Molecular Diversity in Lactuca Species Using Isozymes and RAPD Markers

Background: understanding molecular diversity in Lactuca species is substantial for the management, improvement and ecient uses of their accessions. Therefore, this work aimed to evaluate molecular diversity among and within Lactuca species. Methods and Results: the molecular diversity was assessed using isozymes and RAPD analyses that gave 87.09% and 100% polymorphic percentages respectively, indicating a high genetic variation within and among Lactuca species. The number of alleles were higher in the wild species compared to the cultivated species, reecting a reduction in the richness of alleles in the cultivated species due to domestication that caused a reduction in genetic diversity to meet the demand for high crop productivity. Isozymes and RAPD clustering dendrogrames: (1) separated, L. sativa accessions in more than one cluster conrming their polyphyletic origin; (2) collected the accessions of L. viminea in one cluster revealed its homogeneity; and (3) divided the accessions of L. saligna in two clusters varied in the number of alleles, particularly “A” form. The corresponding analysis associated the accessions of the wild species based on “B” form of the alleles of the tested isozymes, and the cultivated species on the forms “A” and “C”, suggesting that: (1) allele “B” might be the primitive form of these loci that can tolerate the environmental stresses which prevails in the habitats of the wild species, and (2) forms “A” and “C” could be the derived forms. Conclusions: These results are of great interest for the management of Lactuca germplasm and for future breeding programs of lettuce. of L. sativa, (2) the homogeneity of the accessions of L. viminea ruling out the previous study showed that L. viminea has two distinct subspecies, (3) the grouping of the wild accessions based on the form “B” of the studied alleles and the cultivated accessions based on the forms “A” and “C”, suggesting that form “B” might be the primitive form of the alleles of the loci of the assessed isozymes, and “A” and “C” could be the derived forms, and (4) the separating of the accessions of L. saligna in two clusters was due to the variation in the number of alleles, particularly “A” form, suggesting that some accessions could be wild and the other might be subjected to natural mutation or unintended domestication. The accessions of L. sativa x L. serriola group Oilseed, L. indicia, L. saligna were characterized with unique DNA fragments that can be used as markers for identifying these accessions. The association between specic forms of alleles and both cultivated and wild species needs deep insights to be manipulated for lettuce improvement. The considerable genetic variation in the accessions of Lactuca saligna, Lactuca virosa and Lactuca indica open the door for more detailed studies on big number of accessions cover their distribution range using multiple markers.


Introduction
Genus Lactuca L. is a member of subtribe Lactucinae, tribe Lactuceae, family Lactuceae (Asteraceae) (Güzel et al. 2018). It includes annual, biennial or perennial herbs, rarely shrubs with abundant latex. The species of Lactuca distributed mainly in warm and temperate geographical regions of the world (van Herwijnen and Manning 2017). However, the continents Asia and Africa are the center of the diversity of the genus. (Doležalová et al., 2002).
The primary gene pool of L. sativa L. is represented by a worldwide spread L. serriola, further L. aculeata, L. scarioloides, L. altaica, L. azerbaijancia, L. georgica originating in Asia and L. dregeana native to South Africa (Zohary 1991;van Herwijnen and Manning 2017). The secondary gene pool includes L. saligna, whereas the tertiary gene pool includes the species that can be crossed with di culties with L. sativa, e.g. L. virosa (Doležalová et al. 2002;Jemelková et al. 2018).
Genetic diversity is the raw material permitting species to adapt to environmental changes. The genetic structure of accessions varies from niche to niche along the distribution range of a species (Mondini et al. 2009). So, estimating of genetic diversity is crucial for providing information for domestication, propagation, breeding programs, conservation and use of the germplasm of the plant species (Yu et al. 2001). Biochemical and Molecular markers have been proved as valuable tools to assess and evaluate genetic diversity between and within species, populations and accessions (Yang et al. 2013;Khan et al. 2019). Each marker reveals a speci c class of variation, which is dependent on: (1) the fraction of the genome surveyed by that marker, (2) marker distribution throughout the genome and (3) the extent of the DNA target which is analyzed by that marker (Govindaraj et al. 2015;Bhandari et al. 2017).
Isozymes have been used as a reproducible marker in assessing the amount and distribution of genetic variability and systematic relationships within and between Lactuca spp. (Kesseli and Michelmore 1986;Dziechciarková et al. 2004). They exhibited that the genetic variability was lower in intra-species compared with inter-species and suggested that the origin of L. sativa was polyphyletic. The systematic relationship in Lactuca spp. was investigated using isozymes: (1) con rming the genetic closeness between L. aculeata and L. sativa as they are members of L. serriola complex, (2) revealing a wide genetic distance between L. saligna and L. virosa, and (3) identifying L. serriola, L. saligna, L. virosa, and landrace L. sativa as distinct entities (Kesseli and Michelmore 1986).
The use of molecular markers is also important in assessing the level of genetic diversity and in de ning the genetic relationship between and within species, populations and accessions. Randomly Ampli ed Polymorphic DNA marker (RAPD) is one of the class of DNA markers that has received maximum attention in investigating the genetic variability because it is less expensive, less technical, fast and involves no radioactivity and hybridization. It was used to investigate the genetic variability and interspeci c relationship among accessions, cultivars and genotypes of L. sativa indicating wide genetic base (Waycott and Fort 1994;Yoo and Jang 2003;Sharma et al. 2017).
Genetic resources collections of Lactuca species are poorly characterized because they are made up of large groups of numbered accessions of Lactuca that lack descriptive and (or) pedigree (Lebeda et al. 2019). So, little information is available on the genetic variation among different accessions of Lactuca representing different regions in the world. Therefore, the aim of this research was to use isozymes and DNA markers to evaluate the genetic variability and genetic relationships within and between Lactuca species.

