Jing Wng,Chng Liu,Xinrui Guo,Ke Wng,Lipu Du,Zhishn Lin,*,Xingguo Ye,*

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

b State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

Keywords:Wheat Double substitution lines Molecular markers Transmission rate of alien chromosomes Genomic in situ hybridization

A B S T R A C T Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits.In this study we created wheat double substitution lines by hybridizing different wheat-Th.intermedium and wheatbarley disomic alien substitution lines,with the aim of using genes in Th.intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs.As expected,we obtained two types of wheat double substitution lines,2D2Ai#2(2B)2H(2A)and 2A2Ai#2(2B)2H(2D),in which different group 2 wheat chromosomes were replaced by barley chromosome 2H and Th.intermedium chromosome 2Ai#2.The new materials were characterized using molecular markers,genomic in situ hybridization(GISH),and fluorescent in situ hybridization(FISH).GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes,a pair of barley chromosomes,and a pair of Th.intermedium chromosomes.Analysis using specific DNAmarkers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes.Chromosome 2H showed a higher transmission rate than 2Ai#2,and both chromosomes were preferentially transmitted between generations via female gametes.Evaluation of botanic and agronomic traits demonstrated that,compared with their parents,the new lines showed similar growth habits and plant type but differences in plant height,flowering date,and self-fertility.Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis.The novel substitution lines can potentially be used in wheat meiosis research and breeding programs.

1.Introduction

Distant hybridization,in which desirable chromosomes or fragments are transferred from genetically similar plant species to cultivars,is an effective technique for developing new germplasm or varieties[1,2].For example,wild relative species of common wheat(Triticum aestivum L.2n=6x=42,AABBDD)harbor many genes that can be introduced to expand wheat genetic variation and improve wheat biotic and abiotic stress resistance and grain quality.To date,many amphidiploids and addition lines between wheat and its wild relative species have been created via chromosome doubling and stepwise backcrossing,respectively,after distant hybridization. But meiotic recombination between the homoeologous chromosomes of wheat and its wild relatives seldom occurs in amphidiploids and addition lines.A more practical way to take advantage of the traits available in wild wheat species is to develop translocation lines.Developing substitution lines between wheat and its wild relative species is a step toward this goal.For this reason,obtaining stable substitution lines,especially double substitution lines containing multiple pairs of chromosomes from other plant species is very important to wheat breeding.

Thinopyrum intermedium(2n=6x=42,E1E1E2E2StSt)is an allohexaploid species in the Triticeae tribe that has many useful traits that can be used for wheat improvement,such as tolerance to abiotic stresses[3],vigorous growth,and high disease resistance to barley yellow dwarf virus(BYDV),rusts[4-6]and powdery mildew(PM)[7].Many workers have hybridized Th.intermedium with wheat and have bred new lines or cultivars,including addition,substitution,and translocation lines with resistance to BYDV and leaf rust[8-11].

BYDV is one of the most destructive and extensive wheat diseases in the world and causes severe losses in wheat production.Breeding new wheat varieties with high BYDV resistance is the most direct and effective strategy to overcome this disease.The wheat-Th.intermedium disomic chromosome addition line L1[12]and the wheat-Th.intermedium chromosome substitution line P29[13]were found to have desirable resistance to BYDV[13-16],and the chromosome segment carrying the resistance gene Bdv2 was transferred from L1 to wheat via translocation induced by in vitro tissue culture[14,15].The wheat-Th.intermedium disomic addition lines Z1,Z2,and Z6 containing chromosome 2Ai#2 showed high resistance to BYDV[17].Lin et al.[18]developed several wheat-Th.intermedium substitution lines in which wheat chromosomes 2B and 2D were replaced by 2Ai#2.

