dynamics of the centromeric histone cenh3 structure in rye

Research ArticleDynamics of the Centromeric Histone CENH3 Structure inRye-Wheat Amphidiploids (Secalotriticum)

Yulia A Lipikhina1 Elena V Evtushenko1 Oleg M Lyusikov2 Igor S Gordei2

Ivan A Gordei2 and Alexander V Vershinin 1

1 Institute of Molecular and Cellular Biology SB RAS Novosibirsk 630090 Russia2Institute of Genetics and Cytology NAS of Belarus Minsk 220072 Belarus

Correspondence should be addressed to Alexander V Vershinin avershinmcbnscru

Received 21 September 2018 Revised 25 October 2018 Accepted 4 November 2018 Published 26 November 2018

Guest Editor Yuri Shavrukov

Copyright copy 2018 Yulia A Lipikhina et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The centromeres perform integral control of the cell division process and proper distribution of chromosomes into daughter cellsThe correct course of this process is often disrupted in case of remote hybridization which is a stress factor The combinationof parental genomes of different species in a hybrid cell leads to a ldquogenomic shockrdquo followed by loss of genes changes in geneexpression deletions inversions and translocations of chromosome regions The created rye-wheat allopolyploid hybrids whichwere collectively called secalotriticum represent a new interesting model for studying the effect of remote hybridization on thecentromere and its components The main feature of an active centromere is the presence of a specific histone H3 modificationin the centromeric nucleosomes which is referred to as CENH3 in plants In this paper the results of cytogenetic analysis of thesecalotriticum hybrid karyotypes and the comparison of the CENH3 N-terminal domain structure of parent and hybrid formsare presented It is shown that the karyotypes of the created secalotriticum forms are stable balanced hexaploids not containingminichromosomes with deleted arms in full or in part A high level of homology between rye and wheat enables to express bothparental forms of CENH3 gene in the hybrid genomes of secalotriticum cultivarsThe CENH3 structure in hybrids in each crossingcombination has some specific featuresThe percentage of polymorphisms at several amino acid positions is much higher in one ofthe secalotriticum hybrids STr VD than in parental forms whereas the other hybrid STr VM inherits a high level of amino acidsubstitutions at the position 25 from the maternal parent

1 Introduction

Many species of the Triticeae tribe are natural allopolyploidsand attractive objects for obtaining synthetic hybrids whichare promising material for practical breeding Themost com-mon and studied examples of such hybrids are allopolyploidtriticale The synthetic allohexaploid triticale has a genomestructure similar to hexaploid bread wheat except that ithas rye as one of its progenitor instead of the D genomedonor Aegilops tauschii Triticale is an important model forstudying the rapid changes that occur subsequent to poly-ploidization involving genomic remodeling and changes ingene expression [1] However selection and genetic analysisof the triticale gene pool showed that the genetic potentialof rye adaptability is insufficiently realized At the molecular

level it was found that 9 of genes in octoploid triticaleand up to 30 of genes in hexaploid triticale become silent[2] which may be one of the reasons for the incompleterealization of hybrid genetic potential

The synthesis of rye-wheat amphidiploids which havewheat (Triticum L) as the pollinator and rye (Secale L) asthe maternal form can offer better setting for enhancingrye gene expression and for the creation of hybrids valuablefor breeding [3] Crosses like these are normally difficult toachieve due to incompatibility and so rye-wheat amphiploidsare not yet studied Overcoming this barrier is usually associ-ated with severe rearrangements in the genomes of parentalforms which is the most vivid manifestation of the ldquogenomicshockrdquo that occurs when parentsrsquo genomes are combined in

HindawiBioMed Research InternationalVolume 2018 Article ID 2097845 6 pageshttpsdoiorg10115520182097845

2 BioMed Research International

a hybrid cell [4] and is accompanied by various chromoso-mal abnormalities including those affecting the centromerestructure It has been previously determined that using anintermediary species triticale as a source of wheat genomesin crosses with rye proved to be effective for overcomingthe barrier of unidirectional progamic incompatibility ofparent species [3] As a result these rye-wheat amphidiploidhybrids were created by crossing tetraploid rye (SRRRR 2n= 4x = 28) with hexaploid triticale (TRRAABB 2n = 6x =42) and were named secalotriticum (timesSecalotriticum syntimesSecalotriticum = Secale L times Triticum L SRRAABB 2n =6x = 42) The presence of rye maternal cytoplasm in hybridswas proved by analyzing restriction fragments of species-specific mitochondrial DNA18S5S-repeated sequences andthe ndhH-region of chloroplast DNA which showed a pat-tern characteristic of chloroplast DNAand rye mitochondrialDNA [3] The created secalotriticum hybrids are charac-terized by a wider variability range of morphological andeconomically valuable traits compared to the original triticaleforms [3] Secalotriticum creates more advantageous condi-tions for enhancing the expression of rye genetic systems andthe manifestation of its valuable adaptive traits

