Development of Oligo-GISH kits for efficient detection of chromosomal variants in peanut

Pei Du,Liuyng Fu,Qin Wng,To Lng,Hu Liu,Suoyi Hn,Chenyu Li,Bingyn Hung,Li Qin,Xiodong Di,Wenzho Dong,Xinyou Zhng,*

a Henan Academy of Crop Molecular Breeding/State Industrial Innovation Center of Biological Breeding/The Shennong Laboratory/Key Laboratory of Oil Crops in Huang-Huai-Hai Plains,Ministry of Agriculture/Henan Provincial Key Laboratory for Oil Crops Improvement,Henan Academy of Agricultural Sciences,Graduate T & R Base of Zhengzhou University,Zhengzhou 450002,Henan,China

b Institute of Biotechnology and Nuclear Technology,Sichuan Academy of Agricultural Sciences,Chengdu 610061,Sichuan,China

Keywords:Peanut Oligo-GISH Genomic relationship Variants Chromosome identification

ABSTRACT Oligo probe staining is a low-cost and efficient chromosome identification technique.In this study,oligo genomic in situ hybridization(Oligo-GISH)technology was established in peanut.Peanut A and B subgenome-specific interspersed repeat(IR)oligo probe sets were developed based on clustering and electronic localization of tandem repeat sequences in the reference genome of Tifrunner.The Oligo-GISH kit was then used to perform staining of 15 Arachis species.The A-subgenome probe set stained the chromosomes of A-and E-genome Arachis species,the B-subgenome probe set stained those of B-,F-,K-,and E-genome species,and neither set stained those of H-genome species.These results indicate the relationships among the genomes of these Arachis species.The Oligo-GISH kit was also used for batch staining of the chromosomes of 389 seedlings from the irradiated M1 generation,allowing 67 translocation and deletion lines to be identified.Subsequent Oligo-FISH karyotyping,FISH using single-copy probe libraries,and trait investigation identified seven homozygous chromosomal variants from the M3 generation and suggested that there may be genes on chromosome 4B controlling seed number per pod.These findings demonstrate that the IR probe sets and method developed in this study can facilitate research on distant hybridization and genetic improvement in peanut.

1.Introduction

Peanut(Arachis hypogaea L.),a worldwide food and oilseed crop,is an allotetraploid species(AABB genome,2n=4x=40)that likely evolved from a cross between two diploid wild progenitors,A.duranensis(AA genome)and A.ipaensis(BB genome)[1–4].Chromosome engineering can be used to create new materials or to integrate beneficial genes from wild species to improve peanut[5,6].However,the low accuracy and efficiency of chromosome identification limit the detection and utilization of useful chromosomal variants.

In oligo probe–fluorescence in situ hybridization(Oligo-FISH),single-strand oligonucleotide(SSON)probes are designed based on computationally identified repetitive or single-copy sequences in whole genomes and artificially synthesized[7–11].These probes can be used to distinguish subgenomes[12,13],identify chromosomes[5,14],identify homoeologous chromosomes,and characterize meiotic crossovers[15,16].Some oligo probes can invade double-stranded DNA in nondenatured chromosomes[17,18].The invasiveness of an oligo probe is influenced by its length and sequence structure,and highly invasive oligo probes can produce clear signals on chromosomes even at very low concentrations[19,20].Consequently,oligo probe staining has become a lowcost and efficient chromosome identification technique for peanut[20].

Genome sequencing has revealed that＞70% of genome sequences in cultivated peanut are repetitive[21,22].However,the type and content of repetitive sequences vary between the A and B subgenomes of cultivated peanut and between the two parental genomes.Most transposon families are shared by the two parental genomes,and the macroscale positioning of abundant transposon families is similar between the two.However,transposon activity since their divergence has resulted in differences in microscale positioning and relative abundance[23].Cytological evidence also supports differences between the A and B subgenomes.For example,the A subgenome is distinguished from the B subgenome by strong centromeric banding and a pair of small chromosomes[24].

