A century of wheat breeding in Brazil: the origin and inheritance of the Lr34 locus in wheat varieties released from 1922 to 2016

Wheat breeding in Brazil began in 1919. A cross between the varieties Fronteira and Mentana gave rise to Frontana, the most popular ever released, being recommended for more than 40 years. Frontana played an important role as a source of resistance to leaf and stripe rust as well as resistance to preharvest sprouting. Possessing an early maturity cycle, it contributed to change the predominant varietal pattern at that time. The resistance gene Lr34 was first described in Frontana. The gene confers durable adult plant resistance to leaf rust and other biotrophic fungi. Because of its durability, stability across environments, haplotype diversity and wide geographical distribution, Lr34 is probably the most important single resistance gene in wheat. We evaluated 453 wheat varieties that have been released or recommended for cultivation in Brazil from 1922 to 2016 using KASPar markers to detect those varieties carrying the diagnostic mutations corresponding to the presence of the Lr34 resistance allele.

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INTRODUCTION
heat reedin in ra il which e an in 1919 is com letin a centur in 2019 el uca 1999 ousa and aier o 201 he rst lines were selected from amon rimitive wheats introduced uro ean immi rants in the 16 th centur that dis la ed li le ada ta ilit to local rowin conditions armo 1911 n 192 the rst ra ilian wheat varieties ol ss and 1 a ol ss selection were released and soon a er were used intensivel in crosses ivin rise to man descendants amon cultivars in ra il ousa 200 n the 1920s the talian si cultivars entana and rdito released in tal in the earl 1900s or hi 2001 were introduced and su se uentl widel cultivated in ra il on account of their outstandin ada ta ilit and disease resistance the were used as arents in man local reedin ro rams ousa and aier o 201 n 19 0 a cross etween ronteira and entana ave rise to rontana el uca 1999 the most o ular variet ever released now sowed across the countr for more than 0 ears from 19 0 to 19 ousa and aier o 201 rontana is the enot e with the reatest num er of o s rin amon the commercial cultivars in ra il with 1 descendants in total rontana la ed an im ortant role as a source of resistance to leaf and stri e rust resistance to re harvest s routin and as source of the earl maturit c cle inherited MS Chaves et al. 11 that detects the indel in e on 11 the est dia nostic mar er for mar er assisted selection of Lr34 due to its co dominanc and ro ustness a ouri et al 2010 n a centur of wheat reedin in ra il the oint e orts of man u lic and rivate research institutes resulted in the release of more than 0 varieties to farmers raits such as disease resistance aluminum tolerance c cle lant hei ht and architecture as well as ield and technolo ical ualit were the focus of these reedin ro rams ousa and aier o 201 n this a er we evaluated wheat varieties released or recommended for cultivation in ra il from 1922 to 2016 usin ar mar ers to detect those varieties carr in the dia nostic mutation corres ondin to the Lr34 resistance allele ased on the results of oth and edi ree anal sis we discuss the li el inheritance of this locus throu h enerations of varieties released in ra il from the ver e innin of reedin ro rams to the resent da e also resent the reactions to leaf rust in adult lant sta es of a rou of varieties ossessin the Lr34 resistance locus and discuss the sta ilit of the rust reaction conferred across environments

