Characterization and Identification of Seed Storage Protein of Twelve Lettuce Cultivars

We used SDS-PAGE to evaluate and characterize the protein patterns of seed storage proteins in 12 lettuce cultivars. Total protein content of lettuce seeds in all cultivars did not show any significant difference. Results of SDSPAGE pattern of a few protein bands were up regulated whereas some other bands showed down regulation. The identified protein patterns may be used protein marker for lettuce cultivars. The seed storage protein analyses helps in characterization and identification of diversity in lettuce crop varieties, cultivars and their wild varieties and also provides information on phylogenetic relationship of the accessions. It is also known that variation in protein bands provide information on the relationship among the used seeds. SDS-PAGE of seed protein using Tris-glycine buffer was performed to study the relationships within 12 cultivars of Lactuca sativa L. (lettuce). In the present study, SDS-PAGE was used to investigate and characterize the protein patterns of seed storage proteins in order to find protein bands as markers for cultivar characterization. Data were analyzed by clustering method and similarity coefficients using NTSYSpc version 2.02i. Thevalidity of cluster analysis of SDS-PAGE seed proteins profile data was discussed.

The genus Lactuca L. is the one of the economically and medicinally important genera of Asteraceae family, subfamily Cichorioideae, tribe the Lactuceae, lant genetic resources comprise all agricultural crops and their wild relatives of valuable traits 1 .The genus Lactuca L. belonging to the Asteraceae family, is widely distributed in different geographical and ecological areas.Lettuce and most of the other species of the genus Lactuca L. have been cultivated for their economic and medicinal importance.This review summarizes recent knowledge of the application of biochemical (isozymes) and molecular technologies in Lactuca germplasm in order to better understand the genetic variation, interspecific relationships, taxonomy and breeding as a basis for further research studies.This is in order to better understand and improve breeding programmers and biology of crops for future use in agriculture and food security.Hence, advanced molecular genetic technologies including biochemical and molecular markers, have been developed to overcome those limitations of morphological and cytological traits 2,3,4,5,6 .Seed germination is a complex process that is influenced by many environmental factors, such as light, temperature, and moisture 7 .Recently,the genome of Lactuca sativa cv Salinas was sequenced 2 .Genetic variation studies are vital for providing information for propagation, taxonomy, disease resistance, and breeding programs as well as conservation and utilization of Lactuca genetic resources.Genetic diversity can be evaluated based on morphological, cytogenetic, biochemical and molecular markers [8][9][10][11][12] .However, the evaluation of genetic variation based on morphological and cytological traits has the disadvantages of being affected by both genetic and environmental factors and may not provide an accurate measure 13 .
Lettuce (Lactuca sativa) is a temperate annual or biennial plant of the daisy family Asteraceae.It is most often grown as a leaf vegetable.In many countries, it is typically eaten cold, raw, in salads, sandwiches, hamburgers, tacos and in many other dishes.In some places, including China, lettuce is typically eaten cooked and use of the stem is as important as use of the leaf.Both the English name and the Latin name of the genus are ultimately derived from lac, the Latin word for "milk" referring to the plant's milky juice 14 .Mild in flavour, it has been described over the centuries as a cooling counterbalance to other ingredients in a salad.The earliest depiction of lettuce is in the carvings at the temple of Senusret at Karnak, where he offers milk to the god Min, to whom the lettuce was sacred.Lettuce was considered an aphrodisiac food in Ancient Egypt and appears as such in the contendings of Horus and Seth.Later, Ancient Greek physicians believed lettuce could act as a sleep-inducing agent.The Romans cultivated it, and it eventually made its way to the Papal Court at Avignon, France 15 .
There are six commonly recognized cultivar groups of lettuce which are ordered here by head formation and leaf structure; there are hundreds of cultivars of lettuce selected for leaf shape and color, as well as extended field and shelf life, within each of these cultivar groups: (1) Butterhead (L.sativa var.capitata L.) forms loose heads.Its leaves have a buttery texture.Butterhead cultivars are most popular in Europe.Popular varieties include Boston, Bibb, Buttercrunch, and Tom Thumb.(2) Chinese lettuce (L.sativa var.asparagina Baily.)types generally have long, sword-shaped, non-head-forming leaves, with a bitter and robust flavour unlike Western types, for use in stir-fried dishes and stews.(3) Crisphead, also called Iceberg, forms tight, dense heads that resemble cabbage.They are generally the mildest of the lettuces, valued more for their crunchy texture than for flavour.Cultivars of iceberg lettuce are the most familiar lettuces in the USA.The name Iceberg refers to the crisp, cold, clean characteristics of the leaves.(4) Loose-leaf (L.sativa var.crispaL.)has tender, delicate and mildly flavored leaves.This group includes oak leaf and lollorosso lettuces.( 5) Romaine (L.sativa var.romanaLam.), also called Cos, grows in a long head of sturdy leaves with a firm rib down the center.( 6) Summer Crisp, also called Batavian, forms moderately dense heads with a crunchy texture.This type is intermediate between iceberg and loose leaf types.
Lettuces contain antioxidants and Vitamin K, romaine and loose leaf lettuce contain five to six times the Vitamin C and five to ten times the Vitamin A of iceberg.Romaine and butterhead lettuce are good sources of folate.Lettuce naturally absorbs and concentrates lithium 16 .The taxonomicstatus of cultivated lettuce (Lactuca sativaL.), the boundaries among L. sativa and close relatives and the boundaries of the genus Lactuca L., it self have been the subject of controversy among taxonomists for many decades.
Plant storage proteins can be classified into two classes; seed storage proteins (SSPs) and vegetative storage proteins (VSPs).SSPs are a set of proteins that accumulate at high levels in seeds during the late stages of seed development, whereas VSPs are proteins that accumulate in vegetative tissues such as leaves, stems and tubers, depending on the plant species.SSP genes were classic targets for work on plant molecular biology 17 .
The abundant expression of plant storage proteins in seeds allowed for easy detection of the gene transcripts and cDNA cloning during research on plant molecular biology in late 70's to early 80's.Characterization of germplasm using biochemical fingerprinting has got special attention due to its increased used in crop improvement and the selection of desirable genotypes for breeding crops.The use of genetic markers and protein profiling has also been successfully used to resolve the taxonomic and evolutionary problems of several crop plants 18,19,20,21,22 .
Seed protein is highly stable, being unaffected by environmental conditions 23 .Thus electrophoretic banding patterns of total seed protein as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) have provided a valid source of taxonomic evidences and were used to address taxonomic relationships at the generic and specific levels, for example Trifolium 24 , Sesbania 25 , the genus Vicia 26 , the genus Lathyrus 27 ,Lens esculenta 28 , Hordeum vulgare 29 , and Psidium 30 , Rosaceae 31 , Cruciferae 32 , Raphanus L. 33 , Campanulaceae 33 , Gentiana L. and Gentianella Moench, 34 , canola 35 ,Onobrychis 36 and Hordeum Vulgare L. 37 .
The seed storage protein analyses help in identification and characterization of diversity in crop varieties, cultivars and their wild varieties and provides information on phylogenetic relationship of the accessions.It is also known that variation in protein bands provide information on the relationship among the used seeds collected from various geographical regions 38,22,39 .There are different amounts of storage proteins in all plant seeds.They play two main roles including nitrogen and energy source and defense against insects and pathogens such as bacteria and fungi.
The aim of this study is to summarize recent knowledge of the seed storage protein analysis can be a useful tool for identification of species, varieties and cultivars, in the present study, SDS-PAGE was used to investigate and characterize the protein patterns of seed storage proteins in 12 cultivars of Lactuca sativa L. (lettuce) to find protein bands as markers for cultivar characterization.Data were analyzed by clustering method and similarity coefficients using NTSYS-pc version 2.02i.The validity of cluster analysis of SDS-PAGE seed proteins profile data was discussed.