Genomic in situ hybridization(GISH)using genomic DNA from the diploid species as probes can clearly distinguish A-and Bsubgenome chromosomes[25,26].The copy numbers and sites of many repetitive sequences differ between the A and B subgenomes[27].The purpose of the present study was to mine specific interspersed repeats(IRs)in the A or B subgenome from the highquality reference sequences[21]of A.hypogaea cv.Tifrunner,a runner-type peanut widely grown in the USA,and establish a low-cost and efficient Oligo-GISH method for peanut A and Bsubgenome identification.To validate its potential for practical application,the Oligo-GISH kit was used to identify wild species and chromosomal variants in radiation-induced M1and M3seedlings.

2.Materials and methods

2.1.Plant materials

The plant materials used in this study included two peanut cultivars:Tifrunner and Silihong(SLH)(AABB genome),the allotetraploid wild species A.monticola,14 wild diploid species,and an interspecific hybrid,W1824,derived from a cross between A.duranensis and A.ipaensis(Table S1).

To create chromosomal variants,SLH was planted in pots.The flowers of these plants were pinched off until full bloom,and 12 plants each were irradiated using60Co-γ at an intensity of 12 Gy or 16 Gy(Isotope Institute Co.,Ltd.,Henan Academy of Sciences,Zhengzhou,Henan,China).After irradiation,plants were allowed to self-pollinate in isolation.Finally,respectively 175 and 214 M1seeds were obtained from the plants irradiated with the two dosages.The chromosomes of all M1seedlings were identified,and the M1plants with chromosomal variants were selfpollinated for two generations.Homozygous variants were selected from the M3seedlings.

2.2.Chromosome preparation

Chromosomes were prepared following Du et al.[27].First,root tips of seedlings were cut,submerged in 2 mmol L-18-hydroxyquinoline for 3 h at 25 °C,and fixed in 3:1 absolute ethanol:glacial acetic acid.Next,0.3–0.5 mm of root tip was excised and squashed in a drop of 45%acetic acid on a slide.After freezing at-80 °C overnight,slides containing mitotic chromosomes were dehydrated in absolute ethanol and used for FISH or Oligo-GISH.

2.3.Design of oligo probes

For developing subgenome-specific peanut probes,tandem repeats(TRs)were extracted from the genome sequence of Tifrunner using Tandem Repeats Finder 4.09(TRF)[28].The following parameters were applied:match=2;mismatch=7;inDel=7;probability of match=80;probability of inDel=10;min score=50;and max period=2000.Non-redundant TRs(NR-TRs)were obtained by filtering and clustering the TRs using TR-toolkit(https://gitee.com/lhtk/TR-toolkit)scripts process_trf_dat2get_best.pl (--more_coverage enabled), get_all_entries.pl(--min_consensus_size 301)and cluster_gt10_TR.pl(--top 10).The NR-TRs were then aligned in arrays with the Tifrunner reference genome using B2DSC[9](with default parameters)to identify interspersed repetitive sequence NR-TRs.Next,the interspersed repetitive sequence NR-TRs were cut into oligos with Jellyfish 1.1.11[29].Finally,oligo probe sequences enriched in the A or B subgenome were obtained by removing redundant sequences and positioning using respectively CD-HIT(cdhit-est,key parameters:-n 5-c 0.85-d 0-aL 0.8-aS 0.8)and B2DSC(pident=90,qcovhsp=90).Oligo probes were synthesized by the General Biosystems Company(Chuzhou,Anhui,China).

2.4.Oligo-GISH

Each Oligo-GISH kit included 1000 ng μL-1probe mix containing the oligo probes,100 μg mL-1diamino-2-phenylindole(DAPI),20×saline sodium citrate buffer(SSC)and VECTASHIELD mounting medium(VECTOR,CA,USA).Oligo-GISH was performed following method I or method II.

Method I:Staining dye containing 0.01 μL of probe mix,14.5 μL of 20×SSC,and 0.5 μL of DAPI was added to chromosome slides.The slides were stained with the probe dye for 2 h under dim light.Finally,the slides were washed 8–10 times with 2×SSC at room temperature and mounted with VECTASHIELD.

Method II:Dye solution was prepared by combining 40 mL of 2×SSC,5 μL of probe mix and 5 μL of DAPI.The chromosome slides were then stained in the prepared probe dye for 6 h under dim light.Finally,the slides were washed 8–10 times with 2×SSC at room temperature and mounted with VECTASHIELD.

