line.(a,e,i)Pachytene;(b,f,j)metaphase I;(c,d,g,h,k,l)anaphase I.Scale bars,10 μm.

Fig.4.Localization of oligo-3BL signals in PMCs of common wheat Fukuho and WA5P in corresponding meiotic stages.(a,d,g)Pachytene;(b,e,h)Metaphase I;(c,f,i)Anaphase I.Scale bars,10 μm.

Assembly of the clean reads produced 146,776 unigenes,ranging in length from 201 to 15,648 bp,with the N50 size 1091 bp(Table S2).The unigenes were used as reference sequences for mapping the clean reads.Of these unigenes,731 showed differential expression between WA5P and Fukuho,including 716 upregulated and 15 downregulated genes in WA5P relative to Fukuho(Fig.S7;Table S3).We focused on whether the genes were differentially expressed during meiosis.Functional annotations and GO term enrichment(Table S4)identified 252 genes that might be involved in meiosis.However,none of the genes showing differential expression was involved in meiosis initiation,sister chromatid cohesion,centromeric cohesion protection,meiotic DSB formation,meiotic DSB processing,DNA strand invasion or exchange,synaptonemal complex formation,or crossover formation(Fig.S8).Thirteen differentially expressed genes involved in DNA repair or chromatin remodeling were upregulated in WA5P(Fig.5a;Table 1).The results of qRT–PCR were consistent with those of the differential expression analysis(Fig.5b).With the exception of TRINITY_DN42058_c0_g1 and TRINITY_DN26967_c2_g4,the other 11 genes came from A.cristatum chromosome 5P according to sequence alignment using BLASTn searches against the IWGSC RefSeqv1.0 genome and the A.cristatum transcriptome database.Among these genes,TRINITY_DN38985_c1_g1 and TRINITY_DN43253_c1_g8 were annotated as RAD52-like and MSH6 genes,respectively;the first gene is involved in meiotic double-strandrepair and the second in DNA mismatch repair.We developed polymorphic markers based on the sequences of the 11 differentially expressed unigenes,and the markers were specific to A.cristatum and WA5P(Fig.S9;Table S1).This indicated that the 11 genes were all located on chromosome 5P.Thus,chromosome 5P,which caused chromosomal structural variation in the wheat background,influenced the process of meiotic double-strand break repair.

Table 1 Thirteen differentially expressed genes involved in DNA repair or chromatin remodeling.

We used Xwgc2111 and Xwgc2049 to detect the presence of the Ph1 locus in the wheat–A.cristatum 5P addition line.The results showed no deletion of fragments in WA5P that was different from that observed in ph1b mutants(Fig.6).TaZIP4-B2,a candidate gene for Ph1[64],was present in WA5P,as shown using a specific marker(Fig.6).The TaZIP4-B2 gene was not differentially expressed between the transcriptomes of WA5P and Fukuho(Fig.S8).A similar result was found for TaMSH-3D(Fig.S8),the target gene of Ph2[65].Thus,chromosomal structural variation occurred in wheat–A.cristatum 5P derivative lines in the presence of the Ph1 and Ph2 genes.

4.Discussion

4.1.A new type of gene inducing chromosomal structural variations located on A.cristatum chromosome 5P

The effect of chromosome 5P observed in the common wheat background was different from that of Gc chromosome.Not only was the wheat–A.cristatum 5P monosomic addition line shown to cause abnormal meiosis,but the 5P disomic addition line showed a similar effect.Moreover,chromosome 5P generated chromosomal structural variations,especially translocations involving this chromosome.The Ph1 suppressor gene identified in several wheat relatives achieves the effect of ph1 gene deletion mainly by suppressing the Ph1 gene.The effect is different from that of chromosome 5P in the following respects:the Ph1 inhibitor generally induces homoeologous chromosome translocation[14,22,66],while chromosome 5P can induce translocation between nonhomoeologous chromosomes,such as T4B.3A translocation;translocations between wheat chromosomes and alien chromosomes induced by ph1 mutants usually involve alien chromosome monosomic substitution or heterozygous translocation of alien fragments[18,19,67],whereas 5P-induced translocation can occur in addition lines;and the presence of many univalents induced by 5P and the production of nonhomoeologous translocations suggest that the mechanism by which this chromosome influences meiosis differs from that of Ph1 gene suppression.What these mechanisms have in common is the chromosomal structural variation produced in the presence of the Ph1 gene.

Fig.5.Differential expression of genes involved in DNA repair or chromatin remodeling between WA5P and Fukuho.(a)Thirteen differentially expressed genes identified by transcriptomic analysis.(b)qRT–PCR analysis of six differentially expressed genes.

Robertsonian translocations can arise by centromeric misdivision of univalents followed by the segregation of the derived telocentric chromosomes to the same nucleus and the fusion of the broken ends during interkinesis in meiosis II[68].However,Robertsonian translocations were rarely observed in our study,and translocated chromosomes could be found in meiosis I,indicating that the mechanism of misdivision–fusion differed from that of chromosome 5P.The translocation frequency induced by misdivision–fusion was only 2.0%in our previous wheat–A.cristatum 6P substitution line[69].The translocation frequency was much lower than the 8.3%(10/121)translocation frequency induced by chromosome 5P.It was previously[44]found in our laboratory that chromosome 1.4P could induce recombination between some wheat homoeologous chromosomes.In the present study,chromosome 5P not only induced wheat chromosome translocation but also caused translocation involving the 5P chromosome and wheat chromosomes.Collectively,these findings suggest the presence of a new type of gene on chromosome 5P that can induce chromosomal structural variation.

