全 文 ：Vol131 , No12
pp1 177 - 182 　Feb1 , 2005作 　物 　学 　报ACTA AGRONOMICA SINICA第 31 卷 第 2 期2005 年 2 月 　177～182 页
Melon ( Cucumis melo L1) Cultivar Identification by Reversed2phase High
Performance Liquid Chromatography of Seed Proteins
WANG Qiang1 , RUAN Xiao1 , CHEN He2 , SUN Xiao2Lu2 , SHENGJian2Dong3 , YAN Qi2Chuan1
(1 Department of Biological and Pharmaceutical Engineering , Ningbo Institute of Technology , Zhejiang University , Ningbo 315100 , Zhejiang ; 2 Testing Center ,
Xinjiang Academy of Agricultural Science , Urumqi 830050 , Xinjiang ; 3 Department of Environment , Xinjiang Agriculture University , Urumqi 830052 , Xinjiang ,
China)
Abstract :Reversed2Phase High2Performance Liquid Chromatography (RP2HPLC) of cereal grain proteins has been receiv2
ing much attention in the agricultural literature published in recent years1 A study of melon ( Cucumis melo L1) hybrid par2
ents S ♂and S ♀and F1 hybrids of K9628 ×621 , T25 ×red1228 and 94 ×821 was undertaken to determine if RP2HPLC
could be adapted to the cultivar identification of melons1 Chromatograms of the three inbred lines and F1 hybrids of cultivars
were shown to be different and were used to characterize those cultivars1 RP2HPLC was shown to be a quick , replicable and
reliable method of melon cultivar identification for general screening of seed lots1
Key words :Melon( Cucumis melo L1) ; Cultivar identification ; Reversed2Phase High Performance Liquid Chromatography
甜瓜种子醇溶蛋白的反相 HPLC 分析鉴定
王　强1 　阮　晓1 　陈　贺2 　孙晓路2 　盛建东3 　颜启传1
(1 浙江大学宁波理工学院 ,生物与制药工程系 ,浙江宁波 315100 ;2 新疆农业科学院分析测试中心 ,新疆乌鲁木齐 830050 ; 3 新疆农业大
学 ,资源环境学院 ,新疆乌鲁木齐 830052)
摘 　要 :针对目前国内甜瓜的主栽品种 K9628 ×621、T25 ×red1228 和 94 ×821 的自交系及杂交种种子醇溶贮藏蛋白进行反
相 HPLC法分离。结果表明 ,其色谱图明显不同 ,可以用于该品种 (系) 的鉴定 ,认为 RP2HPLC 技术是一种快速、准确、可
信的甜瓜品种鉴定手段。
关键词 :甜瓜 ;品种鉴定 ;反相高效液相色谱
中图分类号 :S652 , O65717
　　Cultivar verification is an important area of study in
seed analysis[1 ] 1 Alcohol soluble seed storage proteins are
genetically stable molecules , which can be analyzed by
electrophoresis or HPLC to identify cultivars1 Electro2
phoresis studies greatly enhanced the investigation and
understanding of cereal proteins1 Brink , Price , Nguyen ,
Fuerst and Martinez (1989) used isoelectric focusing of
zeins and an immunoassay system to evaluate the genetic
purity of maize ( Zea mays L1 subsp1 mays ) [2 ] 1 New
methods such as RP2HPLC and capillary electrophoresis
have been applied to identify cultivars1 Dinelli G and Bonetti A (1992) [3 ] used capillary electrophoresis as anidentification tool for Phaseolus vulgaris L1 cultivars1Both the International Seed Testing Association and theAssociation of Official Seed Analysis are interested in ap2plying new biochemical methods to the analysis of seedsand the standardization of these techniques for cultivarsidentification1Reversed2phase high performance liquid chromatog2raphy ( RP2HPLC) can be employed to separate proteinsbecause of the introduction of relatively large pore ( > 300nm) columns1 Pioneering work in the use of RP2HPLC to
繱基金项目 : 国家自然科学基金 (30470330) 、国家自然科学基金 (30470330) 、浙江大学宁波理工学院青年博士基金 (1050257G304) 和新疆自
治区科技厅自然基金项目 (200121107)资助。
作者简介 : 王强 (1971 - ) ,浙江大学博士 ,多年从事作物种子科学方面的教学与研究工作。E2mail : wangqiangsky @2631net
Received(收稿日期) :2003211228 ,Accepted(接受日期) : 20042042041

separate seed storage proteins and thereby identify culti2
vars and show associations of particular proteins with grain
quality has been carried out by Dr1 Jerold Bietz and co2
workers at the USDA Northern Regional Research Center ,
ARS2USDA , Peoria , Illinois[4～6 ] 1 RP2HPLC has been
used to characterize species and individual inbred lines or
cultivars in wheat ( Triticum aestivum L1 ; T1 durum
Durf1) [7 ] , barley ( Hordeum vulgare L1) [8 ] , oats ( Avena
spp1) [9 ] , and maize ( Zea mays L1) [10 ] , as well as for
the identification of cultivated genotypes and the analysis
of varietal mixtures and hybrid pedigrees1
Melon ( Cucumis melo L1) is one of the most impor2