Method I uses a higher probe concentration(0.67 ng μL-1)than method II(0.125 ng μL-1)but a shorter staining time(2 h vs.6 h).Thus,method I is suitable for rapid chromosome identification when a small number of slides needs to be examined,whereas method II is suitable for chromosome identification when a batch of slides needs to be checked and stain recycling is required.

2.5.Probes,FISH,and GISH

Total genomic DNA of A.duranensis was labeled with biotin-16-dUTP(Roche,Basel,Switzerland)by nick translation and detected with fluorescein anti-biotin,whereas total genomic DNA of A.ipaensis was labeled with digoxigenin-11-dUTP(Roche)and detected with anti-digoxigenin-rhodamine.Repetitive oligo probes for karyotype analysis of peanut from Fu et al.[6]were used;multiplex #3 included FAM-modified TIF-439,TIF-185-1,TIF-134-3 and TIF-165-3,and multiplex #4 included TAMRA-modified Ipa-1162,Ipa-1137,DP-1 and DP-5.

The single-copy oligo libraries L2A-1 and L4A-1 were developed using reference sequences of A.duranensis(PeanutBase,https://www.peanutbase.org/blast)following Du et al.[27].Library L2A-1 was designed based on the two regions 4–9 Mb and 88–93 Mb of chromosome A2,and library L4A-1 was designed based on the 112–114 Mb region of chromosome A4.Libraries were synthesized by MYcroarray(Ann Arbor,MI,USA).The resulting libraries were amplified and labeled with biotin-16-dUTP or digoxigenin-11-dUTP according to the MYcroarray_MYtags(Ann Arbor)labeling protocol.

FISH and GISH were performedfollowing Du et al.[27].Hybridization solution,consisting of 7.5 μL of formamide,1.5 μL of 20×SSC buffer,2 μL of each probe,2.0 μL of 50%dextran sulfate,was made with ddH2O to a final volume of 15 μL.The solution was denatured at 105°C for 13 min,and the spread chromosomes were denatured in 70% formamide at 75 °C for 70 s,followed by hybridization overnight at 37 °C.After hybridization,the slides were washed three times in 2×SSC at room temperature,mounted with VECTASHIELD,and stained with DAPI.

Sequential Oligo-GISH/GISH was performed to map the oligo probe signals.After Oligo-GISH using the A-and B-subgenome probe sets,the slides were denatured,washed to remove all signals,and dried.GISH was then performed using total genomic DNA of A.duranensis and A.ipaensis as probes to identify A-and Bsubgenome chromosomes.

Sequential Oligo-GISH/FISH was performed to identify the chromosomal variants.After Oligo-GISH,the chromosome signals were removed by denaturing and washing.FISH was then conducted using repetitive oligo probes or single-copy oligo libraries.

2.6.Image capture and analysis

FISH and GISH images were captured with a DM6000 fluorescence microscope(Leica,Wetzlar,Germany)with a cooled charge-coupled device camera(Leica).Images were optimized for contrast and brightness using Adobe Photoshop.Approximately 3–5 cells of each material were observed for karyotyping and chromosomal variant identification.Karyotypes were developed from single cells unless overlap of chromosomes was observed.

To characterize the genomic relationship among Arachis species,chromosome signal intensity of Oligo-GISH was divided into four types,strong signal(represented by‘‘+++”),moderate intensity signal(represented by‘‘++”),weak signal(represented by‘‘++”),and no signal(represented by‘‘-”).These labels were then converted into‘‘3”,‘‘2”,‘‘1”,and‘‘0”to calculate the distances among branches with SPSS Statistics 22(IBM,Armonk,NY,USA).

2.7.Trait investigation of pods and seeds

Seed number per pod of all pods and seed coat color were recorded for a single plant of SLH and progenies of the chromosomal variants.Seed number per pod was evaluated following Wang et al.[30],and the numbers of 1-seed pods(1-SPN),2-seed pods(2-SPN),and pods with≥3 seeds(multi-seed pods)were recorded.The ratio of multi-seed pods per plant(RMSP)was calculated as the ratio of the number of multi-seed pods to the total number of pods per plant.The lengths of four mature pods and weights of four well-filled seeds from SLH and the progenies of the chromosomal variants were recorded.