Plant material
he erm lasm collection com rised he a loid wheat enot es that have een under cultivation in ra il from 1922 to 2016 a le 1 he ma orit 1 are im roved varieties roduced local reedin ro rams few varieties were introductions from tal rdito and entana ru ua enten rio ustralia lorence r entina uc anantial e ico iano 6 nia 66 u ateco itic 62 onora 6 anori 1 and nahuac and ara ua ara ua 21 ara ua 2 1 and ta ua ome landraces ranco eadeiros ales amacr nia and olonista and varieties of un nown ori in andeirantes eneral ar as and eladinho that have een cultivated in ra il were also anal ed eeds were rovided the m ra a heat erm lasm an located in asso undo in the state of io rande do ul DNA extraction and marker analysis eeds from each accession were sown in commercial su strate and rown in a cham er under controlled tem erature 22 1 da ni ht at 1 h da len th for 1 da s hen the seedlin s were at the two leaf sta e leaf se ments from one seedlin er accession were cut and laced in 2 m microtu es mmediatel a er the leaf sam les were l o hili ed for h he dried tissue was macerated usin issue ser ia en and the was e tracted accordin to the he adec ltrimeth lammonium romide u er rotocol efort and ou las 1999 concentrations were measured usin a s ectro hotometer hermo cienti c and normali ed to a out 100 n 1 he sam les were laced in 96 well lates 96 en and sent to en en t td orcester outh frica to e anal ed usin the om etitive llele eci c ar mar er rait ased assa s for the resence of Lr34/ Yr18/Sr57/Pm38 alleles del del or ins ins were run as reviousl descri ed reisi ac er et al 2016 he wheat variet arie a was used as ositive control and a no tem late control was used as a ne ative control

Disease scoring
he reaction to leaf rust of some wheat varieties was evaluated under eld conditions from 2006 to 2010 and in 201 ecause of the limited amount of seeds availa le at the m ra a heat erm lasm an onl varieties with ade uate num er of seeds re uired to sta lish eld lots were included enetic stoc s com risin near iso enic lines and varieties from diverse ori ins nown to ossess the Lr34 ene were also evaluated for rust reaction i hl susce ti le ra ilian varieties were used as controls ach enot e was sowed in a ro imatel 1 m rows 20 cm a art and 0 to 0 seeds were lanted er lot reader rows of the susce ti le variet 10 were sowed er endicular to lots and inoculated in earl une with a mi ture of races of P. triticina covering a wide range of the virulence occurring in Brazil.
aces chosen were as follows 2 2 9 9 0 1 2 6 and 9 he mi ture of isolates was sus ended in oltrol 1 0 oil and s ra ed on the s reader rows in earl cto er usin a ac ac s ra er he severit and res onse ratin for adult lant eld resistance was ased on the modi ed o scale eterson et al 19 he host infection t e was rated as resistant ver small uredinia with necrosis moderatel resistant small to moderate uredinia with necrosis moderatel susce ti le small to moderate uredinia with chlorosis and susce ti le lar e uredinia without necrosis or chlorosis oelfs et al 1992 eaf rust severit and resistance res onses were recorded in mid ovem er when the susce ti le control 10 had leaf rust severit of at least 0

RESULTS AND DISCUSSION
n a centur of wheat reedin in ra il more than 0 varieties were released to farmers ecause of the hi hl conducive environments one of the ma or concerns addressed these ro rams was the enhancement of levels of resistance to the common wheat diseases includin leaf rust rom the accessions anal ed in this stud 11 2 6 were found to carr the mutation del del corres ondin to the Lr34 leaf rust resistance allele a le 1 few lines resented a deletion in a sin le allele del ins and therefore the Lr34 locus was not con rmed to e resent in these lines as a functional allele he ori in of the Lr34 resistance allele in ra ilian wheat varieties and its inheritance across decades over one century of wheat breeding is discussed below.

The 1920s and 1930s
oth entana and rdito were found to carr the Lr34 ene usin the dia nostic mar ers which is in accordance with revious results usin other molecular mar ers olmer et al 200 a ouri et al 201 entana is the older recognized source of the Lr34 locus in outh merica olmer et al 200 it was traced to e the most li el donor of the Lr34 ene to the ma orit of varieties detected to carr this ene either directl or indirectl via rontana i ure 1 ranco and eadeiros are the oldest ra ilian cultivars found to carr the Lr34 resistance ene a le 1 oth are landraces and their edi rees are un nown and therefore the ori in of the Lr34 ene in these varieties remains unclear inuano and ur resa are also amon the old cultivars ossessin the Lr34 ene oth were develo ed war ec man from a cross etween ol ss and lfredo haves 6 21 he cultivar lfredo haves 6 21 is a selection from the landrace urco ousa 199 ol ss is a landrace selected from undetermined talian varieties in the earl 1900s ousa 200 ol ss is the most li el donor of the Lr34 ene to inuano and ur resa ecause it carries the Ltn ene causin leaf ti necrosis which is a henot ic mar er associated with the ene Lr34 ousa and arcellos 2000

