American Journal of Plant Sciences, 2010, 1, 87-94
doi:10.4236/ajps.2010.12011 Published Online December 2010 (http://www.SciRP.org/journal/ajps)
87
Databasing Molecular Identities of Sugarcane
(Saccharum spp.) Clones Constructed with
Microsatellite (SSR) DNA Markers
Yong-Bao Pan
Sugarcane Research Laboratory, Houma, USA.
Received October 1st, 2010; revised October 31st, 2010; accepted November 13th, 2010.
ABSTRACT
This paper reports the development of the first SSR marker-based sugarcane (Saccharum spp.) molecular identity da-
tabase in the world. Since 2005, 1,025 sugarcane clones were genotyped, including 811 Louisiana, 45 Florida, 39
Texas, 130 foreign, and eight consultant/seed company clones. Genotyping was done on a fluorescence-capillary elec-
trophoresis detection platform involving 21 highly polymorphic SSR markers that cou ld potentially amplify 144 distinc-
tive DNA fragments. Genotyping data were processed with the GeneMapper™ software to reveal electrophoregrams
that were manually checked against the 144 fragments. The presence (A) or absence (C) of these 144 fragment s in any
sugarcane clone was recorded in an affixed sequence order as a DNAMAN® file to represent its molecula r identity be-
ing achieved into a local molecular identity database. The molecular identity database has been updated annually by
continued genotyping of newly assigned sugarcane clones. The database provides molecular descriptions for new cul-
tivar registration articles, enables sugarcane breeders to identify mis-labeled sugarcane clones in crossing programs
and determine the patern ity of cross progeny, and ensures the desired cultivars are grown in farmers’ fields.
Keywords: Sugarcane (Saccharum spp.), Breeding, SSR Marker, Molecular Identity Database
1. Introduction
Sugarcane (Saccharum spp.) is a complex aneu-poly-
ploidy plant (2n = 8x or 10x = 100-130) that propagates
asexually through planting of vegetative cuttings (setts)
of mature stalks [1,2]. A sugarcane breeding cycle in
Louisiana takes 12 years. This cycle begins with cross
hybri dization and continues with field evaluation and
selection, advancement, and multi-year, multi-location
testing, and ending with the release of a new cultivar [3].
During this cycle, exchange and shipment of elite clones
and breeding lines in the form of stalk cuttings (setts)
across different test locations occur regularly for the
purposes of verifying parental source or desired use of a
clone in an experiment. Traditional tools for sugarcane
breeders to identify different varieties rely on anatomical
and morphological characters [1,4]. In Louisiana, the
morphological descriptors, stalk wax, leaf sheath wax,
leaf sheath margin, leaf sheath hair (pubescence), dewlap
appearance, stalk color, auricle size and color, and other
distinguishing characteristics, are used by Louisiana sug-
arcane breeders [5]. Others may use sugarcane descrip-
tors available under the USDA-ARS GRIN system
(http://www.ars-grin.gov/npgs/descriptors/sugarcane). Al-
though these morphological descriptors may serve
breeders who are directly involved in the evaluation and
selection of those clones, breeders from other locations
or researchers in other disciplines may not be familiar
with these morphological traits, especially traits for
which differential expression is already known to be
strongly influenced by the environment. Therefore, it is
not uncommon that mislabeling or misidentification of
sugarcane clones occurs from time to time, whether on
crossing carts or in the field plots (Jim Miller, personal
communication, 2003). It might be worth noting that
cumulative probability of this error may be high for pa-
rental clones that are propagated many times over the
years (Phil Jackson, personal communication, 2010).
Because of this, sugarcane pedigree information some-
times may not be so reliable (Karl J. Nuss, personal
communication, 2003). Thus, to ensure correct variety
identity and its genetic pedigree, a procedure for accurate
identification using molecular data is urgently needed
[6-8].