3.Results

3.1.Development of subgenome-specific oligo probes for peanut

Using TRF,1,457,800 TRs were extracted from the Tifrunner reference genome.After filtering with TR-toolkit,952,382 NR-TRs were retained.The NR-TRs were further screened and clustered to obtain 40 NR-TR clusters with length＞300 bp and total copy number＞50.When the NR-TRs were aligned to the Tifrunner reference genome using B2DSC,and one interspersed repetitive sequence NR-TR cluster containing 12 NR-TRs,cluster 08–9,was identified(Table S2).The NR-TRs in cluster 08–9 were cut into 9361 oligos with a length of 40 nt using Jellyfish.After removal of redundant sequences and positioning of the oligos using CDHIT and B2DSC,two oligos,oligo A-4 derived from A02-194 and oligo A-7 derived from A05-31,were more evenly enriched in the A than in the B subgenome,whereas oligo B-9 and oligo B-10 derived from B04-207 were more evenly enriched in the B than in the A subgenome(Table S2).

Based on the above oligos,two specific IR oligo sets were developed:multiplex #A,which included FAM-modified oligo A-4 and oligo A-7,and multiplex #B,which included TAMRA-modified oligo B-9 and oligo B-10(Table S3).In silico mapping revealed that the sites of multiplex#A were distributed with high density in the A but low density in the B subgenome(Fig.1A),whereas the sites of multiplex #B were located mainly in the B and not in the A subgenome(Fig.1B).

FISH of Tifrunner chromosomes using FAM-modified multiplex#A and TAMRA-modified multiplex #B yielded clear green signals on 18 chromosomes and clear red signals on 20 chromosomes,respectively(Fig.1C,D).Sequential GISH using A.duranensis and A.ipaensis genomic DNA as probes confirmed that the chromosomes with multiplex #A signals were identical to the chromosomes with A.duranensis genomic DNA probe signals,while the chromosomes with multiplex #B signals were identical to the chromosomes with A.ipaensis genomic DNA probe signals(Fig.S1).

3.2.Development of the peanut Oligo-GISH kit to detect subgenome changes in peanut

Multiplexes#A and#B were used to develop an Oligo-GISH kit for peanut.The Oligo-GISH kit included 100 μL of probe mix(100 ng μL-1)composed of multiplexes#A and#B,200 μL of DAPI(100 μg mL-1),250 mL of 20×SSC,and 500 μL of VECTASHIELD.Staining of Tifrunner chromosomes with the Oligo-GISH kit using methods I and II produced clear green signals for A-subgenome chromosomes and red signals for B-subgenome chromosomes(Fig.S2).

To test the efficiency of the Oligo-GISH kit in peanut chromosome identification,we applied it to three materials with different origins:the wild species A.monticola,the radiation-induced line FS162-16b-35-1,and W1824,which is an interspecific hybrid between A.duranensis(AA)and A.ipaensis(BB).In agreement with the results obtained for Tifrunner,20B-subgenome chromosomes with strong red signals and 18 A-subgenome chromosomes with strong green signals were observed,and two‘‘small chromosomes”with very weak green signals were detected in A.monticola(Fig.2A).In FS2020-2-1,four chromosomal translocations between the A and B subgenomes were identified(Fig.2B).Ten chromosomes with red signals and the other 10 with green signals were detected in W1824,confirming that W1824 is a true interspecific hybrid between A.duranensis and A.ipaensis(Fig.2C).