The process of the hybrid genome formation and itssubsequent stabilization are directly related to the normal-ization of meiosis and proper segregation of chromosomesIncompatibility of centromeres of different species seems tobe the main reason of the chromosome elimination of oneof the parental genomes in hybrids [5 6] The pivotal rolein the proper chromosome segregation during meiosis andmitosis lies with centromeres their identity being defined bythe presence of the centromere-specific variant of histone H3known in plants as centromere-specific histone H3 variantCENH3 [7 8] This is due to the fact that at the molecularlevel the most specialized and universal characteristic of theactive centromere is the presence of CENH3 instead of thecanonical histone H3 in the nucleosomes of centromericchromatin As it has been shown in somemammalian speciesandDrosophila in case of centromeric histone loss there is nokinetochore formation and no correct chromosome segrega-tion during cell division for some reason [9]Unlike canonicalhistone X3 which has a conserved structure CENH3 nor-mally shows considerable variability across species [5 10 11]Different domains of this molecule are diverging differentlyAn extended N-terminal tail (NTT) domain and loop 1 ofthe histone fold domain (HFD) putatively interact with cen-tromeric DNA [12] and show signatures of positive selectionin some animal and plant species [13] while the part of theHFD that lies outside loop 1 is generally conserved [14 15]

Because of a special role CENH3 has in the formationand function of centromeres we explore effects of remotehybridization using an unusual combination of parentalforms on the structure of this protein and perform a cyto-genetic analysis of the karyotypes

2 Materials and Methods

Two secalotriticum combinations (timesSecalotriticum RozenstSRRAABB 2n = 6x = 42) Verasenrsquo timesMikhasrsquo (STr VV) and

Verasenrsquo timesDubrava (STr VD) were developed by hybridizingthe tetraploid rye cultivar Verasenrsquo (119878RRRR 2n=4x=28)with the hexaploid triticale cultivars Mikhasrsquo and Dubrava(119879AABBRR 2n=6x=42) Triticale acted as an intermediaryspecies a source of wheat genomes and an inhibitor ofrye S-RNases which allowed overcoming the rye progamicincompatibility with wheat Stabilization of karyotypes wasfacilitated by a single backcross of rye-triticale pentaploidF1 hybrids (SRRABR 5x=35) on triticale followed by self-pollination within 15 generations and constant selection forcytological stability and phenotypic homogeneity

Karyotyping of secalotriticum identification of the orig-inal species chromosomes was carried out by means ofroot apical meristem squashed preparations and differentialstaining using a Giemsa technique (C-banding) [16]

Molecular analysis was conducted on plants of parentalforms and hybrids grown in a greenhouse The total RNAwas isolated from leaves of 10-12-day-old seedlings from4-5 plants of parental forms RNA of hybrid forms wasisolated from each plant separately Isolation of RNA wasperformed using the TRI Reagent (MRC Ink USA) [17]To avoid contamination with genomic DNA total RNA wastreated with RQ-RNase Free DNase (Promega MadisonUSA) To synthesize cDNA a RevertAidTM H MinusFirst Strand cDNA Synthesis Kit (Thermo Scientific USA)was applied The resulting cDNA was used as a templatein a series of PCR reactions with primers synthesizedspecifically to amplify the CENH3 N-terminal tail (NTT)The primer sequences are 51015840 ndash ATGGCCCGCACCAAGC(F) and 51015840 ndash GAAACTCGACCGACTTCTG (R) Theproduct size was 268 bp PCR products were clonedinto the pTZ57RT plasmid (InsTAclone PCR cloningkit Thermo Scientific bd0) and analyzed by Sangersequencing using BigDye Terminator v31 Cycle SequencingKit (Applied Biosystems USA) The reaction productswere separated on the 3500 Genetic Analyzer (AppliedBiosystems USA) The sequences of individual clonesobtained for each sample were analyzed using the UniProUgene software (httpugenenet) and FASTA softwarepackage [18] The search for the identity of the nucleotidesequences was carried out using the BLAST algorithm inthe NCBI database (httpblastncbinlmnihgovBlastcgi)Amino acids alignments were performed online usingClustal Omega (httpwwwebiacukToolsmsaclustalo)and used for downstream analysis and visualization(httpwwwjalvieworg) Graphic images were preparedusing the Jalview program (httpwwwjalvieworg)