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
88
Microsatellite or simple sequence repeats (SSRs) DNA
markers are short DNA fragments that contain various
numbers of tandem repeat units of di-, tri-, tetra- or
composite-nucleotide motifs [9,10]. SSR markers are
useful for genotyping sugarcane because they are abun-
dant, co-dominantly inherited, and highly reproducible
[11,12]. Since the beginning of the century, a high-
throughput molecular genotyping technology has been
developed for sugarcane (6, 8). By using a fluores-
cence/capillary electrophoresis (CE)-based genotyping
system, a total of 144 distinctive SSR DNA fragments
were consistently amplified among the U.S. sugarcane
germplasm from primer pairs of 21 polymorphic SSR
DNA markers [13]. The 144 DNA fragments were ar-
ranged in a linear order in an Excel spreadsheet, which
was used to score the presence (denoted by A) or absence
(C) of each fingerprint from a sugarcane clone. The
unique sequence of As or Cs was then converted to a
DNAMAN® (Lynnon Biosoft, Vaudreuil, Canada) file to
represent the molecular identity of that clone.
This paper describes the development of the first sug-
arcane molecular identity database that has been used by
the sugarcane breeders as a molecular breeding tool.
Unlike the anatomical and morphological traits that are
influenced by environment, SSR DNA marker-based
molecular identities represent stable genetic fingerprints
that are not affected by geographical region or seasonal
changes. With the advent of this molecular breeding tool
[6], U.S. sugarcane breeders have been able to provide a
molecular descriptor for new variety releases, identify
any sugarcane clone that has been mislabeled [7-8], iden-
tify S. spontaneum cytoplasm-derived hybrids for trait
introgression without violating the noxious weed regula-
tions, and determine paternity of clones derived from
polycrosses [14].
2. Materials and Methods
2.1. SSR Markers and Genotyping Sample
Collection
Primer pairs of 21 highly polymorphic SSR markers de-
veloped by the International Consortium of Sugarcane
Biotechnologists [11] based on the genomic DNA se-
quence of sugarcane cultivars Q124 and R570 were used.
The nucleotide sequences and annealing temperatures of
these primer pairs are listed in Table 1. The 5' ends of
Table 1. Sugarcane SSR markers, anne aling temperatures, and primer seque nc e s.
SSR Name* Anneal (℃) Forward Primer (5' to 3') Reverse Primer (5' to 3')
SMC119CG 58 TTC ATC TCT AGC CTA CCC CAA AGC AGC CAT TTA CCC AGG A
SMC1604SA 58 AGG GAA AAG GTA GCC TTG G TTC CAA CAG ACT TGG GTG G
SMC18SA 62 ATT CGG CTC GAC CTC GGG AT AGT CGA AAG GTA GCG TGG TGT TAC
SMC24DUQ 64 CGC AAC GAC ATA TAC ACT TCG G CGA CAT CAC GGA GCA ATC AGT
SMC278CS 64 TTC TAG TGC CAA TCC ATC TCA GA CAT GCC AAC TTC CAA ACA GAC T
SMC31CUQ 62 CAT GCC AAC TTC CAA TAC AGA CT AGT GCC AAT CCA TCT CAG AGA
SMC334BS 60 CAA TTC TGA CCG TGC AAA GAT CGA TGA GCT TGA TTG CGA ATG
SMC336BS 62 ATT CTA GTG CCA ATC CAT CTC A CAT GCC AAC TTC CAA ACA GAC
SMC36BUQ 64 GGG TTT CAT CTC TAG CCT ACC TCA GTA GCA GAG TCA GAC GCT T
SMC486CG 58 GAA ATT GCC TCC CAG GAT TA CCA ACT TGA GAA TTG AGA TTC G
SMC569CS 62 GCG ATG GTT CCT ATG CAA CTT TTC GTG GCT GAG ATT CAC ACT A
SMC7CUQ 60 GCC AAA GCA AGG GTC ACT AGA AGC TCT ATC AGT TGA AAC CGA
SMC597CS 64 GCA CAC CAC TCG AAT AAC GGA T AGT ATA TCG TCC CTG GCA TTC A