The 1940s
he rst descendants from entana were released in the 19 0s with rontana ein the most im ortant variet amon them rontana is the most o ular variet ever released in ra il ein sowed across the countr for ears from 19 0 to 19 rontana is the variet where the ene Lr34 was rst descri ed ic 19 u se uentl it was included in the rou of enetic stoc s used for the develo ment of the dia nostic molecular mar ers a udah et al

MS Chaves et al.
2009 used in our stud ere we found that rontana is the most li el source of the Lr34 ene of more than 0 wheat varieties released for cultivation in ra il i ure 1 n addition to rontana other varieties released in the 19 0 s were found to possess the Lr34 ene a le 1 namel incana loreana ales and a eadinho itana descends from entana via loreana in our stud it was found to ossess the Lr34 gene as well. Pitana also carries the Lr13 resistance ene identi ed due to the lin a e with the ene Ne2 roducin the h rid necrosis henot e ousa 1999

The 1950s
Of the four varieties released in this decade and found to carry the Lr34 ene usin the dia nostic mar ers three are descendants from rontana a le 1 i ure 1 eran olis ara inho and ortale a eran olis and Carazinho also carry the Ne2 ene even 19 even 19 1 and ma carr the Lr13 resistance ene as well escendin directl from entana the cultivar 11 6 was released in 19 and remained under cultivation for ears ousa and aier o 201 n addition to ossessin the Lr34 resistance allele 11 6 was widel used in the develo ment of several other wheat enot es rimaril due its tolerance to l ousa 199 ur results are in accordance with previous reports in which the presence of the The 1960s he Lr34 ene resent in the varieties released in the 1960s a le 1 was ro a l inherited in one of four wa s 1 irectl or indirectl from rontana i ure 1 descends directl from rontana ova rata and oti or oth descend from eran olis released in 19 0 and ossessin the Lr34 ene as reviousl discussed in this wor oti or has been previously postulated to carry the Lr34 gene due to the presence of the Ltn gene associated with the leaf ti necrosis ousa and arcellos 2000 re im most li el inherited Lr34 from rontana via olotana o re ro a l inherited Lr34 from rontana via olotana or a tana 2 ndirectl from entana i ure 1 ara ua u probably inherited Lr34 from entana via eana ecause loreana descends directl from entana and was detected to possess the Lr34 ene in this stud a le 1 ndirectl from ol ss atriarca most li el inherited Lr34 from inuano as reviousl discussed in section The 1920s and 1930s rom undetermined sources ased on our results and on the edi ree anal sis the ori in of the Lr34 ene could not e traced in the followin varieties ila elha uara uava and 2

