The Use and Development of Molecular Breeding Tools in Cucurbita: A Literature

Review
Rebecca N Brown
Department of Horticulture, Oregon State University, Corvallis, OR 9!!"
#ools for molecular mapping an$ mar%er&assiste$
selection are being increasingly applie$ to t'e genus
Cucurbita( However, muc' of t'e wor% w'ic' 'as
alrea$y been $one 'as remaine$ unpublis'e$, or 'as
been publis'e$ in )ournals of limite$ circulation, suc'
t'at many w'o wor% wit' Cucurbita may not be
aware of it( #'is review of t'e literature to $ate is
inten$e$ to provi$e some bac%groun$ for current
wor%(
#'e $evelopment of molecular tools for use in
bree$ing Cucurbita species is still in t'e early stages,
particularly w'en compare$ to cereal crops suc' as
corn an$ w'eat, an$ ot'er vegetable crops suc' as
tomatoes an$ lettuce( *ven t'e Cucumis species C.
melo an$ C. sativus are far a'ea$ of Cucurbita( One
reason for t'e late $evelopment of molecular tools in
Cucurbita is t'at all species 'ave twenty pairs of
relatively s'ort c'romosomes( Using flow cytometry,
+rumuganat'an an$ *arle ,"- $etermine$ t'at t'e
'aploi$ genome of .ucc'ini ,C. pepo- is
appro/imately 011 million base pairs long( + typical
nucleus ,2n- contains "(13 4 "(15 picograms of DN+(
6ost morp'ological traits appear to be unlin%e$, an$
many mar%ers are re7uire$ to a$e7uately map t'e
genome( Cucurbita species are of limite$ economic
significance in $evelope$ countries, an$ most
Cucurbita researc'ers wor% on several crops( #'e
large si.e of t'e plants ma%es t'em ill&suite$ to
genetics stu$ies, an$ some types re7uire a very long
growing season(
No lin%age map of any Cucurbita species e/iste$
prior to t'e $evelopment of molecular mapping( 8n
t'e "951s t'e use of iso.ymes reveale$ t'at
Cucurbita is an ancient allotetraploi$ ,25-( 8so.ymes
also provi$e$ a mar%er lin%e$ to one of t'e
complementary genes responsible for t'e e/pression
of 96: resistance in crosses between C. maxima
an$ C. ecuadorensis ,2-(
6any of t'e species in Cucurbita can be successfully
crosse$, particularly if embryo rescue is use$(
However, t'e ;" an$ later generations of t'e
interspecific crosses are fre7uently sterile or e/'ibit
re$uce$ fertility ,22-( 8so.ymes 'ave been use$ to
$etermine if t'e re$uce$ fertility was a result of
c'romosomal rearrangement in crosses between C.
maxima an$ C. ecuadorensis( 9all an$ 9'ita%er ,20-
e/amine$ t'e in'eritance of leucine aminopepti$ase
an$ esterase iso.ymes in a set of C( ecuadorensis / C(
maxima crosses an$ conclu$e$ t'at t'e c'romosomal
structure of t'e two species $iffere$ in t'e region of
t'e esterase locus( 9ee$en an$ Robinson ,2<-
e/amine$ t'e in'eritance of twenty iso.ymes in t'e
cross C( maxima / C( ecuadorensis( #'ey use$ t'eir
$ata to buil$ t'e first map of Cucurbita( 8t was base$
on t'e ;2 of t'e cross C( maxima / C( ecuadorensis,
an$ containe$ "" iso.yme loci in five lin%age groups(
;rom t'e iso.yme map 9ee$en an$ Robinson ,2<-
were able to $etermine t'at t'e significant $ecrease in
fertility of t'e ;2 an$ bac%cross generations of
crosses between C( maxima an$ C( ecuadorensis was
