A bulked segregant analysis using RAPD technique was carried out to identify molecular markers linked to apomixis in a Brachiaria humidicola F1 population that segregated 1 : 1 for the mode of reproduction (apomixis and sexual). A marker related to the apo-locus was found. Segregation data, together with this marker were used to generate a map of the region. This marker was located at 4.61 cM of the target locus, and it can be used in deploying marker-assisted selection for mode of reproduction in the hybrid progenies of this species

BITENCOURT, G. de A.

CHIARI, L.

LEGUIZAMON, G. O. de C.

PAGLIARINI, M. S.

SCHUSTER, I.

VALLE, C. B. do

ZORZATTO, C.

P>A bulked segregant analysis using RAPD technique was carried out to identify molecular markers linked to apomixis in a Brachiaria humidicola F(1) population that segregated 1 : 1 for the mode of reproduction (apomixis and sexual). A marker related to the apo-locus was found. Segregation data, together with this marker were used to generate a map of the region. This marker was located at 4.61 cM of the target locus, and it can be used in deploying marker-assisted selection for mode of reproduction in the hybrid progenies of this species.Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Gado Corte, BR-79002970 Campo Grande, MS, BrazilUniversidade Estadual de Maringá (UEM), Dept Biol Celular & Genet, Maringa, Parana, BrazilUniv Estadual São Paulo Julio de Mesquita Filho, Jaboticabal, SP, BrazilCooperat Cent Pesquisa Agr, Cascavel, Parana, BrazilUniv Estadual São Paulo Julio de Mesquita Filho, Jaboticabal, SP, Brazi

Zorzatto, C.

Chiari, L.

de Araujo Bitencourt, G. [UNESP]

do Valle, C. B.

de Campos Leguizamon, G. O.

Schuster, I.

Pagliarini, M. S.

Repositório Institucional UNESP

Plant Breeding

Identification of a molecular marker linked to apomixis in Brachiaria humidicola (Poaceae)

P>A bulked segregant analysis using RAPD technique was carried out to identify molecular markers linked to apomixis in a Brachiaria humidicola F(1) population that segregated 1 : 1 for the mode of reproduction (apomixis and sexual). A marker related to the apo-locus was found. Segregation data, together with this marker were used to generate a map of the region. This marker was located at 4.61 cM of the target locus, and it can be used in deploying marker-assisted selection for mode of reproduction in the hybrid progenies of this species.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

de Araujo Bitencourt, G.