4.2.Chromosome 5P can be used as a new tool for wheat improvement

A.cristatum chromosome 5P not only induces structural variations in wheat chromosomes but also shows the same function as an A.cristatum chromosome introduced into common wheat,and may be used as a new tool for the mutagenesis breeding of wheat.Chromosome 5P could be effectively used for the production of chromosome deletions and especially for addition of alien chromosomes to common wheat.This strategy can be compared to the application of the Gc system for the creation of deletion lines.For example,barley 7H deletion lines were produced by 2C of Aegilops cylindrica[70],and a DNA marker bin map was constructed using the rearranged 7H chromosomes[10].In the present study,we obtained nine deletions involving different sizes of 5P chromosome segments,which were identified using DNA markers.These genetic stocks will be useful for constructing a physical map of chromosome 5P and mapping genes of interest or for creating other novel materials.Another use of the chromosome 5Pinduced chromosomal mutation approach would be to produce wheat–alien translocations with the aim of transferring useful alien chromatin into the wheat genome.Although there are many nonhomoeologous translocations between chromosome 5P and wheat chromosomes,translocation lines with desirable traits as well as good balance and genetic stability may be directly used in wheat improvement.Many nonhomoeologous translocations are produced in wheat breeding programs,such as the 1RS?7DL and 7DS?1BL translocations identified in the founder germplasm‘‘Aimengniu”[71].Investigating the phenotypic effects of these translocations can reveal whether the translocations are favorable or unfavorable.To exploit the genes that induce chromosomal structural variation on chromosome 5P,the production of small segmental translocation lines and the fine mapping and cloning of genes with the aid of the P reference genome will be the focus of the next step of this work.

Fig.6.Detection of the Ph1 locus in a wheat-A.cristatum 5P addition line and its derivative lines using the DNA markers Xwgc2111,Xwgc2049,and TaZIP4-B2.M,DNA marker;lane 1,A.cristatum Z559;lane 2,WA5P,lanes 3–13,wheat-A.cristatum 5P derivative lines;lane 14,CS ph1b mutant.

4.3.Genes on chromosome 5P influence meiotic DSB repair in the wheat background

Meiosis is a specialized form of cell division producing haploid germ cells that is essential for sexual reproduction and ensuring stable chromosome numbers in eukaryotes.During meiosis,crossovers(COs)form between homologous chromosome pairs and ensure accurate chromosome segregation.Homologous recombination(HR)and classical nonhomologous end joining(C-NHEJ)are two basic strategies for DSB repair;the first is key to the generation of COs and the second involves direct ligation of the broken ends[72].In the present study,11 genes involved in DNA repair were more highly expressed in WA5P than in Fukuho based on transcriptome analysis,suggesting that the functional genes on chromosome 5P may either be DNA repair genes or regulate the process of DNA repair.

During meiosis,HR not only occurs between allelic DNA segments on homologous chromosomes but also may occur between nonallelic DNA sequences that share high sequence similarity[73–75].The outcomes of nonallelic homologous recombination(NAHR)events can be channeled into noncrossover events by mismatch repair proteins and/or several helicases[76].The suppression of AtMSH6 does not affect the rate of meiotic recombination but increases the frequency of recombination between two homoeologous repeats of a marker gene by three fold relative to that observed in wild-type plants[77].Although the MSH6 gene was up-regulated in WA5P,whether this up-regulation caused NAHR was unclear.In WA5P,many univalents and chromosome aggregation were observed in metaphase I,along with chromosome bridging in anaphase I and homologous chromosome pairing abnormalities in the pachytene phase;these phenomena were similar to findings reported in the rice Osrad17 Oszip4 double mutant[78].These similar phenomena suggested that the nonhomoeologous chromosomal translocations observed in this study were formed by NHEJ,and the identified chromosome deletions indicated that NHEJ could not complete DNA repair.And the similar phenomena seem to suggest that the function of chromosome 5P is similar to that of the Oszip4Osrad17 double mutation.However,TaZIP4-B2 and TaRAD17 were not differentially expressed in WA5P compared with Fukuho.The mechanism of chromosome 5P-induced chromosomal variation invites further study.

CRediT authorship contribution statement

Haiming Han:Data curation,Formal analysis,Funding acquisition,Investigation,Methodology,Software,Validation,Visualization,Writing–original draft,Writing–review & editing.Xinyuan Ma:Investigation,Methodology,Software,Visualization.Zhen Wang:Investigation,Methodology,Software,Visualization.Kai Qi:Investigation,Methodology,Visualization.Wenjing Yang:Investigation,Methodology,Visualization.Weihua Liu:Investigation,Methodology,Resources.Jinpeng Zhang:Investigation,Methodology.Shenghui Zhou:Investigation,Methodology.Yuqing Lu:Investigation,Methodology.Xinming Yang:Investigation,Methodology.Xiuquan Li:Investigation,Methodology.Lihui Li:Conceptualization,Funding acquisition,Project administration,Supervision,Resources,Writing–review & editing.

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 financially supported by the National Key Research and Development Program of China(2021YFD1200605)and the National Natural Science Foundation of China(32171961).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.06.002.