Barley(Hordeum vulgare L.,2n=2x=14,HH)is one of the most important cereal crops in the world and is a source of feed for livestock and of malt for brewing.For decades,attempts have been made to hybridize wheat with barley,and the first hybrid was made successfully by Kruse[19].Later,six disomic addition lines with the wheat cultivar Chinese Spring(CS)genetic background,each carrying one of the six chromosomes 2H,3H,4H,5H,6H,and 7H,from the barley cultivar Betzes were developed[20].Wheat substitution lines in which barley chromosomes 1H,3H,6H,and 7H replaced the wheat homoeologous chromosomes in groups 1,3,6,and 7,respectively,were reported by Akmr[21]and Efremova[22].Many important genes carried on barley chromosome 2H could be used to improve the characteristics of common wheat.Rar1,which contains a novel eukaryotic protein domain,is activated by race-specific resistance(R)genes and is an aggregation point in the powdery mildew resistance signaling pathway,regulating H2O2accumulation in host cells[23].Cly1 and ppd-H1 control flowering time in barley[24,25].Isa is located on barley chromosome 2HL and encodes a cereal storage protein,bifunctional α-amylase/subtilisin inhibitor(BASI).Isa is expressed in the endosperm during grain development/maturity stage and affects endogenous α-amylase activity and controls preharvest sprouting damage[26].In fact,some wheat varieties containing Isa that show reduced preharvest sprouting have been developed[26].

The above germplasm resources,including wheat-Th.intermedium and wheat-barley disomic alien substitution lines,are important materials for genetic analysis and novel gene discovery.However,wheat double substitution lines have not yet been reported.In the present study,to develop further materials for wheat breeding and cytological studies,two types of wheat double substitution lines were obtained by hybridizing different wheat-Th.intermedium and wheat-barley disomic alien substitution lines.Following five generations of selfing,wheat double substitution lines were identified using molecular markers and cytogenetic assessment,including genomic in situ hybridization(GISH)and fluorescent in situ hybridization(FISH).The results of this investigation will be useful for studying wheat homoeologous recognition during meiosis and for developing new translocation lines between three plant species.

2.Materials and methods

2.1.Plant materials and crosses

The materials used in this study include the wheat-Th.intermedium 2Ai#2(2D)disomic substitution lines N431 and N452,the 2Ai#2(2B)disomic substitution lines N439 and N420[18],the wheat-Th.intermedium 2Ai#2 disomic addition line Z6(2n=44)[5],the wheat-barley 2H(2A),2H(2B),and 2H(2D)disomic substitution lines X99-4,X99-7,and X99-12,the CSBetzes 2H disomic addition line(2n=44)[27],and the wheat cultivar Chinese Spring(CS).All these materials were maintained in our laboratory.Genomic DNA of barley cv.Betzes and Th.intermedium accession 440,001 were provided by Prof.Fangpu Han at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences.In total,six crosses were made in 2008 at the experimental station of the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences in Beijing:N431/X99-7,N452/X99-7,X99-12/N420,X99-12/N439,X99-4/N420,and X99-4/N439.Plants containing target chromosomes in generations F1to F5were identified using chromosome-specific molecular markers.In addition,three types of F1generations were crossed with the wheat line CS to generate BC1populations,which were genotyped using molecular markers specific to the alien chromosomes to calculate the transmission rates of the alien chromosomes through the male and female gametes.

2.2.Chromosome-specific molecular markers

Primers specific to barley chromosome 2H were designed based on the sequences of wheat ESTs located on wheat group 2 chromosomes using Primer Premier 6.0 software(http://www.premierbiosoft.com/primerdesign/).Primers specific to the alien chromosome Th.intermedium 2Ai#2 and to the wheat chromosomes 2A,2B,and 2D,designed based on the sequences of TaARG genes located on wheat group 2 chromosomes,were developed in our previous studies[28,29].Primers P1-2A,P2-2B,and P3-2D specifically identify wheat chromosomes 2A,2B,and 2D,and primers P97 and P79 can be used for specific detection of Th.intermedium chromosome 2Ai#2 and barley chromosome 2H at the same time by amplifying bands of different size(Table 1).

2.3.DNA extraction and polymerase chain reaction(PCR)amplification

Genomic DNA was extracted from one-month-old leaves of F2-F5plants,CS,substitution lines,and addition lines using the NuClean Plant Genomic DNA Kit(CW Bio Inc.,Beijing,China).The DNA pellet was dissolved in sterile water,and then diluted to 100 μg μL-1for use as template for PCR amplification and other detection purposes.Total genomic DNA was extracted from fresh leaves of Th.intermedium,barley,and wheat using the CTAB method.Th.intermedium and barley genomic DNA were used as probes and wheat genomic DNA was used as a block in GISH and FISH analysis.