tant fruits in China1 The Xinjiang Autonomous Region in
northwest China is the biggest production center of both
the fresh fruit and melon seeds1 In China , the planted ar2
eas of cultivars that were identified in this research totaled
over 100 , 000 ha in 2000 , while the planted areas of
greenhouse2cultivated melon measured over 70 000 ha in
20011 The planted areas of the F1 hybrid of 94 ×821
summed over 10 000 ha in 20021 This makes China the
biggest producer of greenhouse2cultivated melon in the
world (2002 ,CSY) [11 ] 1 The pollination characteristics of
melon are open , which makes it difficult to control seed
purity1 The resultant seed contamination can cause heavy
losses for fruit producers1 Identifying inbred lines and de2
termining F1 hybrid purity are important quality require2
ments in fruit breeding and seed production1 Thus , fast ,
simple and reliable methods for the analysis of genetic pu2
rity of melon seeds are essential for both seed suppliers
and fruit growers1 Therefore , the study of this paper ex2
plores the potential of RP2HPLC for the fractionation of
alcohol soluble seed storage proteins from melons grown in
Xinjiang and other parts of China and assesses the possi2
bility of using this method as a means of cultivar identifi2
cation1
1 　Materials and Methods
111 　Plant materials
　　The hybrid parents S ♂and S ♀ and F1 hybrids of
K9628 ×621 , T25 ×red1 - 28 and 94 ×8 - 1 were obtained
from the Department of Horticulture at Xinjiang Academy
of Agricultural Sciences1 All seed lots examined were certi2
fied so that their origin and purity could be documented1
112 　Extraction of protein from melon seeds
A single melon seed was crushed and ground with a
pestle and mortar , then soaked 2 h in 015 mL 70 % ( VΠ
V) aqueous ethanol1 The samples were then ground for a
further 10 min , clarified by centrifugation (12 000 ×g
for 10 min) , and filtered through a 0145μm pore filter
before injection into the column1
113 　Chemicals and reagents
Unless noted otherwise all chemicals were of“Ana2
lar”grade1 Acetonitrile and methol ( spectra analyzed
grade) were from TEDIA Company Inc1 , USA1 Trifluoro2
acetic acid ( spectra analyzed grade) was from British
Drug Houses , London , UK1 Water was doubly distilled
and purified using a Waters Millipore Milli2Q purification
system to a conductivity of 18μω1
114 　Reversed2Phase High Performance Liquid
Chromatography
　　All chromatography data were obtained using a Shi2
madzu Associates Model LC 6A , Shimadzu Class VP510
multi2solvent delivery system and a Shimadzu SCL26A
system controller (Shimadzu , Japan) 1 Samples were in2
jected through a Rheodyne 7000 injection valve connected
to an LKB 2157 auto sampler (LKB , Bromma , Sweden) 1
Eluted proteins were monitored at 210 nm using a Shi2
madzu LC210A TVP photodiode and ray detector (PDA) 1
A Shimadzu CTO26A Column oven was used to maintain
constant column temperature at 60 ℃ during RP2HPLC1
Column was a Waters Delta Pak C18 , 5μparticle size re2
versed2phase column (150 mm ×319 mm) 1
115 　Computation
Data were acquired by means of a Shimadzu CR23A
Chromatopac , using an Intel personal computer with Nel2
son Analytical software that facilitated data manipulation
and storage1 Chromatograms of stored data were displayed
on a video monitor using a Hercules graphics card1 Peak
areas were determined through a manual interactive inte2
gration process on the monitor by aligning the cursor at
positions corresponding to the beginning and the end of an
eluting peak1 The area under this curve was calculated in
a manner that permitted exact comparisons to be made bet
ween the same time intervals for the chromatography of
different samples1 This ensured a reproducible calculation
of peak areas1 Statistical analysis of data was undertaken
871 　　　　作 　　物 　　学 　　报 第 31 卷 　

using the Minitab statistical package (supplied by Minitab