3.3.Characterization of genomic relationships among Arachis species using the peanut Oligo-GISH kit

When the peanut Oligo-GISH kit was applied,five of the seven species carrying the A genome:A.duranensis(Fig.3A),A.herzogii(Fig.3C),A.villosa(Fig.3D),A.simpsonii(Fig.3F),and A.duranensis-2(Fig.3G),showed strong green signals and almost no red signals,and two species:A.diogoi(Fig.3B)and A.microsperma(Fig.3E)showed strong green signals and weak red signals.For the B-genome species A.ipaensis(Fig.3H)and A.valida(Fig.3I),the K-genome species A.batizocoi(Fig.3J),and the F-genome species A.trinitensis(Fig.3K),moderate-intensity red signals and almost no green signals were observed.The E-genome species A.stenophylla(Fig.3L)showed both moderate-intensity red and green signals,whereas the H-genome species A.pusilla(Fig.3M)and A.dardonoi(Fig.3N)showed no chromosome signals(Fig.3;Table S4).Clustering of the 14 diploid wild species and two subgenomes of Tifrunner based on the strength of the signals was performed.Overall,the species were divided into three groups based on their genomic affinity.The first group included two H-genome species;the second group included five species representing the B,K,F,and E genomes and the B subgenome of Tifrunner;and the third group included seven A-genome species and the A subgenome of Tifrunner.These results indicated that the genomes of A.duranensis,A.diogoi,A.herzogii,A.villosa,A.microsperma,A.simpsonii,and A.duranensi-2 were closely related to the A subgenome of peanut,whereas the B,K,and F genomes represented by A.ipaensis and A.valida,A.batizocoi,and A.trinitensis were closely related to the B subgenome.The genome of A.stenophylla showed relatively high similarity with both the A and B subgenomes of peanut,while the A.pusilla and A.dardonoi genomes shared relatively low similarity with both the A and B subgenomes of peanut(Fig.S3).

Fig.1.Confirmation of the subgenome probe sets in peanut cultivar Tifrunner.(A,B)The red lines on the chromosome plot indicate the consensus sequence of multiplex#A or multiplex #B against the reference sequence of Tifrunner.(C–F)The location of multiplex #A(green)or multiplex #B(red)signals by dual-color FISH and DAPI staining(blue).Scale bars,10 μm.

3.4.Identification of chromosomal variants of peanut with the Oligo-GISH kit

Dye solution were used to identify the chromosomes of 175 and 214 M1seedlings produced by irradiation with 12 or 16 Gy,respectively,using method II for the Oligo-GISH kit.Translocations or deletions were detected in 86 chromosomes of 67 seedlings(Fig.4;Table S5).Among the 175 seedlings derived from irradiation with 12 Gy,29 harbored chromosomal variants,corresponding to a rate of variation of 17%;chromosomal variants were detected in 38 of the 214 seedlings from irradiation with 16 Gy,giving a rate of variation of 18%.

Among the 67 seedlings with chromosomal variants,48(72%)contained only one chromosomal translocation,17(25.37%)contained two translocations,one(FS172-84;1.49%)contained three translocations,and one(FS162-6b;1.49%)was monotelosomic(Fig.4).Two types of translocations were observed:simple(FS161-10a)and insertional(FS172-65a,FS162-30b and FS172-33).Most were translocations between A-and B-subgenome chromosomes,but a few were non-homologous translocations between chromosomes of the A subgenome,such as FS171-117a,which contained a potential dicentric chromosome formed by the translocation of two chromosomes of the A subgenome(Fig.4;Table S5).

3.5.Selection and accurate identification of homozygous chromosomal variants from M3 seedlings by sequential Oligo-GISH and FISH

To identify homozygous chromosomal variants,sequential Oligo-GISH and FISH were performed(Figs.S4–S7).Seven homozygous chromosomal structural variants were detected.Karyotyping revealed that FS161-1a-5-6 and FS171-72-9-1 each contained two pairs of translocations between homoeologous chromosomes.These translocations were T1BS-1AS·1AL and T1AS-1BS·1BL in FS161-1a-5-6 and T4AS·4AL-4BS and T4BL·4BS-4AL in FS171-72-9-1.FS172-62-7-1 and FS182-30b-2-1 each had one pair of translocations between homoeologous chromosomes,which were T3AS-3BS·3BL and T5BS-5AS·5AL,respectively.Two pairs of non-homologous translocations were detected in FS162-12a-14-1:T8BS-7AS·7AL and T7AS-8BS·8BL.The sister lines FS171-148-7-1 and FS171-148-7-2 both had the T2BS-2AS·2AL translocation and ditelosomic 4BL(Fig.5).To further confirm the chromosomal variants,we designed two single-copy probe libraries of L2A-1 and L4A-1,which produced signals only on chromosomes 2 and 4 of the A and B subgenomes(Fig.S8).The results of sequential Oligo-GISH/FISH confirmed the identity of these translocated and ditelosomic chromosomes in FS162-12a-14-1,FS162-12a-14-2,and FS171-72-9-1(Figs.5,S5–S7).