Molecular markers specific to alien chromosomes 2H and 2Ai#2 and wheat chromosomes 2A,2B,and 2D were used to identify wheat double substitution lines.Amplification of fragments corresponding to wheat chromosomes 2A,2B,and 2D from CS but not from F2-F5plants indicated that these chromosomes were lost.Amplification of fragments from F2-F5offspring plants using primers specific to alien chromosomes 2Ai#2 and 2H indicated that the wheat chromosomes were replaced by the alien chromosomes.

For PCR amplification,the 15 μL PCR mixture contained 5.9 μL distilled water,1.0 μL template DNA(100 ng μL-1),0.3 μL each primer(10 μmol L-1),and 7.5 μL 2× Taq MasterMix(containing Mg2+and dNTPs,CW Bio Inc.).PCR was performed in a Biometra TProfessional thermal cycler(Life Technologies,Germany),with an initial denaturation at 95°C for 5 min,35 cycles of 30 s at 95°C,30 s at different annealing temperatures dependent on the specific primers(58°C for 2A-,2B-,2D-,and 2Ai#2-specific primers and a temperature gradient from 53 °C to 54 °C for 2H-specific primers),45 s at 72 °C,and a final extension for 8 min at 72°C.Detailed information about these primers is presented in Table 1.PCR products were separated on a 1%agarose gel with a standard TAE buffer and visualized under UV light after ethidium bromide staining.

2.4.Cytological analysis at mitosis and meiosis

Wheat seeds were soaked in water for 24 h and transferred to Petri dishes containing moist filter paper.The root tips were cut from the germinating seeds when they reached about 4-5 cm in length,placed into a nitrous oxide container for 2 h,and then fixed in alcohol.Cell spreading preparations at mitosis were made as previously described[30].The spikes of the wheat double substitution lines at different stages of meiosis were fixed in Carnoy solution and treated in the same way as root tips,after which chromosome spreading slides of pollen mother cells(PMCs)were made.The chromosome configuration in a suitable cell was photographed using a fluorescence microscope(Leica DMR,Germany)equipped with a chargecoupled device(CCD)camera(Zeiss AxioCam HRM,Germany).

2.5.GISH and FISH identification

The putative wheat double substitution lines identified based on molecular markers and chromosome configurations were analyzed by GISH following previously described methods[31].The total genomic DNA of Th.intermedium and barley was labeled with fluorescein-12-dUTP and Texas-Red-5-dCTP,respectively,using the nick translation method according to the manufacturer's instructions.The labeled DNA was used as a probe.Total genomic DNA of wheat was fragmented by ultrasonication for 5 min and used as a block.Hybridization signals were observed under the fluorescence microscope and photographed as described above.

The probe D05 specific to the chromosome arms of Th.intermedium[32],and the oligonucleotide probe(AGGGAG)nspecific to the centromeres of barley chromosomes were used in FISH experiments to detect the presence of alien chromosomes.The probe CRW(Centromere retrotransposon of wheat)and probes specific to wheat centromeres and telomeres were also included in the FISH analysis.The four probes were labeled with digoxigenin-11-dUTP or biotin-16-dUTP using nick translation,and then correspondingly detected with anti-DIG-fluorescein or avidin rhodamine.Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole(DAPI),and FISH images were captured with a CCD camera.

Table 1-Primers used for tracing chromosomes in homoeologous group 2 of wheat,barley,and Th.intermedium.

2.6.Evaluation of agronomic traits

The main growth characteristics of the putative double substitution lines and their parents,including tillering habit,booting date,flowering date,and plant type,were recorded.At the maturity stage,plants from each line were randomly collected to assess the main botanic and agronomic traits,including plant height,spike length,spike shape,1000-kernel weight,grain length and width,and self-fertility.Self-fertility was calculated by dividing the number of seeds set by the total number of florets per spike.

3.Results

3.1.Screening of target plants in five generations using chromosome-specific markers

Six crosses,N431/X99-7,N452/X99-7,X99-12/N420,X99-12/N439,X99-4/N420,and X99-4/N439,corresponding to F1codes A,B,C,D,E,and F,respectively,were made and the progeny were repeatedly selfed.Molecular markers that had been previously verified in different materials including wheat and different wheat substitution lines were used to genotype the plants in the F1to F5generations(Tables 1,2).To verify that the F1plants from the cross combinations were genuine hybrids,two molecular markers,P79 and P97,which specifically amplify fragments from the long arm of barley 2H and the short arm of Th.intermedium 2Ai#2,respectively,were employed to genotype all the F1plants.The F1plants from five of the six cross combinations were found to carryboth alien chromosomes.The F1plants from the remaining cross combination,N431/X99-7(A),carried chromosome 2Ai#2 but not 2H(Fig.1)and were excluded from further experiments.