SMC703BS 62 GCC TTT CTC CAA ACC AAT TAG T GTT GTT TAT GGA ATG GTG AGG A
SMC851MS 58 ACT AAA ATG GCA AGG GTG GT CGT GAG CCC ACA TAT CAT GC
mSSCIR66 48 AGG TGA TTT AGC AGC ATA CAC AAA TAA ACC CAA TGA
mSSCIR3 60 ATA GCT CCC ACA CCA AAT GC GGA CTA CTC CAC AAT GAT GC
SMC1751CL 60 GCC ATG CCC ATG CTA AAG AT ACG TTG GTC CCG GAA CCG
SMC22DUQ 62 CCA TTC GAC GAA AGC GTC CT CAA GCG TTG TGC TGC CGA GT
mSSCIR43 52 ATT CAA CGA TTT TCA CGA G AAC CTA GCA ATT TAC AAG AG
mSSCIR74 54 GCG CAA GCC ACA CTG AGA ACG CAA CGC AAA ACA ACG

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
89
the forward primers were labeled with one of three fluo-
rescent phosphoramidite dyes, FAM, VIC, or NED (Ap-
plied Biosystems, Foster City, CA). For U.S. cultivars
and advanced breeding clones, leaf samples were collected
from healthy younger leaves without disease symptoms
from sugarcane plants maintained on the crossing carts,
breeding nurseries, varietal trials, quarantine facilities, or
commercial fields. For foreign sugarcane cultivars, either
leaf samples collected from clones grown at USDA-ARS,
SRL or genomic DNA samples obtained from foreign
sugarcane breeding programs were used.
2.2. Leaf DNA Extraction
Leaf DNA was extracted by either using CTAB-beta
mercaptoethanol [15] or hot NaOH-Tween 20 buffer [16].
For the CTAB-beta mercaptoethanol buffer procedure,
total nucleic acids were extracted from approximately
200 mg fresh leaf tissue by blending in a 2-ml microfuge
tube containing 1 ml CTAB extraction buffer [2% CTAB,
1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0),
2 μl beta-mercaptoethanol added prior to extraction] and
a 4.5 mm diameter sterile chrome-steel bead by violently
shaking the tube using a Mini-Bead-BeaterTM (BioSpec
Products, Inc., Bartleville, OK) for 1 min. The leaf ho-
mogenate was incubated at 60℃ for 30 min, extracted
once with 0.75 ml chloroform/isoamyl alcohol (24/1) by
centrifuging at 6,000 x g for 10 min at 4℃and transfer-
ring 600 μl aqueous phase to a new microfuge tube that
contained 500 μl of cold isopropyl alcohol. The mixture
was incubated at –20℃for at least 1 hr before centrifug-
ing for 15 min at 12,000 x g. The resulting pellet was
washed with 500 μl of 70% ethanol plus 10 mM sodium
acetate and centrifuged for 10 min at 12,000 x g to col-
lect the nucleic acid pellet. Excess wash solution was
evaporated in a DNA 120 SpeedVac System (Savant
Instruments, Inc., Holbrook, NY) and the pellet was re-
hydrated in 200 μl sterile water. The DNA concentration
was determined using NanoDrop1000 (Thermo Scientific,
Wilmington, DE) and adjusted to 10 μg/μl accordingly.
For the hot NaOH-Tween 20 buffer procedure, small
pieces (about 30 mm2) of leaf tissue were excised from
the youngest fully expanded leaves and dislodged into
sample wells of a 96-well microplate that was pre-loaded
with 50 μl of a freshly prepared denaturing buffer (100
mM NaOH and 2% Tween-20). The plates were sealed
with aluminum sealing tape, incubated at 95℃ for 10
min, placed on ice for three min, and spun at 1,480 x g
for 1 min. Fifty μl of a neutralization buffer (100 mM
Tris-HCl and 2 mM EDTA) were then added to each
well. The plates were re-sealed with aluminum sealing
tape; the buffers were mixed by vortex, and spun at 1,480
x g for 1 min. The resulting supernatants were transferred
to a fresh sterile 96-well microplate.