3 Results and Discussion

The STr VM (Verasenrsquo times Mikhasrsquo) and STr VD (Verasenrsquotimes Dubrava) (SRRAABB 2n=6x=42) secalotriticum kary-otypes were analyzed by differential staining (C-banding)[16] This method reveals the pattern of heterochromaticregions localization which is specific for each chromosomeofrye and wheat thereby allowing identification of each chro-mosome in a hybrid karyotype Figures 1(a) and 1(b) showthat the karyotypes of the STr VM and STr VD hybrids arestable balanced hexaploids not containing minichromosomes

BioMed Research International 3

(a) (b)

Figure 1 The STr VM (a) and STr VD (b) karyotypes of hybrid secalotriticum following C-banding appear with the whole sets of R A andB genomes

with deleted arms in full or in part The hybrids and theoriginal rye and wheat forms have an identical C-bandingpattern

One of the genomic shock manifestations which arisefrom the combination of two parental genomes in a hybridcell in case of remote hybridization is various chromosomalaberrations Deletions and translocations of individual chro-mosomal regions and chromosome arms are among the mostcommon changes and have been found in the cytogeneticanalysis of both wheat-rye substitution and addition lines[19 20] and in triticale and offspring fromcrosses of triticaletimeswheat [21 22]The disorders herewith have been documentedin the chromosomes of both parental forms Stabilization ofsecalotriticum karyotypes was assisted by a single backcrossof rye-triticale pentaploid F1 hybrids (SRRABR 5x=35) ontriticale with subsequent self-pollination during 15 gener-ations and constant selection for cytological stability andphenotypic homogeneity Diploid RR-rye genome in rye-triticale F1 hybrids is a factor of meiosis stabilization andprovides functionality of gametes with different chromosomecomposition The formation of the secalotriticum genometakes place in F1BC1 on the basis of partially unreduced21-chromosome gametes of F1 pentaploids with balancedchromosome sets of the original species haplogenomes (7(R)7(A) 7(B)) Equation division (division into chromatids) ofasynaptic univalents in anaphase I (AI) is the main sourceof chromosomal abnormalities during the second meiosisdivision (MII) individual chromatids are not included in themetaphase plate in MII in anaphase II they often lag behindother chromosomes in division segregate randomly undergofragmentation and form micronuclei in the microsporetetrads which leads to the elimination of genetic materialand aneuploidy and the genomic instability [3 23 24] Sta-bility of the secalotriticum genome and its genetic diversityare determined by the cytoplasm type and some factors

apparently related to the structural and functional state ofcentromeres in its genome Analyzing meiosis in hybridswe observed the formation of (pseudo)univalents mainly asa result of desynapsis [23 24] Desynapsis here means theearly decomposition of bivalents into univalents of desynapticorigin (pseudounivalents) that occur in the late prophase(prometaphase I) of meiosis Unlike the asynaptic univalentscharacteristic of triticale the pseudounivalents maintaintheir unipolar centromere orientation in the reductional (I)division of meiosis The equational (II) division of meiosiswas characterized by regular polar segregation and a low levelof chromosome elimination [23 24]

The secalotriticum forms created underwent rapid stabi-lization inmeiosis during selection for productivity Stabiliza-tion was due to the narrowing of the spectrum and a rapiddecrease in the frequency of the chromosome abnormalitiesin the secondmeiotic division (by 15-50) in F1-F3 includingthe level of chromosome elimination (from30 to 6 tetradswith micronuclei) In F3-5 and subsequent secalotriticumgenerations the normalization of the first division of meiosiswas more pronounced the average level of anomalies in MImetaphase decreased from 573 in F3 to 169 in F5 and alsofrom 25-30 up to 8-10 in AI-AII anaphases and reachedless than 5 at the tetrad stage [3 23 24] The fact thatthe chromosomal changes mentioned above tend to occurquickly andmost intensively in the first generations up to thefifth inclusive and especially in F1 before the chromosomedoubling is probably a general trend in remote hybridizationof cereals [3 21 23 24] Thus continuous cycles of crossesin the secalotriticum production played a significant role instabilizing the hybrid karyotype and replacing possible lostparts of the parental genomes in the first generations afterhybridization