Plant materials
The experimental material was obtained from the CGN (Centre for Genetic Resources, Wageningen, The Netherlands).

Protein Extraction, Electrophoresis And Activity Staining
Protein extraction, electrophoresis and activity staining Isozymes crude extracts were prepared by macerating 20 mg young leaves of fteen days old seedlings with 1 mL of extraction buffer consisted of 0.05M sodium phosphate buffer (pH 7.2), 20% v/v glycerol, 14 mM 2-mercaptoethanol and 0.05% v/v triton X-100 (Manchenko 1994). The clear supernatant was applied directly on 7% PAGE at 4°C in a Mini Protean III unit (BioRad, California, USA), under a constant current of 100 mA for 5 to 6hr, until the tracking dye had moved 5 to 7cm from the cathodal end. The gels were subjected to staining for Phosphorylase, Catalase, α-Esterase and β-Esterase isozymes following the protocols of Pasteur et al. (1988). Phosphorylase gels were stained in solution formed of 10 mM I 2 mixed with 14 mM KI after incubation in 100 ml solution of 0.1M sodium phosphate buffer (pH 5.1) at 37 0 C for 3 to 5 h, developing white bands on a dark blue background. The chromatic or light brown bands appeared at the bottom of the gels were amylase bands. The gels of catalase were stained by immersing in 1:1 mixture of solutions 2% potassium ferricyanide and 2% ferric chloride after incubation in a solution of 3% H 2 O 2 for about 15 min. The gels were then washed and gently agitated for a few minutes in water. Yellow bands of Catalase activity appeared on a blue-green background. The gels of α and -esterases were incubated at 37°C for 15 min in 100 ml staining solution consisted of 0.05 M phosphate buffer (pH 7.2) containing 1% α or β naphthyl acetate for α and -esterases respectively and 50 mg Fast Blue RR until brown colored bands appeared. The stained gels were photographed as quickly as possible and stored in 3% acetic acid. At least 5 and generally 10 plants per accession were examined for isozyme patterns.
Dna Extraction For Rapd Analysis 0.5 g young leaves of fteen days old seedlings were ground with a pestle in liquid nitrogen. The ground sample was suspended in 1 ml preheated CTAB buffer (1.4 M NaCl, 0.2 % 2-mercaptoethanol, 100 mM Tris-Cl and 20 mM EDTA) at 65ºC for one hour. The suspended solution was centrifuge at 1000 rpm and the supernatant was mixed with 0.5 ml of 24:1 chloroform: isomyl, then centrifuge at 14000 rpm (Doyle and Doyle 1990). The aqueous layer was mixed with ice cold isopropanol, then incubated overnight at -20 ºC and centrifuged at 14000 rpm. The supernatant was discarded and the pellet which contained the nucleic acid was carefully washed twice with cold 70% ethanol, dried at room temperature and re-suspended in 100 µl of sterile de-ionized distilled water.