2.3. Semi-Automatic PCR and CE
Fifty-μl aliquots that were either diluted DNA samples
from the CTAB procedure or supernatant from the
NaOH-Tween 20 procedure were transferred into the
wells of 96-well microplates. Plates were sent to the
USDA-ARS, Mid-South Area Genomics Laboratory in
Stoneville, MS for high throughput PCR and CE-based
fragment analyses. A robot machine, the Hamilton’s Mi-
crolab Star Liquid Handling Station (Hamilton Company,
Reno, NV), was used to consolidate the DNA samples
from four 96-well plates into a single 384-well plate and
prepare 384-well PCR amplification reaction plates con-
taining a 5-μl PCR reaction mixture within each well.
The PCR reaction mixture consisted of 0.25 μl of the
DNA sample, 0.5 μl of 10X Buffer, 0.3 μl of 25 mM
MgCl2, 0.1 μl of 10 mM dNTPs, 0.41 μl each of 3 pm/μl
forward and reverse primers, 0.5 μl of 10 mg/ml BSA-V,
0.5 μl of 100 μg/μl PVP-40, 0.025 μl of 5 Units/μl Taq,
and 2.0 μl of PCR water. PCR amplification reactions
were conducted on a DNA Engine Tetra equipped with
four 384-well Alpha blocks with heated lids (Bio-Rad
Laboratories, Hercules, CA) under a program of 95℃ for
15 min, 40 cycles of 94℃ for 15 sec, annealing for 15
sec, and 72℃ for 1 min, final extension at 72℃ for 10
min, and holding at 4℃. When PCR amplification was
complete, the robot was used again to prepare 384-well
CE sample plates by first diluting the amplified SSR
DNA fragments and then mixing in each well one μl of
the diluted products with nine μl Hi-Dye formamide so-
lution premixed with the GeneScan™ Rox™ 500 Size
Standard. The CE sample plates were subjected to auto-
mated fragment analysis by ABI3730XL following
manufacturer’s instruction to produce Genescan files
(Applied Biosystems, Inc., Foster City, CA).
2.4. GeneMapper® Analysis, Construction of
Molecular Identity (ID), and Clone
Identity Check
Genescan files were downloaded online from the file
download site of the MSA Genomics Laboratory home
page (https://msa.ars.usda.gov/computerhelp/upload/) and
archived into individual folders named after sugarcane
clones before being processed with the GeneMapper™
software (Applied Biosystems, Inc., Foster City, CA).
The software calibrated SSR fragments based on the
GeneScan™ Rox™ 500 Size Standard and revealed SSR
fragments in the Sample Plot Window, which were in-
terpreted and scored manually. True SSR fragments that
could be scored exhibited measurable fluorescence peaks.
When both “plus-adenine” and “Minus-adenine” DNA

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
90
fragments were present, only “plus-adenine” DNA frag-
ments were scored. Fragments that showed measurable,
yet inconsistent, fluorescence peaks such as “stutters”,
“pull-ups”, or “dinosaur tails” [6] were not scored either.
For the genotyping project, only 144 distinctive SSR
DNA fragments [8] were targeted during the manual
scoring process (Figure 1). Presence of any SSR frag-
ment was given a score of “A”; while the absence of any
SSR fragment was given a score of “C”. The resulting
linear sequence of “A” or “C” was converted to a
DNAMAN® sequence file to represent the molecular
identity of that particular clone. The DNAMAN® file was
named according to a general formula “Clone
Name_Location_Year” before being stored in a local
molecular identity database.