Differences in the CENH3 structure between the parentsallow estimating the expression level of the parental forms


Verasen MART KHP AVR KT KV PPKK KLG TRP PG GT QRRQ DTD GAG TS AT PRRA GRA AAP GA AE GATG QPK QRK PH RF RMikhas MART KHP AVR KT KV PPKK KLG TRP SG GT QRRQ DTD GAG TS AT PRRA GRA AAP GA AE GATG QPK QRK PH RF RVerasen x Mikhas MART KHP AVR KT KV PPKK KLG TRP PG GT QRRQ DTD GAG TS AT PRRA GRA AAP GA AE GATG QPK QRK PH RF R

Verasen MART KHP AVR KT KV PPKK KLG TRP PG GT QRRQ DTD GAG TS AT PRRA GRA AAP G A AE GATG QPK QR K PH RF RDubrava MART KHP AVR KT KV PPKK KLG TRP SG GT QRRQ DTD GAG TS AT PRRA GRA AAP G A AE GATG QPK QR K PH RF RVerasen x Dubrava MART KHP AVR KT KV PPKK KLG TRP SG GT QRRQ DTD GAG TS AT PRRA GRA AAP G A AE GATG QPK QR K PH RF R

lowast

lowast lowast lowast lowast

Figure 2 The amino acid consensus sequences of 120572CENH3 NTT domain derived from parental forms of rye (Verasenrsquo) triticale (MikhasrsquoDubrava) and secalotriticum amphidiploids (STr VM and STr VD) The asterisks indicate the positions of nonsynonymous amino acidsubstitutions Amino acid polymorphic positions are color-coded according to their frequency white if below 50 light-gray if above 50In the positions 41 54 56 and 66 the percentage of substitutions does not exceed 50 although it has high values for Dubrava and the hybridtherefore the amino acids with the highest percentage are shown in these positions The exact quantification of amino acid substitutions inthe polymorphic positions is provided in Figure 3

0

10

20

30

40

50

60

70

80

25

VerasenMikhasVerasen times Mikhas

(a)

0

5

10

15

20

25

30

35

40

45

41 54 56 66

VerasenDubravaVerasen times Dubrava

(b)

Figure 3 Quantitative determination of amino acid polymorphism in the NTT domain of 120572CENH3 leading to nonsynonymous aminoacid substitutions On the x-axis the position in the amino acid sequence on the y-axis the percentage of clones having substitutions inpolymorphic positions to the total number of clones sequenced (a) Parental forms Verasenrsquo and Mikhasrsquo hybrids Verasenrsquo times Mikhasrsquo (b)Parental forms Verasenrsquo and Dubrava hybrids Verasenrsquo times DubravaNote fifteen cDNA clones were sequenced for each parental plant andthe deduced amino acid sequences were obtained About 40-45 clones were sequenced for each hybrid plant

of the protein in a new genomic environment that arisesin a hybrid cell under remote hybridization The first workon studying possible connection between differences in theCENH3 structure in parental forms and the processes ofparental genomes chromosome segregation during hybridcell division was carried out on hybrids obtained from thecrossing of cultured barley H vulgare and its closest wildrelative H bulbosum [5] The CENH3 molecules were notincorporated in the centromeres of H bulbosum chromo-somes which were herewith inactivated and eliminated fromhybrid embryos Perhaps the absence of CENH3 in thecentromeres of H bulbosum chromosomes is caused by per-ceptible differences in the structure of this protein betweenbarley species especially in the structure of the N-terminaltail (NTT) [5] Unlike barley species DNAsequences of120572 and120573 CENH3 forms of various rye and wheat species have a veryhigh identity (95-99) [25] which significantly complicates

the search for interspecific differences and accordingly theidentification of the nature of their inheritance in hybridgenomes