not a result of minor c'romosomal rearrangements(
Restriction ;ragment =engt' >olymorp'isms
,R;=>s- 'ave not been muc' use$ in Cucurbita
because of t'e time an$ e/pense involve$ in creating
t'e probes( However, ribosomal ,5, 23- an$
c'loroplast DN+ 'as been stu$ie$, an$ t'e
c'loroplast genome 'as been mappe$ ,21-( #'e
c'loroplast map was constructe$ using gene&specific
probes from ot'er species? t'is is possible because
t'e c'loroplast genome is 'ig'ly conserve$( 9ilson
et al( ,29- use$ restriction en.ymes an$ clone$
c'loroplast fragments to stu$y t'e c'loroplast DN+
$iversity of "0 species of Cucurbita( #'eir
conclusions agree$ wit' t'ose obtaine$ t'roug'
interspecific crossing stu$ies an$ stan$ar$ ta/onomy(
#'ey also $etermine$ t'at t'e annual Cucurbita
species evolve$ from t'e perennial species( Havey et
al( ,"1- use$ R;=>s to stu$y t'e transmission of t'e
c'loroplast an$ mitoc'on$rial genomes in cucurbits(
#'ey conclu$e$ t'at bot' organelle genomes were
maternally transmitte$ in Cucurbita, unli%e in
Cucumis w'ere t'e mitoc'on$rial genome is
paternally transmitte$(
=ee et al( ,"- $evelope$ a R+>D map of an ;2
population of a C. pepo / C. moschata interspecific
'ybri$( #'ey screene$ t'e parents wit' 1 "1&mer
primers from t'e University of Britis' Columbia
,UBC 01"&01-? "0 of t'e primers were polymorp'ic
between t'e parents( #'ese fifteen primers were use$
to amplify DN+ from 31 ;2 in$ivi$uals, resulting in
05 R+>D ban$s( ;orty&seven repro$ucible mar%ers
were use$ to buil$ t'e map? 25 mar%ers were mappe$
into five lin%age groups( No morp'ological traits or
ot'er types of mar%ers were inclu$e$ on t'e map( #'e
map of =ee et al( ,"- was small, base$ on a small
number of progeny, an$ was publis'e$ in t'e @orean
Aournal of Horticulture, wit' t'e result t'at it is not
well %nown in t'e Cucurbita community(
;urt'ermore, t'ey selecte$ arbitrary i$entifiers for t'e
mar%ers on t'eir map, rat'er t'an following t'e
stan$ar$ practice of i$entifying t'e mar%ers by t'e
primer an$ t'e ban$ si.e in base pairs( #'is furt'er
prevents comparisons between t'eir map an$ ot'er
Cucurbita maps( #'e map of =ee et al ,"- is
currently t'e only publis'e$ molecular map of
Cucurbita( Data were collecte$ for a ran$om
amplifie$ polymorp'ic DN+ ,R+>D- map on a C.
maxima / C. ecuadorensis population, but t'e map
was never publis'e$ ,N( 9ee$en, personal
communication-(
R+>Ds an$ ot'er molecular mar%er tec'nologies
'ave been use$ to $o DN+ fingerprinting analysis
wit'in an$ between Cucurbita species( Aeon et al(
,""- use$ R+>Ds to $istinguis' among @orean
cultivars of C. pepo an$ C. moschata, w'ile Boun et
al( ,!1- use$ R+>Ds to stu$y t'e genetic
relations'ips among Sout' @orean lan$races of C.
moschata( Stac'el et al( ,2!- use$ R+>Ds to estimate
genetic $iversity among commercial inbre$ lines of
+ustrian oilsee$ pump%in, C. pepo var( styriaca(
Cwanama et al ,9- use$ R+>Ds to $etermine t'e
genetic variability present in t'e C. moschata
lan$races of sout'&central +frica( Barane% et al( ,2-
use$ R+>Ds to stu$y t'e genetic $iversity wit'in an$
between species of C. pepo, C. moschata, an$ C.