Data analysis
The isozymes and RAPD bands were scored as "0" for presence and "1' for absence for a band at a particular locus in each accessions, which were transformed into a binary character matrix. The binary character matrix of each marker was subjected to correspondence and cluster analyses using the software package "PAST", Version 4.02, Natural History Museum, University of Oslo, 1999-2020. The cluster analyses were performed using the unweighted pair-group method with arithmetic mean (UPGMA).

Rapd Analysis
Among 18 random primers tested in this study, 5 primers generated reproducible bands. A total of 186 polymorphic bands were identi ed using the ve primers. Maximum and minimum percentages of polymorphic bands were observed by primers OPA3 (46 fragments) and OPA1 (31 fragments) respectively. The average percentage of polymorphic bands was 100 %. The average size of DNA fragments ranged between 33bp and 600bp (Fig. 3 The clustering dendrogram constructed based on RAPD results showed seven clusters (C1-C7) (Fig. 4). The rst and second clusters (C1 and C2) included L. serriola group Oilseed lettuce CGN04770 and all the accessions of L. sativa except L. sativa group Butterhead CGN04706 which was separated as singleton at far genetic distance. The wild species L. serriola CGN16210 and L. sativa x L. serriola group Oilseed CGN05115 were clustered in C7 which is connected with other accessions at genetic distance 7.5. The accessions of L. viminea, L. dregeana and L. perennis were included in C5. The accessions of Lactuca saligna (C3 and C6), L. virosa (C3 and C4) and L. indica (C4 and C5) were distributed in two clusters. It can be noticed that two accessions of Lactuca saligna (Lactuca saligna CGN13327 from Greece and Lactuca saligna CGN10883 from Portugal) were collected with Lactuca serriola and Lactuca sativa x Lactuca serriola group Oilseed and the third accession (Lactuca saligna CGN13330 from Turkey) was clustered with Lactuca virosa and Lactuca indicia.