Fig.3.Characterization of relationships among Arachis species and subgenomes of cultivated peanut using the peanut Oligo-GISH kit.(A)A.duranensis(AA genome).(B)A.diogoi(AA genome).(C)A.herzogii(AA genome).(D)A.villosa(AA genome).(E)A.microsperma(AA genome).(F)A.simpsonii(AA genome).(G)A.duranensis-2(AA genome).(H)A.ipaensis(BB genome).(I)A.valida(BB genome).(J)A.batizocoi(KK genome).(K)A.trinitensis(FF genome).(L)A.stenophylla(EE genome).(M)A.pusilla(HH genome).(N)A.dardonoi(HH genome).From left to right,blue in the first column is DAPI staining,green in the second column is the multiplex#A signals,and red in the third column is the multiplex #B signals.Scale bars,10 μm.

3.6.Genetic effects of homozygous chromosomal variants on pods and seeds

Fig.4.Detection of chromosomal variants in radiation-induced M1 plants by Oligo-GISH using the A-subgenome probe set(green)and B-subgenome probe set(red).Scale bars,10 μm.

Fig.5.Karyotypes and chromosomal variants of radiation-induced M3 plants.Karyotype staining was performed with Oligo-GISH using the A-subgenome probe set(green)and B-subgenome probe set(red);Single-strand oligos(SSONs)show the FISH karyotype using the SSON probes multiplex #3(green)and multiplex #4(red).Scale bar,10 μm.

To reveal the genetic effects of homozygous chromosomal variants on pods and seeds,we investigated the seed number per pod,pod length,single-seed weight,and seed coat color of SLH and the progenies of seven chromosomal variants(Table 1).Like that of SLH,the seed coat color of all variants was red(Fig.S9).However,the single-seed weight,pod length and RMSP of some variants differed from those of SLH.All variants except FS171-148-7-1 and FS171-148-7-2 had lower single-seed weights than SLH.With respect to RMSP,the pods of SLH,FS161-1a-5-6,FS172-62-7-1 and FS182-30b-2-1 were mainly multi-seed,whereas the pods of FS171-148-7-2,FS162-12a-14-1 and FS171-72-9-1 were mainly 1-seed and 2-seed pods(Figs.6,S10;Table 1).

Table 1 Traits of homozygous chromosomal variants.

Fig.6.Seed number per pod of SLH and the homozygous chromosomal variants FS171-148-7-1.

4.Discussion

4.1.Low-cost identification of chromosomal variants in the A and B subgenomes of peanut

Eukaryotic genomes are rich in interspersed repetitive DNA sequences[31,32].Genome-specific IR oligo probes of the B and D subgenomes of wheat were mined from reference genome sequences to identify subgenomes[13],while genome-specific oligo IR probes for Agropyron cristatum[33],Thinopyrum ponticum[34],and rye(Secale cereal L.)[12]were developed and used to identify the chromosomes of their hybrid progenies with wheat.In this study,we designed the two genome-specific IR oligo probe sets of subgenomes in peanut,indicated that genome-specific IR oligo probe or probe sets may be developed in many plants for Oligo-GISH.

Chromosomal structural variant identification is typically performed with GISH using genomic DNA of A.duranensis and A.ipaensis as probes[25,26],a time-consuming and costly operation.In addition,the probes are difficult to obtain[35].In the present study,GISH using genomic DNA was replaced by Oligo-GISH dye solution to identify the chromosomes of 389 seedlings,demonstrating the efficiency of the Oligo-GISH kit.The Oligo-GISH kit allows low-cost,rapid,recyclable staining with oligo probes and DAPI simultaneously under non-denaturing conditions,thereby enabling large-scale,efficient chromosome analysis.