Consensus amino acid sequences derived fromsequenced cDNA clones obtained from the 120572CENH3NTT of Verasenrsquo and Mikhasrsquo parental forms differ only inone nonsynonymous substitution of amino acid serine toproline at position 25 (Figure 2) It was found in 733 ofthe analyzed clones in the Verasenrsquo variety (Figure 3(a))but only in 77 of the paternal variety Mikhasrsquo clones Thesecalotriticum cultivar Verasenrsquo times Mikhasrsquo inherits a highcontent of proline from the maternal form Verasenrsquo (674Figure 3(a)) However a different cross does not supportthis tendency Verasenrsquo times Dubrava has as low percentage ofsubstitutions (26) as the donor triticale cultivar Dubrava(67) On the other hand Dubrava typically has morepolymorphism than the donor Verasenrsquo at several nucleotide


positions leading to the following amino acid substitutionsalanine to valine alanine to glycine glutamine acid toglutamine and lysine to asparagine (Figures 2 and 3(b))Interestingly although the percentage of substitutions doesnot exceed 50 (of the total number of clones sequenced)this value is much higher in the secalotriticum cultivarVerasenrsquo times Dubrava than in Dubrava something like a caseof heterosis (Figure 3(b)) Thus the results obtained indicatethat both parental genomes participate in the productionof the CENH3 protein in the secalotriticum hybrids At thesame time comparison of the nucleotide and amino acidsequences of the most variable N-terminal tail of the CENH3molecule in parental forms and hybrids does not reveal adefinite tendency in inheritance

Published data on the regulation of the expression ofdifferent genes and other classes of DNA sequences in hybridgenomes obtained by crossing different species or contrastpopulations of one species indicate a complex and hetero-geneous inheritance pattern The phenomenon of nucleolusdominance characteristic for the expression of ribosomalRNA genes in triticale as an example of genomic dominanceconsists in the repression of rye rRNA genes by means ofcytosine methylation [26] However this type of inheritancenamely the genomic dominance of one of the parents is nottypical of most other examplesThe results of gene expressionstudy in hybrids between different ecotypes of Arabidopsisare contradictory In one case a general maternal dominancehas been identified [27] while in the other it has been foundthat the maternal and paternal genomes are transcription-ally equivalent [28] Also it has been demonstrated thatvarious hybrid combinations show significant variations inactivation of parental alleles [29] In some hybrids betweenArabidopsis ecotypes heterosis for biomass and seed yieldhas been documented [30] In these hybrids 95 of activegenes show an intermediate expression level Among mostother nonadditively expressed genes the expression shift wastowards the maternal parent [30] When studying transcrip-tion and epigenetic adaptation of maize chromosomes insupplemented oat-maize lines containing the complete oatgenome and individual maize chromosomes most maizegenes showed transcription specific for maize but repressionof gene activity was a more common trend than activation[31]

The level of parental gene expression in allopolyploidhybrids obtained by crossing wheat and rye species is ofsignificant interest for us We do not know such studiesconducted on secalotriticum however an extensive analysisof rye gene expression using cDNA isolated from varioustissues of hybrid plants has been carried out on allohexaploidtriticale obtained from the T turgidum times S cereale cross [1]The classes of absent or silent rye genes have been identifiedA comparison between diploid rye and hexaploid triticalerevealed 112 rye cDNA contigs (sim 05 of the total amount)which were not determined by expression analysis in any ofthe triticale tissues though their expression was relativelyhigh in rye tissues The average DNA sequence identitybetween rye genes not detected in triticale and their mostsimilar contigs in the A and B genomes in T aestivum wasonly 81This degree of identity was significantly lower than

the global average of 91 identity between the set of 200randomly selected rye genes and their best matches in theA and B genomes of T aestivum Thus rye genes with alow similarity to their homeologs in Triticum genomes havea higher probability of being repressed or absent due todeletions in the genomes of allopolyploids This conclusionis in good agreement with our results High identity of ryeand wheat CENH3 sequences (99) enables a high level ofexpression of both parental forms in secalotriticum hybridgenomes

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Thework was supported by the Russian Foundation for BasicResearch (Project 18-54-00013) the Russian FundamentalScientific Research Program (Project 0310-2018-0010) andthe Belarusian Republican Foundation for FundamentalResearch (Project 218`-170)

References

[1] H B Khalil M-R Ehdaeivand Y Xu A Laroche and P JGulick ldquoIdentification and characterization of rye genes notexpressed in allohexaploid triticalerdquo BMCGenomics vol 16 no1 2015