Discussion
Estimating the genetic diversity between and within accessions of crop species and its wild relatives assist in decision-making to select the best accessions for selection of parents for hybridization and is crucial for providing information for domestication and propagation (Yu et al. 2001;Khan et al. 2019). It is also enhancing the understanding of the plant germplasm for germplasm management and potential users, and helping in producing new varieties best adapted to regional environmental conditions (El-Esawi, et al. 2017;van Herwijnen and Manning 2017).
In the present study, the estimation of genetic diversity with isozyme markers showed high polymorphism between the examined accessions (87.09%) which was attributed to outcrossing of the majority of the species and infrequent interspeci c hybridization (Jemelková, et al. 2018). It also showed that the polymorphism between L. sativa accessions (74.2%) was not with same magnitude of the polymorphism between the wild species, re ecting the impact of domestication (successive rounds of selection) on reducing the genetic diversity of the domesticated species, leaving them with less allelic richness than their wild progenitors and other crop wild relatives (Abbo et al. 2014;Dempewolf et al. 2017).
The highest number of alleles in the wild species (31) compared with the cultivated species L. sativa (27) re ected a reduction in the richness of alleles due to domestication which was reported to modify agronomic phenotypes and genetic signature of the domesticated species, resulted in the reduction in genetic diversity to meet the demand for high crop productivity and crop uniformity in the eld and the marketplace (Dempewolf et al. 2017;Zhang et al. 2017). The variation between cultivated and wild species was not only limited to the number of alleles, but also it extended to the mean frequency of alleles which was found to be higher in the cultivated species (0.52) comparing with the wild ones (0.48). This might be attributed to the absence of selection pressure on the wild species, as the existing variations in these species are natural (Das 2011). The allele with low mean allele frequency (0.07) or what was known as rare alleles was observed only for the allele CAT-1A in L. perennis. The presence of this allele could be due to deleterious mutations or may be due to evolutionary relics (Sammour et al. 2019). The detection of rare allele in combination with high allelic frequency of other loci leads to the conclusion that the studied accessions had wide genetic differentiation.
The separation of L. dregeana with L. virosa in the same cluster in isozymes and RAPD cluster analyses con rmed their closely relatedness as they belong to the same section Lactuca ( conspeci c based on AFLP ngerprints, nuclear and plastid DNA sequence comparison, isozymes and RAPD results of the present research recognized differences between them adequate to maintain them as distinct species, con rming the previous morphological, karyological, historical occurrence, and ecological studies (Zohary 1991;El-Esawi and Sammour 2014;van Herwijnen and Manning 2017).
The distribution of the accessions of L. sativa in more than one cluster in the isozymes and RAPD dendrograms con rmed the polyphyletic origin of L. sativa. Furthermore, the clustering of some of the accessions of L. sativa with L. serriola group Oilseed lettuce based on isozymes data and the clustering of the rest of the accessions with L. dregeana con rmed that L. serriola and L. dregeana were members of the primary gene pool of L. sativa (Zohary 1991) and was consistent with the work of Kesselli and Michelmore (1986) who also suggested that domestication of L. sativa may be due to the repeated domestication from wild progenitors or may be due to the use of interspeci c hybridizations in breeding programmes to introduce characters of interest into cultivated lettuce. However, the prevailing of the allele "B" in the wild species and alleles "A" and "C" in the cultivated species and its progenitors, as it has been shown in the corresponding analysis, indicating that the cultivated species was rstly domesticated from wild progenitors and later breeders used interspeci c hybridizations to introduce characters of interest into cultivated lettuce. The prevailing of the allele "B" in wild species and alleles "A" and "C" in cultivated ones also suggested that: (1) allele "B" might be the primitive form of studied alleles that enable the wild species to resist the environmental stresses which prevails in their habitats, and (2) alleles "A" and "C" could be derived forms that evolved as a result of domestication.
The separation of the accessions of L. saligna in two clusters in the cluster analysis of the isozymes data was consistent with the works of Esawi et al. (2017). The heterogeneity of this species attributed to the difference in the number and types of alleles in the studied accessions. The accessions Lactuca saligna CGN13327 from Greece and Lactuca saligna CGN10883 from Portugal did not have the allele "A" of the loci ACP-2, CAT-2, CAT-4, αEST-2, βEST-3, βEST-4 which characterize the cultivated species, whereas the accession Lactuca saligna CGN13330 from Turkey had these alleles. This lead to infer that the accession from Turkey might be subjected to a natural mutation or a sort of unintended domestication. Although Güzel et al. (2018) observed several samples in the eld and herbaria belonging to L. viminea that easily fell into two distinct subspecies according to their habits and morphological traits, the clustering dendrograms of isozymes and RAPD analyses separated the accessions of L. viminea in one cluster which was inconsistent with their observation. The variation observed by Güzel et al. (2018) could be attributed ecogeographical conditions in which the accessions of L. viminea were originated; the conditions that cause a signi cant variation in the phenotypic characters. The clustering dendrogram based on RAPD data indicated a close genetic relationship between L. sativa, L. serriola, L. saligna and L. virosa which was consistent with the phylogenetic trees based on chloroplast DNA sequence comparison (Wei et al. 2017), chromosomal studies (Matoba et al. 2007), nrITS1 and AFLP ngerprints (Koopman et al. 2001). So, L. serriola, L. saligna and L. virosa can be considered important resources for L. sativa breeding.

Conclusion
Genetic variability was considerable high within and among Lactuca species based on isozymes or RAPD analyses, opened the path for the improvement of the cultivated species. The number of alleles were lower in the cultivated species compared to the wild species, re ecting a reduction in the richness of alleles in the cultivated species due to domestication. The cluster and corresponding analyses revealed: (1) the polyphyletic origin of L. sativa, (2) the homogeneity of the accessions of L. viminea ruling out the previous study showed that L. viminea has two distinct subspecies, (3) the grouping of the wild accessions based on the form "B" of the studied alleles and the cultivated accessions based on the forms "A" and "C", suggesting that form "B" might be the primitive form of the alleles of the loci of the assessed isozymes, and "A" and "C" could be the derived forms, and (4) the separating of the accessions of L. saligna in two clusters was due to the variation in the number of alleles, particularly "A" form, suggesting that some accessions could be wild and the other might be subjected to natural mutation or unintended domestication. The accessions of L. sativa x L. serriola group Oilseed, L. indicia, L. saligna were characterized with unique DNA fragments that can be used as markers for identifying these accessions. The association between speci c forms of alleles and both cultivated and wild species needs deep insights to be manipulated for lettuce improvement. The considerable genetic variation in the accessions of Lactuca saligna, Lactuca virosa and Lactuca indica open the door for more detailed studies on big number of accessions cover their distribution range using multiple markers. Declarations