Inc1 Pennsylvania , USA) 1 Data in the text are expressed
as Mean ± SD1
2 　Results
211 　Optimum separation conditions for the
chromatography
　 　 Chromatographic conditions were optimized by
variation in both run time and gradient conditions by using
water and acetonitrile containing 011 % ( VΠV )
trifluoroacetic acid as solvents A and B , respectively1 A
linear gradient of 22 % - 50 % solvent B with a flow rate
of 1 mL·min - 1 and a run time of 67 min were found to
give optimum separation of melon alcohol soluble
proteins1 Under these conditions , more than 25 different
constituents could be identified in ethanol extracts from a
single kernel of the cultivars1 At the end of each gradient
program , solvent B was increased to a final concentration
of 60 % over a period of 4 min before returning to initial
conditions1 After this , the column was treated by
periodically washing with a 0 - 100 % acetonitrile gradient
for 15 min to remove any strongly absorbed proteins that
might have accumulated on the column from run to run1
212 　The reproducibility of column retention time for
the methods
　　In order to test the reproducibility of column reten2
tion times for different extracts of the same cultivars ,
ethanol extracts of the cultivars K9628 ×621 ♀ and ♂
were analyzed on different days1 In our research , 94 ×82
1 ♂samples were also analyzed on the same day1 When
tested on the same day , the percentage coefficient of
variation for five chosen peaks (Fig11) was found to vary
very little —average value 1197 (Table 1) 1 This is similar
to the results of Bietz using wheat gliadins and Marchylo
and Kruger using hordein proteins extracted from cultivar
of Canadian barley1 When tested on different days ,
reproducibility of retention times for the five chosen peaks
(Fig12 ) tended to be more subject to variation with
average coefficients of variation 4153 and 4156 for the two
different inbreeding lines ( Table 2 ) 1 Comparison of
elution profiles indicated that this level of reproducibility
was higher than acceptable for identification of the
different cultivars1
Table 1 Reproducibility of retention times ( within one day) for
reversed2phase separation of alcohol soluble protein(94 ×821 ♂)
Peak number Mean retention time ± SD(min)
Coefficient of variation
( %)
a 2188 ±0110 3147
b 3187 ±0110 2158
c 7166 ±0112 1157
d 13147 ±0118 1134
e 16115 ±0115 0193
Average ( n = 5) 1197
Table 2 Reproducibility of retention times ( from day to day) for reversed2phase separation of alcohol soluble proteins
K9628 ×621 ♂
Peak number
Mean retention
time ± SD
(min)
Coefficient of
variation
( %)
K9628 ×621 ♀
Peak number
Mean retention
time ± SD
(min)
Coefficient of
variation
( %)
a 2120 ±0115 6182 a 3194 ±0121 5133
b 3194 ±0118 4157 b 7172 ±0145 5183
c 7172 ±0132 4115 c 10122 ±0155 5138
d 20103 ±0135 4174 d 21158 ±0175 3148
e 41196 ±0135 2138 e 37170 ±1105 2179
Average ( n = 5) 4153 Average ( n = 5) 4156
213 　Identif ication results for three cultivars of melon
The RP2HPLC profiles for the hybrid parents’♀
and ♂, and F1 hybrids K9628 ×621 , T25 ×red1228 and
94 ×821 are shown in Fig11 , 2 , and 31 Repeated
analyses of the extracts under similar conditions gave
nearly identical elution patterns , which were characteristic
for each parent1 Some major qualitative differences among the resulting chromatograms will be noted1 In the elutionprofile , characteristic peaks eluting between 0 - 12minutes are recognizable which contain proteins commonto each parent and hybrid F11 For example , in Fig11 , thepeaks eluting at 0160 min , 2120 min , 2190 min , 3194min , 5173 min , 7172 min , 35163 min and 37170 minrepresent proteins common to the hybrid parents and F1
971　第 2 期 WANG Qiang et al . : Melon ( Cucumis melo L1) Cultivar Identification by Reversed2phase High Performance111 　　　

hybrids of K9628 ×621 , while the peaks eluting at 1160
min , 32119 min , 41196 min and 50169 min represent