Whenever there is need for clone identity, the identity
of the clone in question is aligned with all other identities
available from the database using DNAMAN® software
(Lynnon Biosoft, Vaudreuil, Canada). The algorithm first
produces a homology matrix based on the sequence
variability among molecular identities and then applies a
correction method [17] before aligning all sequences
progressively. Dynamic Alignment Method is used with
analytical parameters set at “10” for gap open penalty,
“5” for gap extension penalty, and “40%” for delay di-
vergent sequences. Bootstrap values were obtained upon
1,000 trials.
SMC119CG SMC1604SA SMC18SA SMC24DUQ
106 112 118 128 131 109 112 115118121 124137140 144 147150126 128 131 135 137142
1 2 3 4 5 6 7 89 1011121314151617 18 19 20 2122
I
5 6 5 6
SMC278CS SMC31CUQ
140 153 166 168 170 174 176 178182138 150160162 163 165167171 173 177 179
23 24 25 26 27 28 29 30313233343536373839 40 41 42
II
9 11
SMC334BS SMC336BS SMC36BUQ
146 149 151 161 163 164 141 154164166 167169171 173 175177183 112 118 121
43 44 45 46 47 48 49 50515253545556575859 60 61 62
III
6 11 3
SMC486CG SMC569CS SMC7CUQ
224 227 237 239 241 167 170 210219222 158162164 166 168170
63 64 65 66 67 68 69 707172737475767778
IV
5 5 6
SMC597CS SMC703BS
144 148 154 157 159 161 163 164165168 174201206 208 210212214 216 220 222
79 80 81 82 83 84 85 86878889909192939495 96 97 98
V
11 9
SMC851MS mSSCIR66 mSSCIR3
128 130 132 134 136 141 127 130132134 141145171 173 175177178 180 182 187
99 100 101 102103 104 105 106107108109110111112113114115 116 117 118
VI
6 4 10
SMC1751CL SMC22DUQ mSSCIR43
140 144 147 151 154 125 148 151154157 160163206 209 233235237 239 248 250 252
119 120 121 122123 124 125 126127128129130131132133134135 136 137 138 139
VII
5 7 9
mSSCIR74
217 220 223 226229
140 141 142 143 144
VIII
5
Figure 1. A definition of sugarcane molecualr identity. Within each section (I, II, III, IV, V, VI, VII, and VIII), name of the
SSR marker (first row), allele size (base pairs) (second row), sequential numerical order (third row), and number of allele per
marker (fourth row) are shown. There are a total of 144 SSR alleles amplifiable from the primer pairs of 21 SSR markers.
The molecular identity of any sugarcane clone is defined by a linear sequence of A (presence) or C (absence) of each of the
144 SSR alleles in the order shown.

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
91
3. Results
3.1. Number of Clones Genotyped
From 2005 to 2008, a total of 1,004 samples were geno-
typed targeting the 144 specific DNA fragments that
were potentially amplifiable from the primer pairs of 21
SSR markers. These included 237 samples in 2005, 238
in 2006, 339 in 2007, and 190 in 2008. Most of the
genotyping (803 samples or 78.3%) was conducted on
cultivars and newly assigned breeding lines from the
Louisiana breeding programs. In addition, 45 (4.4%)
Florida, 39 (3.8%) Texas, 130 (12.7%) foreign, and eight
(0.8%) cultivar samples from consultants and seedcane
companies were also genotyped (Table 2). Genotyping
continues annually for the Louisiana sugarcane breeding
program and on request for Florida, Texas, or foreign
sugarcane breeding programs. Depending upon the needs
for rigor identification, multiple samples are collected
from the same clone grown at up to four different loca-
tions, in the same or different years.