Table 2-Identification of plants in the F1 to F5 generations containing different combinations of chromosomes.

In the F2plants derived from cross combinations B,C,and D,substitution events involving wheat chromosomes 2B and 2D and the two alien chromosomes 2H and 2Ai#2 would be expected because these chromosomes are present in these lines and should behave as univalents during meiosis.Similarly,substitutions among chromosomes 2A,2B,2H,and 2Ai#2 would occur in the F2offspring of cross combinations E and F.A total of 151 F2seedlings from the five crosses were identified using markers specific to the five targeted chromosomes.The genotyping results were different for different crosses(Table S1).Seedlings with the desired genotype were recovered from each cross,although at different frequencies.Ultimately,those plants with both alien chromosomes were grown to produce the next generation.

To obtain homozygous substitution lines,the F3to F5generations derived from the targeted F2plants were analyzed using markers specific to the group 2 homoeologous chromosomes.In the F3progeny,a plant derived from cross C,C-30-5,was shown to harbor alien chromosomes 2H and 2Ai-2 and to lack wheat chromosomes 2B and 2D(Table 3),whereas four plants derived from cross E,E-2-6,E-2-7 E-2-8,and E-19-3,and one plant derived from cross F,F-27-4,harbored 2H and 2Ai-2 and lacked 2A and 2B(Table 3).The genotypes of these plants were confirmed in subsequent generations;randomly selected plants in the F5population were genotyped using chromosome-specific markers and shown to contain both alien chromosomes,indicating that these substitution lines are stable(Figs.2 and 3).In summary,the two alien chromosomes barley 2H and Th.intermedium 2Ai#2 were stably inherited,and the target plants containing the two alien chromosomes were obtained in subsequent generations(Tables 2 and 3).

3.2.Transmission rates of the two alien chromosomes via male and female gametes

To determine whether substitution line background and gamete type influenced the transfer of the alien chromosomes,reciprocal crosses were made between wheat cultivar CS and the three substitution lines 2Ai#2(2D)/2H(2A),2Ai#2(2B)/2H(2D),and 2Ai#2(2B)/2H(2A).Molecular markers specific to the two alien chromosomes were then used to genotype plants in the BC1generation derived from these crosses.The transmission rates of the two alien chromosomes in the BC1generation were found to differ for different crosses(Table 4):the average transmission rates for alien chromosome 2H via the male and female gametes were 24.7%and 32.7%respectively,whereas those for the alien chromosome 2Ai#2 were 19.3%and 21.8%.The alien chromosome 2H thus appeared to be transmitted to the next generation at a higher rate than the alien chromosome 2Ai#2 and the two alien chromosomes were preferentially transmitted via the female gametes.Therefore,it is likely that more plants with alien chromosomes will be obtained when the materials harboring alien chromosomes are used as the female parent.

Fig.1-Identification of six F1 hybrids using molecular markers P79(top panel)and P97(bottom panel).Lane 1,Z6;lane 2,CS+2H;lane 3,CS;lanes 4 and 10,F1 hybrids of cross A;lanes 5,11,and 12,F1 hybrids of cross B;lane 6,F1 hybrid of cross C;lane 7,F1 hybrid of cross D;lane 8,F1 hybrid of cross E;lane 9,F1 hybrid of cross F.The F1 hybrid plants from cross combination A carry only chromosome 2Ai#2,and the F1 hybrid plants from the other five cross combinations(B,C,D,E,and F)carry both alien chromosomes.

3.3.Verification of wheat double substitution lines using GISH

To further confirm the identity of the wheat double substitution lines genotyped using molecular markers,chromosomes in root tip cells from these lines were observed.Every cell observed contained the expected number(42)of chromosomes(Fig.4a,b).In GISH analysis of randomly selected root tip cells from the double substitution lines using total genomic DNA of Th.intermedium and barley as a probe,two red hybridization signals and two green hybridization signals were visible(Fig.4c,d).Thus,in agreement with molecular marker analysis,the two double substitution lines 2A2Ai#2(2B)2H(2D)and 2D2Ai#2(2B)2H(2A)carry two chromosomes from Th.intermedium and two chromosomes from barley.