The 1970s
his eriod was mar ed the e ansion of wheat acrea e in ra il and the creation of man research centers for wheat im rovement includin the ational heat esearch enter of m ra a m ra a heat located in asso undo urin this decade a lon lastin colla orative wor etween m ra a heat and was esta lished an varieties from e ico and some varieties from ara ua r entina and ru ua were introduced for cultivation in outh ra il and these were used in crosses the local reedin ro rams as well ousa and aier o 201 he Lr34 ene was detected in 19 varieties released in the 19 0s a le 1 where it was ro a l inherited in one of four wa s 1 ndirectl from rontana i ure 1 ncru ilhada most li el inherited Lr34 from rontana via ortale a released in 19 and ossessin Lr34 as discussed reviousl in this a er and acaria most li el inherited Lr34 from rontana via eran olis released in 19 0 which descends directl from rontana and was detected to ossess the Lr34 ene as discussed reviousl in this a er iano 6 is one of the e ican varieties introduced in ra il in the 19 0s and recommended for cultivation in 19 6 which carries a com ination of adult lant resistance enes to leaf rust and stem rust as a Lr13 Lr34 and Sr2 com le ishra 1992 t was widel used in crosses man reedin ro rams and a num er of its descendants released in the 19 0s were found to carr the Lr34 resistance allele i ure 1 as will e further discussed in this a er iano 6 most li el inherited Lr34 from rontana via hris a orth merican variet derivative of rontana and nown to carr Lr34 olmer et al 200 1 orena released in 19 descends directl from iano 6 and is assumed that the share the same source of the Lr34 ene l ria ro a l inherited Lr34 from rontana via e anos into riollo lthou h we have not found an a er re ortin that e anos into riollo possesses Lr34 el ir has een characteri ed as carr in the csLV34a allele and a no and we assume it inherited Lr34 from entana as discussed ara ua 2 1 is a ara ua an variet descended from entana and recommended for cultivation in 19 1 avantes descends from 11 6 released in the 19 0s which was a ver im ortant variet in ra il as discussed reviousl in this a er ources other than rontana or entana ome varieties have com le edi rees ma in the source of the Lr34 ene less than o vious he ra ilian varieties 1 and 2 most li el inherited Lr34 from the e ican varieties a ui and orte o 6 oth ossessin this resistance ene ishra 1992 olmer et al 200 nevertheless the source is not clear considerin their edi rees 2019 rom undetermined sources ased on our results and on the edi ree anal sis the ori in of the Lr34 ene could not e traced in the followin varieties 6 ulha e ra Peladinho and 1 9 ta ua is a ara ua an variet released for cultivation in ra il in 19 9 t ossesses a com le edi ree in which entana rdito and rontana are resent 2019 nevertheless none of its arental varieties are reported to carry the Lr34 gene.

The 1980s
his decade was considera l roli c with 10 varieties released local reedin ro ram ousa and aier o 201 hirt ve varieties were found to carr the Lr34 resistant allele a le 1 the ma orit from reedin ro rams led m ra a varieties ri o the ronomic nstitute of am inas varieties the ronomic nstitute of aran varieties a ar and the former r ani ation of oo eratives of the tate of aran varieties ce ar currentl the oo erative for echnolo ical and conomic evelo ment oodetec t is ro a le that the Lr34 gene detected in the varieties released in the 19 0s had een inherited in one of three wa s 1 ndirectl from rontana i ure 1 ri o 2 most li el inherited Lr34 from rontana via oti or with eran olis ein a direct descendant from Frontana that carries the Lr34 ene as discussed reviousl in section The 1950s he variet ri o 2 was reviousl reported to carry the Ltn ene causin leaf ti necrosis a henot ic mar er associated with the ene Lr34 ousa and arcellos 2000 t also carries the Lr13 resistance ene identi ed due to the lin a e with the ene Ne2 producing the h rid necrosis henot e ousa 1999 iano 6 i ure 1 and e anos into reco oth descendin from rontana and possessing Lr34 as reviousl discussed in section The 1970s are most li el the donors of Lr34 to the following varieties av o ce ar atu ra utu ri o 11 uarani ce ar acuco ce ar 10 ar a ce ar 11 uriti 162 uiui ri o 1 aiu ri o 1 erena a ar 21 a uari a ar 0 irat ri o 29 ava ce ar 1 a ar 1 itacor l ato 22 a ar 1 aet a ar 29 acatu 2 a a s ri o uar and ce ar 16 he edi ree anal sis a le 1 indicates that these two varieties and their descendants includin lue ird ldan i is in i o ari 66 aral i and lue a all ossessin Lr34 artici ated in hi h fre uenc in the crosses ori inatin the a ove mentioned varieties therwise the derived varieties alidad and en amo 62 also artici ated in the cross ori inatin av o a ar 21 a uari and 162 uiui and therefore are li el donors of Lr34. 2 ndirectl from entana hree of the a ove mentioned varieties namel ce ar atu ra 162 uiui and a ar 21 a uari also could have inherited Lr34 from av a a uro ean winter wheat descendin from rdito a si cultivar to entana reported to carry the csLV34b allele olmer et al 200 ri o 9 errados and errano most li el inherited Lr34 from entana via 11 6 released in the 19 0s i ure 1 as reviousl discussed ce ar 1 ro a l inherited Lr34 from entana via lmond ce ar 1 also has ac round from ocora ue a e ican variet carr in the csLV34b allele olmer et al 200 released for cultivation in ra il in 19 1 ousa and aier o 201 rom undetermined sources ased on our results and on the edi ree anal sis the ori in of the Lr34 gene could not be traced in the followin varieties a ar racatu andaia a ar 6 a e ara 21 ua u inuano 2 2 ocantins 1 ni iraiaras 2 antaneiro and ri o ri o has een reviousl ostulated to carr a com ination of Lr13 Lr34 resistance enes due to the resence of the Ltn and Ne2 enes causin leaf ti necrosis and the h rid necrosis henot e res ectivel ousa 1999 ousa and arcellos 2000