3.2. A SSR Molecular Identity Database
A local SSR identity database was constructed in 2005
by creating a folder named as “Genotyping Database”
inside the “C:/My documents/Breeding” folder located in
the hard drive disk of a desktop PC operated by the Mi-
crosoft Windows program. The “C:/My documents/
Breeding/Genotyping Database” folder is expandable by
creating additional sub-folders that are named after cal-
endar year, for example, sub-folders <2005>, <2006>,
<2007>, etc. (Figure 2). Within each sub-folder are mo-
lecular identity files or DNAMAN® files of sugarcane
clones that are genotyped in that year. For example, the
molecular identity of HoCP 00-950 [18], “CACA-
CAACCCCCCAAAAAACCACCAACCC CCCCCAC-
ACCCCCACACACCCCACACCCCCCAA ACAAAC-
CCCACCAACCCCCACAACCACCACAAA ACCCA-
CAACCACCCCCCCACCCACAAAACAAAA ACCA-
CACCAAAAAAAA”, can be found in subfolders
<2005> as “HoCP00-950_H_05” or “HoCP00-950_
Q_05”, <2006> as “HoCP00-950_H_06” or “HoCP00-
950_Q_06”, and <2008> as “HoCP00-950_CP_08”,
where H = Houma, Q = quarantine, CP = Canal Point.
4. Discussion
Conventional sugarcane breeding takes 12 years from
initial cross hybridization to a new cultivar release [3].
This paper reports the development of the first SSR
marker-based molecular identity database in sugarcane
that can serve as an additional tool to ensure that breed-
ers have the correct clones involved in their crosses as
well as varietal trials. Unlike the anatomical and mor-
phological traits, SSR DNA marker-based molecular
identities represent stable genetic fingerprints that are not
affected by location or seasonal changes [7-8]. Since its
initial establishment in 2005, the database has been
Table 2. Number of sugarcane clones genotyped with SSR
markers (2005-2008).
Sample Collection Site 2005 2006 2007 2008TOTAL
USDA at Houma, FL 57 41 126 131355
USDA at Canal Point, FL 78 97 91 49 315
LSU 52 46 28 7 133
Florida 3 34 6 2 45
Texas 32 7 0 0 39
Consultants/Companies 2 0 3 3 8
Foreign 13 20 85 12 130
TOTAL 237 245 339 2041,025
Figure 2. A local genotyping database at C:\My documents\Breeding\Genotyping database, in which there are five folders,
namely, 2005, 2006, 2007, 2008, and New Folder. Part of the Folder 2006 is shown listing molecular identity files of a few
sugacrane clones that were genotyped in 2006.

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
92
updated each year with newly acquired molecular identi-
ties. It has also been demonstrated that if a Louisiana
sugarcane clone was correctly labeled, then the same
molecular identity would be produced using the same
fluorescence- and CE-based SSR genotyping protocol
and the molecular identity would group together with
those produced in prior years or different field plots from
the same sugarcane clone. One recent example is shown
in Figure 3 that deals with identity checks of three sug-
arcane clones, namely, ST-283, ST-299, and ST-950.
After these clones were genotyped using the standard
protocol, the resulting molecular identities were blindly
aligned with those of all other Louisiana sugarcane
clones constructed in 2005, 2006, and 2007 using the
multiple sequence alignment program of DNAMAN®
software (Lynnon Biosoft, Vaudreuil, Canada). The re-
sults verified that ST-283 was indeed cultivar L 01-283
(Panel A) and ST-299 was cultivar L 01-299 (Panel A).
However, ST-950 was not cultivar HoCP 00-950 but
clone Ho 01-564 (Panel B).
There are three other demonstrated applications of the
reported molecular identity database. The primary and
most important application of the molecular identity da-
tabase is to protect sugarcane breeders’ rights by provid-
ing a molecular descriptor in their cultivar registration.
These include Louisiana sugarcane cultivar Ho 95-988
[18], HoCP 96-540 [19], Ho 00-950 [20], HoCP 91-552
[21], and Ho 00-961 [22]. In addition, molecular de-
scriptors were also included in sugarcane cultivar regis-
tration articles from the Florida sugarcane breeding pro-
gram, including CPCL 97-2730 [23], CP 00-1101 [24],
CP 88-1165 [25], CP 00-1446 [26], and CP 00-2180 [27].