Plant materials
In the present study, sample of 12 cultivars representing Lactuca sativa L. collected from different botanic garden and from the ministry of agriculture in Egypt were used Table (1).

Methods of Analysis Protein Extraction and Electrophoresis
Seed proteins of the studied samples were analyzed using cont-SDS PAGE based on the method of Weber & Osbon 40 .The electrophoretic analysis was carried out using Tris-glycine (pH 8.2) as an extraction buffer.0.1 g of seeds was mixed with an equal weight of pure, clean, sterile fine sand and powdered using a mortar and pestle.Extraction was performed overnight.After centrifugation at 15.000 rpm for 10 min, the supernatant was firstly mixed with equal volume of Tris-HCl pH 6.8 (as a digestion buffer) and boiled for 5 min in water path 41 , then the supernatant was taken for loading on 12% polyacrylamide gels.Electrophoresis was carried out in a Tris-glycine buffer pH 8.3 at 150 V for 2 -2.5 h, using a low molecular weight protein mixture as a marker in each run.Gels were then stained in Comassie brilliant blue R-250 for 2 h, distained, photographed and the number of bands revealed in each gel lane were counted and compared with each other.Gel Pro-Analyzer software version 2.0 was used to determine the molecular mass (MM) of each protein band.

Numerical Analysis
The data of SDS-PAGE seed protein banding patterns were scored as present (1) and absence (0).The data was analyzed to compare various similarity coefficients and clustering methods and to study the relationships among the cultivars detail.Clustering methods and similarity coefficients were tested using the procedures SIMQUAL, SAHN and TREE from the program NTSYS-pc version 2.02i 42 .