To determine whether the two double substitution lines were cytologically stable,54 randomly selected PMCs were observed at meiotic metaphase I.GISH using total genomic DNA of Th.intermedium and barley as a probe revealed that 40 of these PMCs harbored one bivalent and two univalents with strong GISH hybridization signals(Fig.4e,f).This finding indicated that in most of the double substitution lines,the chromosomes from wheat were substituted by a pair of chromosomes from Th.intermedium and a pair from barley.Of the remaining PMCs,ten cells harbored two bivalents and four cells only a single univalent.Thus,most of the double substitution lines were not cytogenetically stable as expected in the selfing progeny.

3.4.Verification of wheat double substitution lines using FISH

Fluorescent in situ hybridization was employed to verify the replacement of two pairs of wheat group 2 chromosomes by Th.intermedium chromosome 2Ai#2 and barley chromosome 2H.The chromosomes in the double substitution lines were visualized at mitotic metaphase by hybridizing cells with the wheat centromere probe CRW labeled with digoxigenin-11-dUTP(red),the Th.intermedium chromosome arm probe D05 labeled with biotin-16-dUTP(green),and a barley centromere probe also labeled with biotin-16-dUTP(green).In the root tip cells of the double substitution lines,there were red hybridization signals in the centromeric regions of 38 wheat chromosomes,green hybridization signals in the centromeric regions of 2 barley chromosomes,and faint green hybridization signals on both arms of 2 Th.intermedium chromosomes(Fig.5).This result was consistent with those from molecular marker analysis and GISH.

3.5.Evaluation of the agronomic traits of wheat double substitution lines

The wheat double substitution lines and their parents grown in different environments displayed similar growth habits,but differences in flowering date,plant type,and other agronomic characteristics.Briefly,the wheat-Th.intermedium substitution lines flowered 10 days earlier than the wheat-barley substitution lines,and substitution line 2Ai#2(2B)flowered earliest.The double substitution lines flowered latest,on average 15 days later than the parents.The double substitution lines were distinctly shorter,by 50-60 cm,than their parents and also had spikes that were 1.8-3.5 cm shorter than those of their parents.The 1000-kernel weights of the double substitution lines were lower,by 15-34 g,than those of their parents.The kernel length and width of the double substitution lines were also significantly different from those of their parents(Fig.6).The spikes of the double substitution lines were spindle-shaped,but those of their parents were rod-shaped(Fig.6).The homozygous double substitution lines had a self-fertility rate of only 15%,much lower than that(80%-90%)of their parents.

Table 3-Screening of putative wheat double substitution lines in the F3 generation using specific markers.

Fig.2-Identification of the wheat double substitution line 2D2Ai#2(2B)2H(2A)using molecular markers specific to the alien chromosomes(marker P97)and wheat chromosomes 2A(marker P1)and 2B(marker P2).M,marker 2000+;lane 1,CS;lane 2,2H(2A);lane 3,2Ai#2(2B);lanes 4-10,plants derived from the homozygous double substitution line 2D2Ai#2(2B)2H(2A).

3.6.Genetic behavior before and during meiosis

Fig.3-Identification of the wheat double substitution line 2A2Ai#2(2B)2H(2D)using molecular markers specific to the alien chromosomes(marker P97)and wheat chromosomes 2B(marker P2)and 2D(marker P3).M,marker 2000+;lane 1,CS;lane 2,2H(2B);lane 3,2Ai#2(2D);lanes 4-10,plants derived from the homozygous double substitution line 2A2Ai#2(2B)2H(2D).

Table 4 - Transmission rates of the two alien chromosomes in the BC1 generation derived from reciprocal crosses between different substitution lines and CS, determined using marker P97.

To observe the genetic behavior of the alien chromosomes in the wheat double substitution lines before and during meiosis,and to determine whether these chromosomes behave the same as wheat endogenous chromosomes,GISH and FISH analysis using different probes was carried out.First,the centromeres and telomeres of the alien chromosomes and wheat chromosomes were visualized in anthers at a suitable stage by FISH,and this analysis revealed the relative change of behavior in the centromeres and telomeres at the premeiotic and meiotic stages(Fig.7a-f).Centromeres grouped at the opposite pole with respect to telomeres,which conformed to the basic Rabl configuration.Centromere pairing or clustering in the double substitution lines appeared to occur prior to telomere bouquet formation,based on the observation of chromosome dynamics in a large number of PMCs.