maxima( +ll t'e researc'ers foun$ R+>Ds to be
effective for $etermining t'e relate$ness of $ifferent
Cucurbita accessions( @at.ir et al( ,"!- use$
microsatellite&anc'ore$ se7uences as primers ,8SSR-
to classify cultivars of C. pepo( No microsatellites
specifically $esigne$ for Cucurbita 'ave been
publis'e$, but polymorp'isms among Cucurbita
pepo accessions 'ave been $etecte$ using SSRs
$evelope$ for Cucumis ,"3, "0-(
>olymorp'ism levels in Cucurbita are mo$erate(
Stac'el et al( ,2!- foun$ ""< polymorp'ic mar%ers by
screening a set of 21 inbre$ lines of oilsee$ pump%in
,C. pepo var( styriaca- wit' !3 R+>D primers(
Barane% et al(,2- foun$ 32(0D mar%er polymorp'ism
among si/ +ustrian C. pepo genotypes( @at.ir et al(
foun$ t'at "3D of t'e Cucumis SSR primers ,"0- an$
52D of 8SSR mar%ers ,"!- were polymorp'ic among
cultivar groups of C. pepo( Boun et al( ,!1- foun$
t'at "5(<D of mar%ers were polymorp'ic among C.
moschata lan$races( Brown an$ 6yers ,3- foun$
"3D mar%er polymorp'ism between temperate an$
tropical C. moschata lines( Barane% et al( ,2- foun$
<3("D mar%er polymorp'ism among a
geograp'ically $isparate collection of C. moschata
accessions( #'ey foun$ 00(9D mar%er polymorp'ism
among a $isparate collection of C. maxima(
6ar%er assiste$ selection is little&use$ in Cucurbita
bree$ing( Brown an$ 6yers 'ave i$entifie$ R+>D
mar%ers lin%e$ to several morp'ological traits in an
interspecific cross between a C. pepo summer s7uas'
an$ C. moschata ENigerian =ocalF ,see article in t'is
issue- but t'ey are not yet in a form t'at will be
$irectly useful to bree$ers( #wo groups 'ave been
wor%ing on i$entifying molecular mar%ers lin%e$ to
GB6: resistance from ENigerian =ocalF introgresse$
into C. pepo ,2, "9- but no mar%ers 'ave been
publis'e$(
+ number of Cucurbita genes 'ave been
c'aracteri.e$ at t'e molecular level an$ clone$(
However, t'is wor% 'as been $one entirely by
molecular geneticists intereste$ in t'e control an$
functioning of pat'ways common to many plants(
#'us t'e genes clone$ 'ave been of little $irect use to
s7uas' bree$ers( Researc'ers in t'e laboratory of C(
+( #'ompson at t'e University of +ri.ona 'ave
stu$ie$ an$ clone$ t'e genes enco$ing important
proteins in t'e p'loem transport system of C. maxima
,!, <, "5-( Ot'er recently clone$ genes in Cucurbita
inclu$e a calcium&$epen$ent protein %inase from
.ucc'ini ,-, a class&! c'itinase ,"<-, a glyo/ysomal
malate $e'y$rogenase ,"2-, an$ an anionic
pero/i$ase ,0-( Cloning of t'ese types of pat'way&
controlling genes from Cucurbita 'as been greatly
facilitate$ by t'e availability of probes an$ se7uence
information from ot'er species w'ere genes wit' t'e
same function 'ave alrea$y been clone$(
8t is apparent from t'e literature t'at molecular
genetics is )ust beginning in Cucurbita( +s t'e
genome is mappe$, it s'oul$ be possible to i$entify
mar%ers for many useful traits( 6ar%ers coul$ be
particularly useful for tagging t'e complementary
virus&resistance genes, suc' as Zym-2 an$ Zym-3 ,2"-
an$ ensuring t'at t'ey are transferre$ $uring
bac%crossing( 6olecular maps will also allow furt'er
investigation into t'e evolution an$ species
relations'ips in Cucurbita, an$ between Cucurbita
an$ ot'er cucurbits(
Literature Cited
"( +rumuganat'an, @ an$ *D *arle, "99", Nuclear
DN+ content of some important plant species,
>lant 6olecular Biology Reporter 9H215&2"5(
2( Barane%, 6(, C( Stift, A( :ollmann an$ #( =elley,
2111, Cenetic $iversity wit'in an$ between t'e
species Cucurbita pepo, C. moschata, an$ C.