The 1990s
MS Chaves et al. owever there was decrease in the num er of varieties detected to carr the Lr34 ene 11 varieties a le 1 ro a l inherited in one of three wa s 1 ndirectl from rontana i ure 1 ri o 9 ara ma a ar a ar 60 and 2 aco have edi rees that trace ac to the cultivars iano 6 hris and e anos intos reco which were im ortant sources of the Lr34 ene in the varieties released in the 19 0s as reviousl discussed in this a er he e ican variet u ateco see section The 1970s is the most li el donor of Lr34 to m ra a 22 and is ossi l the source of Lr34 in a ar 60 2 ma also have inherited Lr34 from ocora ue which also carries the csLV34b allele olmer et al 200 as reviousl discussed in section The 1980s 2 ndirectl from entana he a ove mentioned variety a ar alternativel ma have inherited Lr34 from entana via av a as reviousl discussed in section The 1980s 22 nhumas and ri o 1 fa most li el inherited Lr34 from entana via 11 6 i ure 1 as reviousl discussed in the section The 1950s 10 ro a l inherited Lr34 from ce ar 1 released in 19 which edi ree has een discussed in the corres ondin section rom undetermined sources ased on our results and on the edi ree anal sis the ori in of the Lr34 ene could not e traced in three varieties est halen anito a 9 and a ar 0 irim olmer et al 200 included arin in the rou of varieties ossessin the csLV34b allele and then this variet could be considered as the donor of Lr34 to a ar 0 owever in our stud was not found to ossess the dia nostic deletion that unam i uousl identif the resence of Lr34.

The 2000s
e tested varieties released for cultivation etween 2000 and 2009 f these 19 were found to ossess the Lr34 allele a le 1 all of which were ro a l inherited in one of three wa s 1 ndirectl from rontana i ure 1 he variet ri o 2 was released in the 19 0s lease refer to this section for edi ree anal sis and was widel cultivated The 2010s es ite the reat ro ress of wheat reedin o served in the current decade when 62 varieties were released 11 u lic and rivate com anies there was a stee decrease in the num er of varieties carr in the Lr34 onl the least since the landmar re resented ontana his reduction ma e ecause the Lr34 mediated resistance im oses hi h ener etic demands that lead to the induction of multi le meta olic res onses to su ort cellular ener re uirements olton et al 200 he ener etic cost of the resistance rovided the Lr34 ene ma ro a l a ect the ield so as to ne ativel a ect the selection of lines carr in the Lr34 locus urthermore the selection ressure for cultivars with e er rain ualit could have decreased the intensit of selection for resistance to rust n the varieties released in the 2010s in which the Lr34 ene was found inheritance ma have occurred in one of two wa s 1 ndirectl from rontana i ure 1 a i and o ave descend from varieties 210 released in 19 and 2 aco released in 1991 res ectivel n our stud oth 210 and 2 aco were found to carr the Lr34 gene with ori in traced ac to rontana as discussed in the res ective sections see sections The 1980s and The 1990s 2 rom undetermined sources ased on our results and on the edi ree anal sis the ori in of the Lr34 gene could not e traced in the followin varieties eleto risma and ertero lthou h arcante and u ra most ro a l inherited Lr34 from uamirim released in 200 nevertheless the source of the resistant allele in this variet could not e traced in our wor MS Chaves et al.