All the molecular descriptors of newly released Louisi-
ana sugarcane cultivars are produced from SSR DNA
marker-based genotyping that are stored in the local mo-
lecular identity database.
The second application of the molecular identity data-
base is to facilitate the exploration of S. spontaneum cy-
toplasm through conventional breeding and more general,
to determine whether progeny are from proposed parents
for any type of sugarcane cross, in particular, cross in-
volving related wild species. Prior to the advent of SSR
genotyping technology, there was no report on the use of
the cytoplasmic genome of S. spontaneum clones in sug-
arcane breeding. Also, no genetic stock with S. sponta-
neum cytoplasm had ever been released. This is because
S. spontaneum clones are designated as regulated nox-
ious weeds with substantial self-pollination and vigorous
Figure 3. Molecular identity verification of three sugarcane clones, ST-283, ST-299, and ST-950 conducted in 2007. The
molecular identities of ST-283, ST-299, and ST-950 were aligned with those of all Louisiana sugarcane clones that were
genotyped in 2005, 2006, and 2007 using the multiple sequence alignment program of DNAMAN® software (Lynnon Biosoft,
Vaudreuil, Canada). Results showed that ST-283 was cultivar L 2001-283 (Panel A), ST-299 was cultivar L 2001-299 (Panel
A), and ST-950 was clone Ho 01-564 (Panel B). The dynamic alignment method is used with analytical parameters set at “10”
for gap open penalty, “5” for gap extension penalty, and “40%” for delay divergent sequences. The numerical values on the
branches are bootstrapping (confide nce) values based on 1,000 trials.

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones
Constructed with Microsatellite (SSR) DNA Markers
93
rhizomes [28]. With the advent of SSR genotyping tech-
nology, sugarcane breeders have been able to use DNA
marker information to identify true F1 progeny from
selfs arising from crosses in which S. spontaneum clones
were maternal parents before evaluation in the field en-
suring the noxious weed regulations were not violated. A
few S. spontaneum cytoplasm-derived clones have been
reported, of which US 99-51 [29] and Ho 02-113 (un-
published data) produced consistently high yields of total
dry mass.
A third but potential use of the molecular identity da-
tabase is to determine the paternity of sugarcane progeny,
particularly those from polycrosses [14]. When only a
few tassels are available from desirable parents, sugar-
cane breeders must decide whether to make a limited
number of bi-parental crosses or intersperse the tassels in
a polycross to obtain a greater number of crosses and
more seeds. Without the molecular identity information
for the parental clones, breeders are not able to defini-
tively determine the paternity information for polycross
progeny. Using seven highly polymorphic SSR markers
that produced parent-specific SSR alleles, Tew and Pan
[14] were able to determine the paternity for 79 to 99%
of the progeny from a seven-parent polycross, depending
upon the maternal parent. The ability to identify paternity
of polycross progeny with SSR DNA markers can be
used in sugarcane breeding to maximize the number of
desirable crosses from a limited source of flowers with
minimal loss of pedigree information.
5. Acknowledgements
The high-throughput PCR and fragment analysis were
conducted by Sheron Simpson at the USDA-ARS, Mid-
South Area Genomics Laboratory, Stoneville, MS, di-
rected by Brian E. Scheffler. Lionel Lomax and Jennifer
Shaw provided technical assistance. The study was par-
tially supported by the sugarcane grower/miller check-off
funds administered by the American Sugar Cane League
and the Florida Sugar Cane League.
Product names and trademarks are mentioned to report
factually on available data; however, the USDA neither
guarantees nor warrants the standard of the product, and
the use of the name by USDA does not imply the ap-
proval of the product to the exclusion of others that may
also be suitable.
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