4.2.Application of genome-specific IRs in peanut distant hybridization

As a result of plant genome divergence and evolution,some TRs are shared among related plants,while others are very different.These similarities and differences reflect relationships and divergence of kinship among species[36].The A-and B-subgenome probe sets were used to reveal the genomic relationships between the peanut subgenome and the genomes of wild species and possible divergence points,suggesting that A,B and H genomes might have diverged earliest.The B,K and F genomes showed high similarity and may have been derived from the same genome in a relatively late period.For the E genome there are two possibilities.It may be an older genome and an ancestor of the A and B genomes,accounting for the high similarity with the A and B genomes.Or the E genome is a relatively young genome that evolved from the natural interbreeding of A-and B-genome species.Our findings also show that genome-specific IRs are useful for studying the evolution of the genomes of peanut species.These results demonstrate that Oligo-GISH can be successfully used for the characterization of similarity among peanut subgenomes and genomic identity among Arachis species.Genome-specific IR probes are useful for identifying alien chromosomes resulting from distant hybridization[33,34].In the present study,a true interspecific hybrid between A.duranensis and A.ipaensis was identified using subgenome-specific IRs.These IRs can serve as a reference for developing Arachis species genome-specific IR probes for the rapid identification of alien chromosomes in distant hybridization of peanut.

4.3.Value of efficient chromosomal variant identification for peanut genetic improvement

Chromosomal variants such as translocations and deletions are valuable materials for peanut chromosome engineering.Such variants can be used for translocation or deletion mapping and gene mapping[37].Early study[38]indicated that pods containing≥3 seeds are dominant over 1-seed and 2-seed pods.Wang et al.[30]identified 43 epistatic quantitative-trait loci(QTL)associated with multi-seed pods in peanut and described two main-effect QTL.In the present study,the three homozygous chromosomal variants FS171-148-7-1,FS171-148-7-2 and FS171-72-9-1 had lower RMSP than SLH.FS171-148-7-1 and FS171-148-7-2b contain translocations between chromosomes 2A and 2B and deletions of chromosome 4B.FS171-72-9-1 does not have variants of chromosomes 2A and 2B but has two translocations that swap the long and short arms of chromosomes 4A and 4B.In summary,they all had chromosome 4B,suggesting that this chromosome may contain genes regulating seed number per pod.Of course,the possibility of other chromosomal gene mutations cannot be ruled out.

Translocations between partially homologous chromosomes or heterologous chromosomes promote the exchange of DNA beyond homologous chromosomes in conventional breeding,which may lead to new beneficial traits.Finding chromosomal variants in large populations using previously available techniques is difficult.Oligo-GISH staining provides an effective technique for detecting and selecting of chromosomal variants in large populations.Oligo-GISH staining does not always reveal which chromosomes harbor variations.Single copy Oligo-FISH painting[39]will address this deficiency.It has been successfully used in many crops,including maize(Zea mays L.)[40].Precise identification of peanut chromosomal variants by Oligo-GISH staining combined with single-copy Oligo-FISH painting will facilitate the genetic improvement of peanut.

CRediT authorship contribution statement

Pei Du:Visualization,Data curation,Writing–original draft,Funding acquisition.Liuyang Fu:Visualization,Data curation.Qian Wang:Data curation.Tao Lang:Methodology,Software.Hua Liu:Investigation.Suoyi Han:Visualization.Chenyu Li:Data curation.Bingyan Huang:Writing–review & editing.Li Qin:Formal analysis.Xiaodong Dai:Visualization.Wenzhao Dong:Writing–review & editing,Funding acquisition.Xinyou Zhang:Conceptualization,Funding acquisition,Project administration,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 supported by National Natural Science Foundation of China(31801397),Fund for Distinguished Young Scholars of Henan Academy of Agricultural Sciences(2020JQ03),Independent Innovation Foundation of Henan Academy of Agricultural Sciences,China(2022ZC69),China Agriculture Research System(CARS-13),Key Scientific and Technological Project of Henan Province(201300111000),and Henan Provincial Agriculture Research System(S2012-5).The funding agencies played no role in the design of the study and collection,analysis,and interpretation of data or in writing the manuscript.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.03.014.