SDS-PAGE Electropherogram of the Storage Seed Proteins
Seed protein analysis was carried out on 12 cultivars of lettuce represented the four type of lettuce and the electropherograms produced from seed protein analysis using Tris-glycine buffer revealed great polymorphism among these cultivars as illustrated in Table (2) and Fig. (1).A total number of 73 protein bands were observed within the studied cultivars (57 unique bands and 16 polymorphic bands).Each cultivar was characterized by the presence of unique bands (ranged from 3 bands, recorded in cv.no; 2&3 to 7 bands observed in cv.no; 1).Cultivar no; 10 was found to have six bands and all of them were unique for this cultivar.Cv. no; (11) was found to have the highest number of bands (10), while the lowest number of bands (6) was observed in the sample of cv.no; (10).The highest molecular weight protein band (71.579KDa) among the studied samples was recorded in cv.no; (12), while the lowest one (09.570KDa) was detected in the cv.no; (5).
Seed protein banding patterns as revealed by SDS-PAGE produces reproducible band pattern (profile) when proteins are prepared in a standard method and hence have valid value in taxonomic purposes.Consequently, proteins with identical electrophoretic mobility are deemed to represent the same unit character.Therefore characters derived from seed proteins have been utilized in plant taxonomy at different levels to construct phonetic classifications 43,44,45 .Hence they can be considered as traits to study genetic variation among the plant taxa.However, for the study of the taxonomic relationships among species and higher taxonomic ranks more valid assessment should necessarily be obtained when these data are compiled with other lines of evidence from morphology and cytology.

Numerical analysis
The phenogram produced by the analysis of the studied cultivars based on coding of 73 attributes obtained from Tris-glycine extracted proteins are shown in Fig. (2).This phenogram shows that the examined samples have a total similarity coefficient of about 1.52.At this level, cv.no.(1) was split off from the other cultivars, then at 1.48 level cv.no.(11) was split off from the remaining cultivars.At the levels 1.43 and 1.40, cv.no.(9 & 10) were also split off from the remaining cultivars (respectively).
At the level 1.38, the two cultivars (5 & 6) were separated together in a small group from the other cultivars, then they distinguished from each other at the 1.35 level.At the level 1.322, the two cultivars (4 & 12) were separated together from the other cultivars and then distinguished from each other at the 1.31.At the level 1.313, cv.no; (3) was split off from the remaining cultivars.Then at the level 1.29, cv.no; (8) was separated from the remaining cultivars.Finally, at the level of 1.22, cultivars number (2 & 7) were found to be close to each other.
Electrophoresis of proteins is a powerful tool for identification of genetic diversity and the SDS-PAGE is particularly considered as a reliable technology because seed storage proteins are highly independent of environmental fluctuations 46,47 .Seed protein patterns can also be used as a promising tool for distinguishing cultivars of particular crop species 48,49 .However, only a few studies indicated that cultivar identification was not possible with the SDS-PAGE method 50 .The SDS-PAGE is considered to be a practical and reliable method for species identification 51 .
According to the results of the SDS-PAGE, the overall pattern of seed storage-proteins showed the diversity of lettuce cultivars.The electropherogram produced from seed protein analysis of the 12 cultivars revealed great polymorphism.The diversity in seed storage proteins has also been reported by Khan et al. 52 for wheat varieties.Moreover, identification of three wheat genotypes including ILC-195, CM-2000 and CM-98/99 has also been reported by protein markers 53 .
Since in mature seeds, type and amount of proteins are more constant than other plant tissues 54 therefore, the SDS-PAGE pattern of seed storage proteins of pistachio showed polymorphism based on difference in protein intensity among genotypes.The presence or absence of protein bands has also been applied for detection of polymorphism of Brassica cultivars 55 .
The present investigation revealed variation in different cultivars of lettuce seeds with regard to their total seed protein profiles.These results of SDS-PAGE seed protein patterns show of a few protein bands (6-10) and identified protein patterns may be used protein marker for lettuce cultivars.Regarding inter specific variation among cultivars this investigation revealed some variations.The genetic affinities within cultivars of the same species generally corroborated the morphological analysis.Similar to our finding the result of differentiation of yellow sarson and brown seeded types of Brassica clearly separated the yellow seeded and brown seeded varieties by SDS-PAGE 56 .However, we can conclude that, SDS-PAGE can reveal the differences among seed storage proteins of lettuce cultivars.

Fig. 1 .Fig. 2 .
Fig. 1.Electrophoretic banding profiles of seed proteins extracted in Tris-glycineBuffer of the Studied different lettuce Plants for 12 cultivars

Table 2 .
The molecular weights of protein bands extracted in Tris-glycine Buffer of the Studied different lettuce Plants for 12 cultivars

Table 1 .
The Names of Lettuce Plant (Lactuca sativa) Cultivar and The Sources in This Study