maxima as reveale$ by R+>D mar%ers, CCC
Rpt( 2!H!&(
!( Bostwic%, D*, 68 S%aggs an$ C+ #'ompson,
"993, Organi.ation an$ c'aracteri.ation of
Cucurbita p'loem lectin genes( >lant 6ol( Bio(
2<H55&59(
3( Brown, R( an$ AR 6yers, 2111, Searc'ing for
molecular mar%ers lin%e$ to GB6: resistance in
s7uas'( CCC Rpt, 2!H<9&1(
0( Carpine, S(, 6( Crevecoeur, H( Crepin an$ C(
>enel, "999, 6olecular cloning an$ tissue&
specific e/pression of an anionic pero/i$ase in
.ucc'ini, >lant >'ysiol( "21H99&5"1(
<( Clar%, +6, @R Aacobaen, D* Bostwic%, A+
Dannen'offer, 68 S%aggs an$ C+ #'ompson,
"99, 6olecular c'aracteri.ation of a p'loem&
specific gene enco$ing t'e filament protein,
>'loem >rotein " ,>>"-, from Cucurbita maxima(
>lant Aournal "2H39&<"(
( *llar$&8vey, 6, RB Hop%ins, #A 9'ite an$ #=
=oma/, "999, Cloning, e/pression an$ N&
terminal myristoylation of CpC>@", a calcium&
$epen$ent protein %inase from .ucc'ini
,Cucurbita pepo L(-, >lant 6ol( Bio( !9H"99&215(
5( Canal, 6( an$ :( Hemleben, "95<, Comparison
of t'e ribosomal RN+ genes in four closely
relate$ Cucurbitaceae( >l( Syst( *vol( "03H<!&(
9( Cwanama, C, 6# =abusc'agne an$ +6 Bot'a,
2111, +nalysis of genetic variation in Cucurbita
moschata by ran$om amplifie$ polymorp'ic
DN+ ,R+>D- mar%ers, *up'ytica ""!H"9&23(
"1( Havey, 6A, AD 6cCreig't, B( R'o$es an$ C(
#auric%, "995, Differential transmission of t'e
Cucumis organellar genomes, #'eor( +ppl(
Cenet( 9H"22&"25(
""( Aeon, HA? CC Been? @H Hong? BH Om an$ BD
@im, "993, 8$entification of Cucurbitaceae
cultivars by using R+>D mar%ers( A( @orean Soc
Hort Sci !0H339&30<(
"2( @ato, +(, B( #a%e$a&Bos'i%awa, 6( Hayas'i, 6(
@on$o, 8( Hara&Nis'imura an$ 6( Nis'imura,
"995, Clyo/ysomal malate $e'y$rogenase in
pump%inH Cloning of a cDN+ an$ functional
analysis of its prese7uence, >lant Cell >'ysiol(
!9H"5<&"90(
"!( @at.ir, N, *( =es'.es'en, C( #.uri, N( Reis, B(
Danin&>oleg an$ HS >aris, "995, Relations'ips
among accessions of Cucurbita pepo base$ on
8SSR analysis, in AD 6cCreig't, e$(
Cucurbitaceae F95, +SHS >ress( pp(!!"&!!0(
"3( @at.ir, N, B( Danin&>oleg, C( #.uri, G( @arc'i,
U( =avi an$ >B Cregan, "99<, =engt'
polymorp'ism an$ 'omologies of microsatellites
in several Cucurbitaceae species, #'eor( +ppl(
Cenet( 9!H"252&"291(
"0( @at.ir, N, B( #a$mor, C( #.uri, *( =es'.es'en, N(
6o.es&Daube, B( Danin&>oleg an$ HS >aris,
2111, ;urt'er 8SSR an$ preliminary SSR analysis
of relations'ips among accessions of Cucurbita
pepo( +cta Hortic( 0"1H3!!&3!9(
"<( @im, 6C, @O =ee, N* C'eong, BO C'oi, AH
Aeong, 6A C'o, SC @im an$ SB =ee, "999,