Acervo Digital da Universidade Estadual Paulista

Acervo Digital da Unesp

RCAAP - Repositório Científico de Acesso Aberto de Portugal

Repository Open Access to Scientific Information from Embrapa

Short CommunicationIdentiﬁcation of a molecular marker linked to apomixis in Brachiaria humidicola(Poaceae)C. ZORZATTO1 ,L. CHIARI2 , 5 ,G. DE ARAU´JO BITENCOURT3 ,C. B. DO VALLE2 ,* ,G. O. DE CAMPOS LEGUIZAMO´N2 ,I . SCHUSTER4 ,* andM. S. PAGLIARINI 1 ,*1Departamento de Biologia Celular e Gene´tica, Universidade Estadual de Maringa´, Maringa´, Parana´; 2Embrapa Gado de Corte,BR 262 Km 4 Caixa Postal 154, CEP 79002-970, Campo Grande, Mato Grosso de Sul; 3Universidade Estadual de Sa˜o PauloJu´lio de Mesquita Filho, Jaboticabal, Sa˜o Paulo; 4Cooperativa Central de Pesquisa Agrı´cola, Cascavel, Parana´, Brazil;5Corresponding author, E-mail: lchiari@cnpgc.embrapa.brWith 1 ﬁgure and 1 tableReceived April 30, 2009/Accepted December 18, 2009Communicated by T. Lu¨bberstedtAbstractA bulked segregant analysis using RAPD technique was carried out toidentify molecular markers linked to apomixis in a Brachiariahumidicola F1 population that segregated 1 : 1 for the mode ofreproduction (apomixis and sexual). A marker related to the apo-locuswas found. Segregation data, together with this marker were used togenerate a map of the region. This marker was located at 4.61 cM ofthe target locus, and it can be used in deploying marker-assistedselection for mode of reproduction in the hybrid progenies of thisspecies.Key words: apospory — linkage map — RAPD — tropicalforage grassBrazil is the worlds largest beef exporter and also has thebiggest cattle herd, numbering about 200 million heads. Thislarge herd feeds almost exclusively on pastures. Cultivatedpastures in Brazil cover an estimated 120 million ha, andabout 85% of this area is planted to Brachiaria species(Macedo 2006).This paper describes the work carried out with a hybridpopulation of B. humidicola (Rendle) Schweickert, also knownas koronivia grass, a perennial savanna grass native to Africa.This species is well adapted to poorly drained and infertile acidsoils (Keller-Grein et al. 1996). Most B. humidicola ecotypesare polyploid and reproduce by apomixis, an asexual mode ofreproduction through seeds (do Valle and Savidan 1996).Apomixis in B. humidicola is facultative, pseudogamous andembryo sacs show apospory of the Panicum type, where theapospore develops into a diploid, monopolar four-nucleateembryo sac with a three-celled egg apparatus and a central cellwith one nucleus (Savidan 1982). Pseudogamy means that forendosperm development the secondary nucleus of the embryosac needs to be fertilized by a male gamete (Alves et al. 2001).Facultative apomictic plants develop both aposporous andmeiotic sacs within the same inﬂorescence and even within thesame pistil (Nogler 1984). The two reproductive modes are notmutually exclusive, therefore, some segregation may beobserved in the progeny of an apomictic Brachiaria genitor.In the main Brachiaria species apomixis is simply inherited(do Valle and Savidan 1996), thus apomictic and sexualhybrids are generated at each sexual · apomictic cross in theproportion of 1 : 1.The objective of this work was to identify RAPD markerstightly linked to apomixis in B. humidicola hybrids to improveeﬃciency and accuracy in the determination of the reproduc-tion mode on the breeding programme of this species.Materials and MethodsPlant material: One hundred and seven plants belonging to anF1 population obtained by crossing a polyploid apomictic cultivar(B. humidicola cv. BRS Tupi) with a polyploid sexual parent(BRA005811-H31) were analysed. The hybrid nature of the F1 plantswas conﬁrmed using RAPD markers.Analysis of mode of reproduction: Flowers of these hybrids werecollected at anthesis. Sixty ovules were dissected from each hybrid andthen clariﬁed using methyl salicylate according to the Young et al.(1979) procedure. At least 50 cleared ovules were analysed per plantusing an interference contrast microscope. A chi-squared test wasperformed on this progeny to verify whether the genetic segregation forapomictic to sexual plants ﬁt the expected model for a tetrasomicmonogenic inheritance.Molecular analysis: The DNAs were extracted from young leavesusing the Bonato et al. (2002) method. The polymerase chain reactions(PCRs) and thermal conditions were performed according to theBonato et al. (2002) procedure.A bulked segregant analysis (Michelmore et al. 1991) using 270RAPD primers was carried out to identify RAPD markers linked toapomixis. Five apomictic bulks (AB) and ﬁve sexual bulks (SB) wereprepared, each one containing equimolar quantities of DNA from 10phenotyped plants. Some plants were not used on the bulks, but wereindividually analysed.Linkage analysis: Linkage analysis was conducted with the GQMOLcomputer software (Cruz and Schuster 2007) and a map wasconstructed. The LOD score used was 3.0. Kosambis mappingfunction was used to convert recombination units into geneticdistances (Kosambi 1944). The goodness of ﬁt of segregation wastested by the chi-squared test.