[2] X-FMa P Fang and J PGustafson ldquoPolyploidization-inducedgenome variation in triticalerdquo Genome vol 47 no 5 pp 839ndash848 2004

[3] I A Gordey N B Belko and O M Lyusikov Sekalotritikum(timesSecalotriticum) the genetic basis for the creation and forma-tion of the genome vol 214 Minsk Belaruskaya Navuka Publ2011

[4] B McClintock ldquoThe significance of responses of the genome tochallengerdquo Science vol 226 no 4676 pp 792ndash801 1984

[5] M Sanei R Pickering K Kumke S Nasuda and A HoubenldquoLoss of centromeric histone H3 (CENH3) from centromeresprecedes uniparental chromosome elimination in interspecificbarley hybridsrdquo Proceedings of the National Acadamy of Sciencesof the United States of America vol 108 no 33 pp E498ndashE5052011

[6] T Ishii R Karimi-Ashtiyani and A Houben ldquoHaploidiza-tion via Chromosome Elimination Means and MechanismsrdquoAnnual Review of Plant Biology vol 67 pp 421ndash438 2016

[7] D J Amor P Kalitsis H Sumer and K H A Choo ldquoBuildingthe centromere From foundation proteins to 3D organizationrdquoTrends in Cell Biology vol 14 no 7 pp 359ndash368 2004

[8] L Comai S Maheshwari and P A Marimuthu ldquoPlant cen-tromeresrdquoCurrent Opinion in Plant Biology vol 36 pp 156ndash1672017


[9] P B Talbert T D Bryson and S Henikoff ldquoAdaptive evolutionof centromere proteins in plants and animalsrdquo Journal ofBiology vol 3 no 4 article no 18 2004

[10] S Maheshwari E H Tan A West F C H Franklin L Comaiand S W L Chan ldquoNaturally Occurring Differences in CENH3Affect Chromosome Segregation in ZygoticMitosis ofHybridsrdquoPLoS Genetics vol 11 no 1 p e1004970 2015

[11] P Neumann Z Pavlikova A Koblizkova et al ldquoCentromeresoff the hook massive changes in centromere size and structurefollowing duplication of CenH3 gene in Fabeae speciesrdquoMolec-ular Biology and Evolution vol 32 pp 1862ndash1879 2015

[12] D Vermaak H S Hayden and S Henikoff ldquoCentromeretargeting element within the histone fold domain of CidrdquoMolecular and Cellular Biology vol 22 no 21 pp 7553ndash75612002

[13] H S Malik and S Henikoff ldquoAdaptive evolution of Cid acentromere-specific histone in Drosophilardquo Genetics vol 157no 3 pp 1293ndash1298 2001

[14] P B Talbert R Masuelli A P Tyagi L Comai and SHenikoff ldquoCentromeric localization and adaptive evolution ofan Arabidopsis histone H3 variantrdquo e Plant Cell vol 14 no5 pp 1053ndash1066 2002

[15] CDHirsch YWuH Yan and J Jiang ldquoLineage-specific adap-tive evolution of the centromeric protein cenh3 in diploid andallotetraploid oryza speciesrdquo Molecular Biology and Evolutionvol 26 no 12 pp 2877ndash2885 2009

[16] E D Badaeva L F Sozinova N S Badaev O V Muravenkoand A V Zelenin ldquoldquoChromosomal passportrdquo of Triticumaestivum L em Thell cv Chinese Spring and standardizationof chromosomal analysis of cerealsrdquoCereal Research Communi-cations vol 18 pp 273ndash281 1990

[17] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[18] W R Pearson and D J Lipman ldquoImproved tools for biologicalsequence comparisonrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 85 pp 2444ndash24481988

[19] A G Alkhimova J S Heslop-Harrison A I Shchapova andA V Vershinin ldquoRye chromosome variability in wheat-ryeaddition and substitution linesrdquo Chromosome Research vol 7no 3 pp 205ndash212 1999

[20] S Fu Z Lv X Guo X Zhang and F Han ldquoAlteration ofTerminal Heterochromatin and Chromosome Rearrangementsin Derivatives of Wheat-Rye Hybridsrdquo Journal of Genetics andGenomics vol 40 no 8 pp 413ndash420 2013

[21] R Appels J P Gustafson and C E May ldquoStructural variationin the heterochromatin of rye chromosomes in triticalesrdquoeoretical and Applied Genetics vol 63 no 3 pp 235ndash2441982