proteins common to the ♂and F1 1 The peaks eluting at
8168 min , 9113min , 10122 min , 13142 min and 14166
min represent proteins common to ♀and F1 1 The peaks
eluting at 47133 min are specific to F1 hybrids1 The peaks
eluting at 21158 min , 45174 min , 52108 min 60194 min and 65117 min are specific to hybrid parent ♀, and thepeaks eluting at 1132 min , 18153 min , 20103 min ,23181 min , 29139 min and 54186 min are specific tohybrid parent ♂1 In Fig12 , the peaks eluting at 0155min , 2120 min , 2179 min , 3189 min , 5183 min , 8106min , 8173 min , 10140 min , 11126 min , and 11193 minrepresent proteins common to the hybrid parents and F1
Fig12 RP2HPLC elution profiles of the T25 ×red1228 hybrid parents’♀and ♂, and F1 hybrids
Fig11 RP2HPLC elution profiles of the K9628 ×621 hybrid parents’♀and ♂, and F1 hybrids
081 　　　　作 　　物 　　学 　　报 第 31 卷 　

Fig13 RP2HPLC elution profiles of the 94 ×821 hybrid parents’♀and ♂, and F1 hybrids
hybrids 725 ×red1228 ,while the peaks eluting at 7125
min , 9192 min , 12144 min , 27113 min , 29141 min ,
54178 min and 57170 min represent proteins common to
the ♂and F1 1 The peaks eluting at 13148 min and 15194
min represent proteins common to the ♀ and F1 1 The
peaks eluting at 44103 min , 61196 min and 63136 min
represent proteins specific to F1 hybrids , while the peak
eluting at 13184 min represents protein common to the
hybrid parent1 In Fig13 , the peaks eluting at 0170 min ,
2146 min , 2188 min , 3187 min , 7166 min , 9178 min ,
10129 min , 11137 min and 12169 min represent proteins
common to the hybrid parents and F1 hybrids of 94 ×8211
The peaks eluting at 1114 min , 10195 min , 35161 min
and 58178 min represent proteins common to the ♂and
F1 , and the peaks eluting at 1142 min , 18120 min ,
26157 min , 31182 min and 47169 min represent proteins
common to the ♀ and F1 1 The peaks eluting at 12194
min , 14102 min , 15180 min , 16115 min , 33102 min ,
50144 min and 63120 min represent proteins common to
the hybrid parent , but not appearing in F1 hybrids1 The
peaks eluting at 30199 min , 37198 min , 38130 min ,
52171 min , 53148 min and 54121 min represent proteins
appearing in F1 hybrids but not in hybrid parents1 The
peaks eluting at 13147 min , 14179 min , 28163 min and
40141 min also represent proteins specific to the hybrid
parent ♂1 The peaks eluting at 23168 min , 32111 min ,
42131 min and 47113 min represent proteins specific only
to the hybrid parent ♀1
3 　Discussion
The accurate description and identification of inbred
lines and hybrids are prerequisite to genetic studies , as
well as success in breeding and the production of both
pure foundation and hybrid seeds[12 ] 1 RP2HPLC separates
proteins based on the different interactions of hydrophobic
sites on the surface of the protein molecule with a silicon
coated column support material and a mobile organic
phase that moves through the column1 A mixture of
proteins that are deposited onto the column support can be
sequentially solublised into the organic solvent pumping
through the column as the organic ( hydrophobic )
constitution of the solvent1 Proteins of different
hydrophobiuties can therefore be displaced from the
column by different percentages of organic solvent1 Thus ,
RP2HPLC yields data that is both qualitative and
quantitative in describing cultivars in terms of retention
times on the column and relative amounts for each protein
or proteins1 The method is rapid , highly reproducible and
highly accurate1 Data from RP2HPLC can be readily
181　第 2 期 WANG Qiang et al . : Melon ( Cucumis melo L1) Cultivar Identification by Reversed2phase High Performance111 　　　

compared and analyzed by computer1
RP2HPLC separation of gliadins has been used suc2
cessfully for wheat[13 ,14 ] , maize[15 ] , rye[16 ] and rice[17 ] 1
Data in this research indicate that this separation
technique can be used for the identification of melon
cultivars1 The reproducibility and resolution efficiency of