To further investigate the genetic behavior of alien chromosomes in the double substitution lines,the association between homologue synapsis and telomere clustering for the two pairs of alien chromosomes was observed using GISH,in which the cells were simultaneously hybridized with probes generated by labeling total genomic DNA of Th.intermedium and barley with different fluorescent labels.Different arrangements of chromosome at the pre-meiotic interphase and prophase stages of meiosis were observed(Fig.7g-l).Both types of alien homologous chromosome initially separated during the premeiotic stages of meiocyte development,and then were associated along nearly the entire length of the chromosome(Fig.7g-h),but the presence of two visible telomeric heterochromatin knobs on Th.intermedium chromosome 2Ai#2 indicated that the chromosomes were not completely synapsed.These observations of wheat material containing two types of alien chromosomes also confirmed the finding of previous studies that premeiotic pairing occurs in wheat.During the prophase of meiosis,both pairs of alien chromosomes randomly stretched to the two poles of the cell where the telomeres and centromeres,respectively,were collectively located and then they were partially or completely synapsed at different stages of telomere clustering(Fig.7i-l).Two heterochromatin knobs were visible within the developing cluster(Fig.7i),while in some cells a tight telomere bouquet was formed.The two knobs fused together when the two homologs were synapsed at the distal end or in the distal half,but in some chromosomal regions there was clearly more than one visible strand(Fig.7j-k).Later,the homologs were completely colocalized and formed one thick thread,but both the centromeres and telomeres were completely dispersed in the entire cell(Fig.7l).Based on these observations,we concluded that the alien chromosomes in the double substitution lines behaved similarly to endogenous wheat chromosomes.

4.Discussion

Th.intermedium and barley are relatives of common wheat and possess traits that positively affect wheat breeding.Hybridization is a key method for transferring valuable traits or genes from wild species,including Th.intermedium and barley,to wheat and has been successfully applied in wheat improvement efforts[33].In this study,six different crosses were made between different wheat-Th.intermedium and wheatbarley disomic alien substitution lines,and different transmission rates of the alien chromosomes were observed for each cross.Two double substitution lines were ultimately obtained in the F5generation through consecutive selfing combined with marker-assisted selection.

Molecular markers are widely used to reveal genetic differences among plant germplasms and to select the target genes controlling invisible phenotypes in segregating populations.Molecular markers can be especially useful for detecting subtle divergence between plant genomes[34].To date,molecular markers specific to the chromosomes or chromosome arms of some wild species have been developed for creating alien translocation lines[35,36].Thus,molecular markers are very convenient for tracing the transmission of alien chromosomes,segments or gene loci in the genetic background of a cultivated variety [34,37].Currently,expressed sequence tag(EST)-sequence-tagged site(STS)markers are widely used to detect the inheritance and structural changes of alien chromosomes or arms[38-40].Unlike other PCR-based markers,EST-PCR markers are designed based on coding sequence and may represent genes that are associated with a phenotype.To efficiently identify alien chromosome substitution lines from the huge segregating populations in this study,we used specific primers to genotype different generations.In addition to using markers specific to alien chromosomes,we designed markers specific to wheat chromosomes 2A,2B,and 2D based on the sequences of the TaARG genes located on these three chromosomes[29].Finally,we successfully obtained two types of double substitution lines in the wheat CS background by using molecular markers specific to the alien chromosomes 2H and 2Ai#2 and wheat group 2 chromosomes.

Fig.4-Cytogenetic analysis of the wheat double substitution lines 2D2Ai#2(2B)2H(2A)and 2A2Ai#2(2B)2H(2D).a,b:Chromosome constitution at mitosis in 2D2Ai#2(2B)2H(2A)(2n=42)and 2A2Ai#2(2B)2H(2D)(2n=42),respectively;c,d:genomic in situ hybridization analysis ofmitotic chromosomes in 2D2Ai#2(2B)2H(2A)and 2A2Ai#2(2B)2H(2D),respectively,using genomic DNAof Th.intermedium and barley as probes.e,f:Genomic in situ hybridization analysis of meiotic chromosomes in 2D2Ai#2(2B)2H(2A)and 2A2Ai#2(2B)2H(2D),respectively,using genomic DNA of Th.intermedium and barley as probes.One rod bivalent and two univalents with different fluorescent signals are visible.The Th.intermedium 2Ai#2 chromosomes and barley 2H chromosomes are distinguishable based on green and red fluorescence,respectively.Scale bar,10 μm.