[22] X-F Ma and J P Gustafson ldquoTiming and rate of genomevariation in triticale following allopolyploidizationrdquo Genomevol 49 no 8 pp 950ndash958 2006

[23] O M Lyusikov and I A Gordei ldquoAnalysis of meiotic genomestabilization in the rye-wheat amphidiploid secalotriticum(timesSecalotriticum SRRAABB 2n = 42)rdquo Russian Journal ofGenetics vol 50 no 7 pp 693ndash700 2014

[24] O M Lyusikov and I A Gordei ldquoCytogenetic stabilizationof secalotriticum (timesTriticosecale derzhavinii secalotriticumRozenst et Mittelst 119878RRAABB 2n = 42) genomerdquo Cytologyand Genetics vol 49 no 2 pp 118ndash124 2015

[25] E V Evtushenko E A Elisafenko S S Gatzkaya Y ALipikhina A Houben and A V Vershinin ldquoConserved molec-ular structure of the centromeric histone CENH3 in Secale andits phylogenetic relationshipsrdquo Scientific Reports vol 7 no 12017

[26] J Lima-Brito H Guedes-Pinto and J S Heslop-Harrison ldquoTheactivity of nucleolar organizing chromosomes in multigenericF1 hybrids involving wheat triticale and tritordeumrdquo Genomevol 41 no 6 pp 763ndash768 1998

[27] D Autran C Baroux M T Raissig et al ldquoMaternal epigeneticpathways control parental contributions to arabidopsis earlyembryogenesisrdquo Cell vol 145 no 5 pp 707ndash719 2011

[28] M D Nodine and D P Bartel ldquoMaternal and paternal genomescontribute equally to the transcriptome of early plant embryosrdquoNature vol 482 no 7383 pp 94ndash97 2012

[29] G Del Toro-De Leon M Garcıa-Aguilar and C S Gill-mor ldquoNon-equivalent contributions of maternal and paternalgenomes to early plant embryogenesisrdquo Nature vol 514 no7524 pp 624ndash627 2014

[30] M M Alonso-Peral M Trigueros B Sherman et al ldquoPatternsof gene expression in developing embryos of Arabidopsishybridsrdquoe Plant Journal vol 89 no 5 pp 927ndash939 2017

[31] Z Dong J Yu H Li et al ldquoTranscriptional and epigeneticadaptation ofmaize chromosomes inOat-Maize addition linesrdquoNucleic Acids Research vol 46 no 10 pp 5012ndash5028 2018

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a hybrid cell [4] and is accompanied by various chromoso-mal abnormalities including those affecting the centromerestructure It has been previously determined that using anintermediary species triticale as a source of wheat genomesin crosses with rye proved to be effective for overcomingthe barrier of unidirectional progamic incompatibility ofparent species [3] As a result these rye-wheat amphidiploidhybrids were created by crossing tetraploid rye (SRRRR 2n= 4x = 28) with hexaploid triticale (TRRAABB 2n = 6x =42) and were named secalotriticum (timesSecalotriticum syntimesSecalotriticum = Secale L times Triticum L SRRAABB 2n =6x = 42) The presence of rye maternal cytoplasm in hybridswas proved by analyzing restriction fragments of species-specific mitochondrial DNA18S5S-repeated sequences andthe ndhH-region of chloroplast DNA which showed a pat-tern characteristic of chloroplast DNAand rye mitochondrialDNA [3] The created secalotriticum hybrids are charac-terized by a wider variability range of morphological andeconomically valuable traits compared to the original triticaleforms [3] Secalotriticum creates more advantageous condi-tions for enhancing the expression of rye genetic systems andthe manifestation of its valuable adaptive traits