RP2HPLC analysis have been used for cultivars
identification in other horticultural crops such as Brassica
oleracea L1[18 ] In addition to the high sensitivity and
resolution of the method (HPLC theoretical plates = 105 vs
103 for SDS2PAGE) , it is worth mentioning that the
rapidity of analysis (67 min1 ca) and the possibility of
monitoring the separations allow for possible modifications
in real2time1 Automatic sample handling is another
advantage of modern chromatographic systems1 The
incorporation of an automatic sampler into a system would
enable sequential analyses to be run continuously1 But ,
the disadvantages of HPLC system are : a ) only one
sample can be tested at one time , while SDS2PAGE
allows for fifteen or more samples to be analyzed
simultaneously; b) it is very difficult to determine the
molecular weight and the isoelectric point of the analyzed
proteins ; c) it is difficult to predict their elution order1
In the light of these considerations , RP2HPLC and
SDS2PAGE need not be seen as competitive methods , but
as complements to each other1 SDS2PAGE can be used to
screen analyses because of its rapidity and simplicity ,
whereas RP2HPLC can be used to differentiate varieties
not well characterized or indistinguishable by use of
electrophoretic banding patterns1
Acknowledgements :James Gills and Samuel Kwok for their
assistance with grammatical revisions1
References
[1 ] Association of Official Seed Analyses1 Cultivar Purity Testing Handbook1
Contribution No 33 to the handbook on seed testing1 1991
[ 2 ] Brink D E , Price S C , Nguyen H , Martinez C1 Genetic purity assessment
of commercial single cross maize hybrids : isoelectric focusing of zein1
Seed Science and Technology , 1989 , 17 : 91 - 98
[3 ] Dinelli G, Bonetti A1 Capillary electrophoresis in species and cultivar
determination1 Seed Science and Technology , 1992 , 20 : 241 - 249
[4 ] Bietz J A , Burnouf T , Cobb L A , Wall J S1 Wheat varietal identification
and genetic analysis by reversed2phased high2performance liquid
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[ 5 ] Bietz J A , Burnouf T , Cobb L A , Wall J S1 Gliadin analysis by reversed2
phase high2performance liquid chromatography : Optimization of extraction
conditions1 Cereal Chemistry , 1984b , 61 : 124 - 129
[6 ] Burnouf T , Bietz J A1 Reversed2phase high performance liquid
chromatography of durum wheat gliadines : Relationships to durum wheat
quality1 Journal of Chromatography , 1984 , 2 : 3 - 14
[7 ] Batey I L1 Wheat varietal identification by rapid ion2exchange
chromatography of gliadins1 Journal of Cereal Science , 1984 , 2 : 241 -
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[8 ] Marchylo B A , Kruger J E1 Identification of Canadian barley cultivars by
reversed2phase high2performance liquid chromatography1 Cereal
Chemistry , 1984 , 61 : 295 - 301
[9 ] Lookhart GL , Pomeranz Y1 Characterization of oat species by
polyacrylamide gel electrophoresis and high performance liquid
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[11 ] China Statistics Yearbook220021 Sown areas of major fruit1 Beijing :
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using isoenzyme electrophoresis1 35th Annual Corn and Sorghum
Research Conference , 1980 , 35 : 10 - 31
[13 ] Bietz J A , Cobb L A1 Improved procedures for rapid wheat varietal
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[14 ] Ram C Chandgi , Huebner F R , Bietz J A1 Identification of Indian wheat
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[17 ] Huebner F R , Bietz J A , Webb B D , Juliano B O1 Rice cultivar
identification by high2performance liquid chromatography of endosperm
proteins1 Cereal Chemistry , 1990 , 67 : 129 - 135
[ 18 ] Mennella G, Iori A , Onofaro Sanaja V , Magnifico V1 Broccoli and
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Acta Horticulture , 1996 , 407 : 115 - 121
281 　　　　作 　　物 　　学 　　报 第 31 卷