6olecular cloning an$ c'aracteri.ation of a class
888 c'itinase in pump%in leaves, w'ic' strongly
bin$s to regenerate$ c'itin affinity gel( >lant Sci(
"3H"0&"<!(
"( =ee, BH, HA Aeon, @H Hong an$ BD @im, "990,
Use of ran$om amplifie$ polymorp'ic DN+s for
lin%age group analysis in interspecific 'ybri$ ;2
generation of Cucurbita( A( @orean Soc( Hort(
Sci( !<H!2!&!!1(
"5( =eineweber, @(? +( Sc'ul., an$ C+ #'ompson,
2111, Dynamic transitions in t'e translocate$
p'loem filament protein( +ust( A( >lant >'ysiol(
2H!!&3"(
"9( =elley, # an$ S( HenglmIller, 2111, Bree$ing for
GB6: tolerance of see$&oil pump%in ,Cucurbita
pepo var( styriaca- in +ustria using molecular
mar%ers, CCC Rpt( 2!H""&""9(
21( =im, H, 8( Counaris, RC Har$ison an$ CD Boyer
"991( Restriction site an$ genetic map of
Cucurbita pepo c'loroplast DN+( Curr Cenet
"5H2!&20(
2"( >aris, HS an$ S( Co'en 2111 Oligogenic
in'eritance for resistance to Gucc'ini yellow
mosaic virus in Cucurbita pepo, +nn( +ppl( Biol(
"!<H219&2"3(
22( Robinson, R9 an$ Dec%er&9alters, DS( "99(
Cucurbits( C+B 8nternational(
2!( Stac'el, 6(, C( Csana$i, A( :ollmann an$ #(
=elley, "995, Cenetic $iversity in pump%ins
,Cucurbita pepo =(- as reveale$ in inbre$ lines
using R+>D mar%ers( CCC Rpt( 2"H35&01(
23( #orres Rui., R+, an$ :( Hemleben, "99", Use of
ribosomal DN+ spacer probes to $istinguis'
cultivars of Cucurbita pepo =( an$ ot'er
Cucurbitaceae( *up'ytica 0!H""&"(
20( 9all, AR an$ #9 9'ita%er, "9", Cenetic
control of leucine aminopepti$ase an$ esterase
iso.ymes in t'e interspecific cross Cucurbita
ecuadorensis / C. maxima( Bioc'em( Cenet(
0H22!&229(
2<( 9ee$en, N; an$ R9 Robinson, "95<, +llo.yme
segregation ratios in t'e interspecific cross
Cucurbita maxima / C. ecuadorensis suggest t'at
'ybri$ brea%$own is not cause$ by minor
alteration in c'romosome structure( Cenetics
""3H09!&<19(
2( 9ee$en, N;, R9 Robinson an$ ;( 8gnart, "953,
=in%age between an iso.yme locus an$ one of t'e
genes controlling resistance to watermelon
mosaic virus 2 in Cucurbita ecuadorensis, CCC
Rpt( H5<&5(
25( 9ee$en, N;( "953( 8so.yme stu$ies in$icate t'at
t'e genus Cucurbita is an ancient tetraploi$(
CCC Rpt( H53&50(
29( 9ilson, HD, A( Doebley an$ 6( Duvall, "992,
C'loroplast DN+ $iversity among wil$ an$
cultivate$ members of Cucurbita
,Cucurbitaceae-, #'eor +ppl( Cenet( 53H509&5<0(
!1( Boun, SA? HD C'ung? SA Boun? an$ HD C'ung,
"995, Cenetic relations'ip among t'e local
varieties of t'e @orean native s7uas'es
,Cucurbita moschata- using R+>D tec'ni7ue( ( A(
@orean Soc( Hort( Sci( !9H0"&02"( ,in @orean
wit' *nglis' summary-