The process of the hybrid genome formation and itssubsequent stabilization are directly related to the normal-ization of meiosis and proper segregation of chromosomesIncompatibility of centromeres of different species seems tobe the main reason of the chromosome elimination of oneof the parental genomes in hybrids [5 6] The pivotal rolein the proper chromosome segregation during meiosis andmitosis lies with centromeres their identity being defined bythe presence of the centromere-specific variant of histone H3known in plants as centromere-specific histone H3 variantCENH3 [7 8] This is due to the fact that at the molecularlevel the most specialized and universal characteristic of theactive centromere is the presence of CENH3 instead of thecanonical histone H3 in the nucleosomes of centromericchromatin As it has been shown in somemammalian speciesandDrosophila in case of centromeric histone loss there is nokinetochore formation and no correct chromosome segrega-tion during cell division for some reason [9]Unlike canonicalhistone X3 which has a conserved structure CENH3 nor-mally shows considerable variability across species [5 10 11]Different domains of this molecule are diverging differentlyAn extended N-terminal tail (NTT) domain and loop 1 ofthe histone fold domain (HFD) putatively interact with cen-tromeric DNA [12] and show signatures of positive selectionin some animal and plant species [13] while the part of theHFD that lies outside loop 1 is generally conserved [14 15]

Because of a special role CENH3 has in the formationand function of centromeres we explore effects of remotehybridization using an unusual combination of parentalforms on the structure of this protein and perform a cyto-genetic analysis of the karyotypes

2 Materials and Methods

Two secalotriticum combinations (timesSecalotriticum RozenstSRRAABB 2n = 6x = 42) Verasenrsquo timesMikhasrsquo (STr VV) and

Verasenrsquo timesDubrava (STr VD) were developed by hybridizingthe tetraploid rye cultivar Verasenrsquo (119878RRRR 2n=4x=28)with the hexaploid triticale cultivars Mikhasrsquo and Dubrava(119879AABBRR 2n=6x=42) Triticale acted as an intermediaryspecies a source of wheat genomes and an inhibitor ofrye S-RNases which allowed overcoming the rye progamicincompatibility with wheat Stabilization of karyotypes wasfacilitated by a single backcross of rye-triticale pentaploidF1 hybrids (SRRABR 5x=35) on triticale followed by self-pollination within 15 generations and constant selection forcytological stability and phenotypic homogeneity

Karyotyping of secalotriticum identification of the orig-inal species chromosomes was carried out by means ofroot apical meristem squashed preparations and differentialstaining using a Giemsa technique (C-banding) [16]

Molecular analysis was conducted on plants of parentalforms and hybrids grown in a greenhouse The total RNAwas isolated from leaves of 10-12-day-old seedlings from4-5 plants of parental forms RNA of hybrid forms wasisolated from each plant separately Isolation of RNA wasperformed using the TRI Reagent (MRC Ink USA) [17]To avoid contamination with genomic DNA total RNA wastreated with RQ-RNase Free DNase (Promega MadisonUSA) To synthesize cDNA a RevertAidTM H MinusFirst Strand cDNA Synthesis Kit (Thermo Scientific USA)was applied The resulting cDNA was used as a templatein a series of PCR reactions with primers synthesizedspecifically to amplify the CENH3 N-terminal tail (NTT)The primer sequences are 51015840 ndash ATGGCCCGCACCAAGC(F) and 51015840 ndash GAAACTCGACCGACTTCTG (R) Theproduct size was 268 bp PCR products were clonedinto the pTZ57RT plasmid (InsTAclone PCR cloningkit Thermo Scientific bd0) and analyzed by Sangersequencing using BigDye Terminator v31 Cycle SequencingKit (Applied Biosystems USA) The reaction productswere separated on the 3500 Genetic Analyzer (AppliedBiosystems USA) The sequences of individual clonesobtained for each sample were analyzed using the UniProUgene software (httpugenenet) and FASTA softwarepackage [18] The search for the identity of the nucleotidesequences was carried out using the BLAST algorithm inthe NCBI database (httpblastncbinlmnihgovBlastcgi)Amino acids alignments were performed online usingClustal Omega (httpwwwebiacukToolsmsaclustalo)and used for downstream analysis and visualization(httpwwwjalvieworg) Graphic images were preparedusing the Jalview program (httpwwwjalvieworg)

3 Results and Discussion

The STr VM (Verasenrsquo times Mikhasrsquo) and STr VD (Verasenrsquotimes Dubrava) (SRRAABB 2n=6x=42) secalotriticum kary-otypes were analyzed by differential staining (C-banding)[16] This method reveals the pattern of heterochromaticregions localization which is specific for each chromosomeofrye and wheat thereby allowing identification of each chro-mosome in a hybrid karyotype Figures 1(a) and 1(b) showthat the karyotypes of the STr VM and STr VD hybrids arestable balanced hexaploids not containing minichromosomes


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dynamics of the centromeric histone cenh3 structure in rye

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