Stable transmission of alien chromosomes in the wheat background is the basis for applying useful genes located on these chromosomes in wheat breeding.Many studies have shown that alien chromosomes in a wheat background always exhibit transmission behavior different from that of the native wheat chromosomes[41,42].For example,the recombinant chromosomes in both translocation lines NAU419 and NAU418 could be transmitted through both the male and female gametes,but the transmission rate was markedly higher through female gametes than through male gametes[43].Similarly,the Agropyron elongatum chromosome 6Ags in a wheat background was transmitted at a higher rate through the female gametes(40%-50%)than through the male gametes(3%)[44].In the present study,the transmission rates of the two alien chromosomes 2H and 2Ai#2 were different for different reciprocal cross combinations.The transmission of the two alien chromosomes to the next generation via female gametes was also biased.Interestingly,alien chromosome 2H showed a higher transmission rate than alien chromosome 2Ai#2 in the wheat background.

Fig.5-Fluorescent in situ hybridization signals observed for the double substitution lines 2D2Ai#2(2B)2H(2A)(a)and 2A2Ai#2(2B)2H(2D)(b)using probes specific to the centromeres of wheat and barley chromosomes.The DIG-labeled wheat centromere probe was detected with anti-DIG-fluorescein(red)and the biotin-labeled barley centromere and Th.intermedium chromosome arm probes were detected with FITC(green).Chromosomes were counterstained with DAPI.The two 2H chromosomes are indicated by thin arrows,the two 2Ai#2 chromosomes are indicated by bold arrows,and the 38 wheat chromosomes are distinguishable based on red fluorescence signal in the centromere region.Scale bar,10 μm.

In addition to identification using molecular markers,plant materials containing alien chromosomes also need to be confirmed using other cytogenetic techniques.Genomic in situ hybridization and FISH,which allow the direct visualization of chromosomes,are effective and reliable techniques for tracing alien chromosomes and fragments introgressed into the wheat background.In the present study,genomic DNA of Th.intermedium and barley or centromere sequences were used as probes to observe the alien chromosomes in the double substitution lines in which wheat chromosomes were substituted by a pair of Th.intermedium 2Ai#2 chromosomes and a pair of barley 2H chromosomes.Genomic in situ hybridization and molecular marker analysis were used as complementary approaches to verify the presence of the alien chromosomes and the absence of the endogenous wheat chromosomes.

GISH and FISH were performed using different probes to observe the genetic behavior of the alien chromosomes in the double substitution lines.Common wheat has seven homologous groups,and each group has six chromosomes.For this reason,it is difficult to study homologous chromosome pairing during meiosis.Many studies[45-47]have revealed that interactions between centromeres in early meiotic prophase play a role in chromosome pairing during meiosis.Centromere interactions were first described in onion(Allium fistulosum L.)as the clustering of centromeres during premeiotic interphase and prophase visualized by electron microscopy[48].Later,this type of interaction was further described in a few additional plants[49,50],which have various connections among centromeres and telomeres.In common wheat,centromere interaction occurs earlier than telomere bouquet formation at meiotic prophase[45,50].Chromosome pairing is well understood in yeast and Drosophila[51,52],but it is impossible to study chromosome pairing in hexaploid wheat because of the presence of multiple genomes.Alien chromosome materials derived from crosses between wheat and other related species such as wheat-rye substitution lines or wheat-barley addition lines can be used to overcome this problem.In the two types of double substitution lines developed in the present study,a pair of 2H chromosomes and a pair of 2Ai#2 chromosomes substituted wheat chromosomes 2B and 2D or 2A,respectively,and the different chromosomes in homoeologous group 2 can be distinguished by different specific probes.These probes are useful for studying chromosome pairing during meiosis.