Adult plant field resistance
he resistance henot e of Lr34 is characteri ed an increased latent eriod reduced infection fre uenc smaller uredinia and a decreasin radient of uredinia from leaf ase to ti olton et al 200 enetic anal sis of rontana and man wheats showin hi h levels of artial resistance to leaf rust worldwide indicates that this adult lant resistance is ased on the additive interaction of Lr34 and two or three additional slow rustin enes in h and a aram 1992 n e ico leaf rust severit on most cultivars can e related to the num er of slow rustin enes the carr hile susce ti le cultivars can show 100 of leaf rust severit cultivars carr in Lr34 alone can dis la 0 of rust severit cultivars with Lr34 lus one or two minor enes dis la u to 0 of severit and cultivars with Lr34 and two or three additional enes show a sta le res onse across environments with nal leaf rust ratin s lower than 10 in h and a aram 2002 n ra il wheat roducin areas are in environments ver conducive to leaf rust e idemics and revalent races chan e dramaticall over time accordin to the resistance enes resent in the commercial cultivars erman et al 200 n our wor the susce ti le varieties dis la ed hi h levels of severit mostl 0 to 100 while the Lr34 carr in enot es ra ilian varieties and Lr34 enetic stoc s showed reactions com ati le with the henot es descri ed a ove a le 1 mon the varieties itana and 11 6 li el ossess the Lr34 ene alone ecause the leaf rust reaction ran ed from 1 to 0 of severit with susce ti le infection t es hese varieties have een re orted to carr additional Lr enes a le 1 however the are not e ective a ainst the revalent races occurrin in the outhern one of merica erman et al 2011 he eld reaction of incana atriarca inuano 2 ri o 1 erena ri o 2 ri o m ra a 22 10 20 uatam u am oat 2 and o ave ran ed from to 0 a le 1 com ati le with the resence of Lr34 associated with one or two additional resistance enes he eld reaction of est halen 210 uamirim an ar 296 arcante and a i ran ed across ears from 1 to 20 su estin that in these varieties the resistance probably is due to the presence of Lr34 and two three or more additional enes he Lr34 carr in enetic stoc s dis la ed low levels of disease severit a le 1 ran in from to 20 in 60 0 611 60 hris  ocora ue o er 9 uc uc smeralda 6 lenlea am a arula and onichi 1 smeralda 6 and lenlea showed ma imum leaf rust ratin s of 2 and res ectivel he results o tained in this wor con rm the presence of the Lr34 resistant allele in man ra ilian wheat varieties which has een mostl inherited directl and indirectl from entana and rontana and the enetic varia ilit o served in the wheat erm lasm in ra il is e uall distri uted across the local wheat reedin ro rams as descri ed cherlos et al 201 t can e noticed that this im ortant locus has een maintained over time even thou h unconsciousl durin the c cles of henot ic selection dditionall our results con rm the hi h level and sta ilit of the artial resistance conferred Lr34 across years under a ver conducive environment and across a wide diversit of enetic ac rounds as re orted illemo et al 2011