*Pesquisador do CNPq Scholar.Plant Breeding 129, 734—736 (2010) doi:10.1111/j.1439-0523.2010.01763.x 2010 Blackwell Verlag GmbHwileyonlinelibrary.comSelection eﬃciency: Selection eﬃciency (SE) of the markers linked tothe apomixis locus was based on the comparison between thephenotypic and the genotypic (markers) evaluations and was calcu-lated according to Silva et al. (2007).Results and DiscussionThe Panicum type of aposporous embryo sac can be diﬀeren-tiated from the Polygonum (meiotic) type by the absence ofantipodal cells and the presence of only one polar nucleusupon examination with interference contrast microscopy.Besides, aposporous sacs are rarely alone. The most commonis to ﬁnd two to three aposporous sacs within one ovule of theapomictic plant. The sexual plant will display only onePolygonum type sac (meiotic, or reduced sac) containing theegg cell, two polar nuclei and several large, multinucleatedantipodal cells (Fig. 1). The proportion of meiotic andapomictic embryo sacs is given in Table 1.Determination of mode of reproduction done on the 107hybrids resulted in a proportion of apomictic to sexual plantsnot signiﬁcantly diﬀerent from the 1 : 1 ratio (P = 0.7718)with 55 apomictics plants and 52 sexual plants. The resultscorroborate the proposed hypothesis of a single dominant genecontrolling apomixis in this hybrid population (do Valle andSavidan 1996).Of the 217 RAPD primers tested on the parents, 32(14.75%) ampliﬁed poorly or did not amplify. Of the remain-ing 185 primers, 176 (81.10%) were polymorphic on theparents and only seven (3.98%) ampliﬁed polymorphic mark-ers between contrasting bulks.Only one molecular marker of 650 bp obtained by ampli-ﬁcation with the primer 64, named 64_650, resulted in a goodﬁt considering a 1 : 1 segregation ratio (P = 0.4986) in the F1population and the linkage analysis revealed that this markerco-segregated with apomixis in B. humidicola. The distancebetween the 64_650 marker and the apomixis locus (apo-locus)was 4.61 cM and the value of LOD score was 23.44. Fiverecombinant individuals were found between the 64_650marker and the apomixis locus. The assisted selection eﬃ-ciency for this marker linked to the apo-locus was 95.6%.Although, apomixis in B. humidicola does not exhibitcomplete penetrance, as observed by the percentage of meioticembryos in apomictic plants (Table 1), the possibility ofincomplete penetrance was not included in the molecularmarker analysis. The number of embryos analysed for each F1plant enabled a precise identiﬁcation of its mode of reproduc-tion. The molecular marker identiﬁed in this work may be usedfor assisted selection of the apomixis trait; however, it isimportant to consider that meiotic embryos may be producedby apomitic plants.The use of this molecular marker for detection of apomixisin B. humidicola can certainly increase the ability to accuratelyclassify progenies. This technology oﬀers the prospect of rapidand early screening for the trait. Furthermore, the identiﬁca-tion and characterization of the genomic regions involved inthe phenomenon of apomixis in the diﬀerent species where thetrait has been described is an initial and essential step in theprocess of introgression of this trait into valuable crops.AcknowledgementsThis work was supported by a grant from FUNDECT-MS. C.Zorzatto was the recipient of a scholarship from CNPq (BrazilianGovernment).ReferencesAlves, E. R., V. T. C. Carneiro, and A. C. G. Arau´jo, 2001: Directevidence of pseudogamy in apomictic Brachiaria brizantha (Poa-ceae). Sex. Plant Reprod. 14, 207)212.Bonato, A. L. V., C. B. do Valle, L. Jank, R. M. S. Resende, and G. O.C. Leguizamo´n, 2002: Extrac¸a˜o de DNA genoˆmico de Brachiaria ePanicum maximum. Comunic. Tec. Embrapa Gado de Corte 79,(a)(c)5 µm5 µm 5 µm5 µm(b)(d)Fig. 1: Embryo sacs from Brachi-aria humidicola hybrids observedunder interference contrast micros-copy. (A) Meiotic embryosac – SEX from a sexual hybridcontaining two polar nuclei – Pnand several multinucleated anti-podal cells – A. (B) Sterile embryosac – S. (C) Apomictic embryosac – APO with only one polarnucleus and absence of antipodalcells. (D) Multiple apomicticembryo sacs – MSTable 1: Variation on the percentage of the types of embryo sacsfound in Brachiaria humidicola hybridsReproductionmodeNumberof plantsTypes of embryo sacs (%)MeioticAposporous(single and/or multiple) Sterile AbnormalSexual 55 23–100 0 0–64 0–66Apomictic 52 0–65 18–91 0–28 0–70Total 107RAPD marker for apomixis in Brachiaria 7351)4.Cruz, C. D., I. Schuster, 2007: GQMOL: aplicativo computacionalpara ana´lise de dados moleculares e de suas associac¸o˜es comcaracteres quantitativos. Versa˜o 2007.0.0 Vic¸osa, MG: UFV. http://www.ufv.br/dbg/gqmol/gqmol.htmdo Valle, C. B. do., Y. H. Savidan, 1996: Genetics, cytogenetics, andreproductive biology of Brachiaria. In: J. W. Miles, B. L. Maass, andC. B. do Valle (eds), Brachiaria: Biology, Agronomy and Improve-ment, 147)163. CIAT, Colombia.Keller-Grein, G., B. L. Maass, and J. Hanson, 1996: Natural variationin Brachiaria and existing germplasm collections. In: J. W. Miles,B. L. Maass, and C. B. do Valle (eds), Brachiaria: Biology,Agronomy, and Improvement, 16)42. CIAT, Cali.Kosambi, D. D., 1944: The estimation of map distance fromrecombination values. Ann. Eugen. 12, 172)175.Macedo, M. C. M., 2006: Aspectos eda´ﬁcos relacionados com aproduc¸a˜o de Brachiaria brizantha cultivar Marandu. In: R. A.Barbosa (ed.), Morte de Pastos de Braquia´rias, 35)63. Embrapa,Campo Grande.Michelmore, R. W., I. Paran, and R. V. Kesselli, 1991: Identiﬁcationof markers linked to disease-resistance genes by bulked segregantanalysis: a rapid method to detect markers in speciﬁc genomicregions by using segregating populations. Proc. Natl Acad. Sci. USA88, 9828)9832.Nogler, G. A., 1984: Gametophytic apomixis. In: B. M. Johri (ed.),Embryology of Angiosperms, 475)518. Springer-Verlag, Berlin.Savidan, Y., 1982: Nature et he´re´dite´ de lapomixie chez Panicummaximum Jacq. Trav. et Docum. ORSTOM 153, 1)161.Silva, M. F., I. Schuster, J. F. V. Silva, A. Ferreira, E. G. Barros, andM. A. Moreira, 2007: Validation of microsatellite markers forassisted selection of soybean resistance to cyst nematode races 3 and14. Pesqui. Agropecu. Bras. 42, 1143)1150.Young, B. A., R. T. Sherwood, and E. C. Bashaw, 1979: Cleared-pistyland thick-sectioning techniques for detecting aposporous apomixisin grasses. Can. J. Bot. 57, 1668)1672.736 C. ZORZATTO , L . CH IAR I , G. DE ARAU´ JO BITENCOURT e t a l .

Identification of a molecular marker linked to apomixis in Brachiaria humidicola (Poaceae).

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Identification of a molecular marker linked to apomixis in Brachiaria humidicola (Poaceae)

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