Previous study[53]indicated that homoeologous chromosomes are highly conserved among cereal species and share similar features,including gene number,sequence and order.In addition,homoeologous chromosomes can also be complementary in terms of the genome composition[54].To date,many chromosome substitution lines in which homoeologous chromosomes are substituted by chromosomes from other cultivars or species have been developed in different wheat backgrounds[14,55].In the present study,we developed two types of double substitution lines by crossing disomic substitution lines 2H(2A)and 2H(2D)with disomic substitution line 2Ai#2(2B).The substitution of two pairs of wheat chromosomes by two pairs of alien chromosomes in these lines had negative effects on the plants.For example,compared with the disomic substitution line parents,the substitution lines exhibited delayed development,shorter plant height,shorter spikes,decreased fertility,and decreased grain weight.These differences might be due to the fact that the genetic contributions of the two missing pairs of wheat group 2 chromosomes cannot be completely compensated by one pair of barley 2H chromosomes and one pair of Th.intermedium 2Ai#2 chromosomes and that the wheat genetic balance at the DNA level is disrupted because of chromosome replacement.In addition,by observing the configuration of 2Ai#2 chromosomes in PMCs at metaphase,we found univalent chromosomes in most of the cells,suggesting that chromosomes were lost in later stages of meiosis.Statistical analysis of alien chromosome transmission rates in the double substitution lines also indicated that chromosome 2Ai#2 had a lower transmission rate than 2H in the BC1population.These results suggest that the abnormal behavior of chromosome 2Ai#2 during meiotic metaphase might disturb the well-balanced segregation of chromosomes and lead to lower fertility in the selfing progeny.It is thus desirable to develop translocation lines carrying the genes and traits on barley 2H and Th.intermedium 2Ai#2 by crossing the double substitution lines with commercial wheat varieties and then generating somatic variation via tissue culture.We have successfully used this strategy to obtain a heterozygous line with a translocation between barley 2H and Th.intermedium 2Ai#2(Fig.S1).In the future,we plan to simultaneously transfer useful genes on barley 2H and Th.intermedium 2Ai#2,including Bdv2,Isa,Rar1,Cly1,and ppd-H1,into wheat via the translocation of small fragments for wheat improvement.

Fig.6-Morphological characteristics of the wheat double substitution lines 2D2Ai#2(2B)2H(2A)(a,b,c)and 2A2Ai#2(2B)2H(2D)(d,e,f).a and d,Adult plants;b and e,spikes;c and f,seeds.1,2Ai#2(2B);2,2D2Ai#2(2B)2H(2A);3,2H(2A);4,2Ai#2(2B);5,2A2Ai#2(2B)2H(2D);6,2H(2D).

Fig.7-Genetic behavior before and during meiosis observed in the wheat double substitution lines using specific probes(a-l).a-f:Fluorescent in situ hybridization analysis of centromeres(green)and telomeres(red)in double substitution lines at different stages of meiosis.(a)At the premeiotic stage,centromeres paired randomly and nuclei exhibited a Rabl configuration.(b-e)At stages later than a,the telomeres formed a bouquet,while grouped centromeres started to disperse.(f)At a stage later than that shown in e,both centromeres and telomeres dispersed.g-l:Synapsis behavior of homologous chromosomes at different stages of telomere bouquet formation.In meiocytes at different stages,different numbers of homologous chromosomes were separated,partially synapsed,and completely synapsed.A photo showing telomere behavior in the same meiocyte is shown in the inset in the bottom right corner of every image in g-k.

5.Conclusions

Two types of wheat double substitution lines 2D2Ai#2(2B)2H(2A)and 2A2Ai#2(2B)2H(2D),in which different group 2 wheat chromosomes were replaced by barley chromosome 2H and Th.intermedium chromosome 2Ai#2 were obtained.Chromosome 2H showed a higher transmission rate than 2Ai#2,and both chromosomes were preferentially transmitted between generations via female gametes.The double substitution lines showed normal genetic behavior before and during meiosis.Even though the double substitution lines displayed negative botanic and agronomic traits,they can potentially be used in wheat meiosis research and in breeding programs.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.11.003.

Acknowledgments

We thank Prof.Fangpu Han at the Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,China,for providing Th.intermedium accession 440001 and barley variety Betzes as well as the experimental facilities for GISH and FISH analysis,and Prof.Xueyong Zhang at the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,China,for providing the CRW and D05 probes and oligonucleotide probe(AGGGAG)n.We are grateful to Yuzhe Niu,Lu Liu,Hao Lei,Haili Ma and Zhiwen Shen at the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,China,for their useful contributions to this study.This research was financially supported by the National Key Research and Development Program of China(2016YFD0102001 and 2016YFD0102002),the National Natural Science Foundation of China(31771788),and the Agricultural Science and Technology Innovation Program(ASTIP)of the Chinese Academy of Agricultural Sciences.