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Analysis of genetic diversity in Agave tequilana var. Azul using RAPD markers
Abstract and Figures
By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production of any tequila. Our objective was to assay levels
of genetic variation in field populations of A. tequilana var. Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four different
fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A. tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130
markers, of which 20 (15.4%) were polymorphic betweenA. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of124 RAPD products (0.8%) was polymorphic and 39 of 40 plants were completely isogenic. This is one of
the lowest levels of polymorphism detected to date for the analysis of a crop species, and is proposed to be the result of
the promotion of a single conserved genotype over many years due to an exclusive reliance on vegetative propagation for the
production of new planting materials. The significance of these results is discussed in relation to breeding programs focused
on the improvement of A. tequilana var. Azul.
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... However, the consequences of clonal propagation on the genetic diversity of A. tequilana are still not clear. Some authors reported no genetic diversity (Gil-Vega et al., 2001;Trejo et al., 2018), which may make the species particularly vulnerable to pathogens (Gil-Vega et al., 2001;Dalton, 2005). Other studies have suggested a less pronounced reduction of the genetic variation (Gil-Vega et al., 2006;Vargas-Ponce et al., 2009;Rivera-Lugo, García-Mendoza & Simpson, 2018;Cabrera-Toledo et al., 2022). ...
... However, the consequences of clonal propagation on the genetic diversity of A. tequilana are still not clear. Some authors reported no genetic diversity (Gil-Vega et al., 2001;Trejo et al., 2018), which may make the species particularly vulnerable to pathogens (Gil-Vega et al., 2001;Dalton, 2005). Other studies have suggested a less pronounced reduction of the genetic variation (Gil-Vega et al., 2006;Vargas-Ponce et al., 2009;Rivera-Lugo, García-Mendoza & Simpson, 2018;Cabrera-Toledo et al., 2022). ...
... Previous genetic studies on A. tequilana have reported a broad range of genetic diversity estimates, but we must point out that they used very different molecular methods, not SNP based analysis. For instance, the highest expected heterozygosity H E = 0.205 was reported using AFLPs by Rivera-Lugo, García of 124 RAPD products (0.8%) was polymorphic, and 39 of 40 plants were completely isogenic (Gil-Vega et al., 2001). In other less-intensively managed populations around Tequila town, different levels of genetic diversity have been detected with microsatellites in the varieties A. tequilana "Sigüin" H E was 0.409 and in A. tequilana "Chato" H E was 0.435 (Trejo et al., 2018). ...
Background: Genetic diversity is fundamental for the survival of species. In particular, in a climate change scenario, it is crucial that populations maintain genetic diversity so they can adapt to novel environmental conditions. Genetic diversity in wild agaves is usually high, with low genetic differentiation among populations, in part maintained by the agave pollinators such as the nectarivorous bats. In cultivated agaves, patterns of genetic diversity vary according to the intensity of use, management, and domestication stage. In Agave tequilana Weber var. azul (A. tequilana thereafter), the plant used for tequila production, clonal propagation has been strongly encouraged. These practices may lead to a reduction in genetic diversity.
Methods: We studied the diversity patterns with genome-wide SNPs, using restriction site associated DNA sequencing in cultivated samples of A. tequilana from three sites of Jalisco, Mexico. For one locality, seeds were collected and germinated in a greenhouse. We compared the genomic diversity, levels of inbreeding, genetic differentiation, and connectivity among studied sites and between adults and juvenile plants. Results: Agave tequilana presented a genomic diversity of HT = 0.12. The observed heterozygosity was higher than the expected heterozygosity. Adults were more heterozygous than juveniles. This could be a consequence of heterosis or hybrid vigor. We found a shallow genetic structure (average paired F ST = 0.0044). In the analysis of recent gene flow, we estimated an average migration rate among the different populations of m = 0.25. In particular, we found a population that was the primary source of gene flow and had greater genomic diversity (HE and HO), so we propose that this population should continue to be monitored as a potential genetic reservoir.
Discussion: Our results may be the consequence of more traditional management in the studied specific region of Jalisco. Also, the exchange of seeds or propagules by producers and the existence of gene flow due to occasional sexual reproduction may play an important role in maintaining diversity in A. tequilana.
... Unfortunately, this industrialization also brought expensive social and environmental consequences, including the erosion of the diversity of traditional landraces [11,[12][13][14][15]. Since the DOT established that only blue agave can be used to make tequila, producers shifted their efforts to grow only blue agave landrace, severely mining the diversity of landraces that used to be part of traditional tequila agroecosystems 400 years ago [8,11,16]. In addition, to preserve the integrity of the blue agave, producers have historically recurred almost exclusively to asexual propagation, a strategy that has also been adopted for the cultivation of other landraces, resulting in an extreme loss of genetic diversity [17,18]. These current conditions urge the development and promotion of traditional and new landraces locally adapted to regional climatic regimes and conditions [11,14,15,19,20]. ...
... A. tequilana, A. angustifolia and A. rhodacantha belong to a morphological and genetic complex of species known as A. angustifolia complex [24,25]. Previous studies using traditional molecular markers have proposed wild populations of A. angustifolia in the state of Jalisco as possible wild closest relatives of the blue agave [17,18,26]. Ethnobotanical evidence shows that Southern Jalisco is the nucleus of the greatest diversity of spirit producing landraces within the A. angustifolia complex, and it is likely that this area was where traditional farmers initiated the selection of Agave germplasm for spirit production [8]. ...
... Given its importance in tequila production, A. tequilana and the suggested landraces have been studied in more depth than A. angustifolia. The study of the genetics of A. tequilana with traditional genome-wide genetic techniques such as AFLPs and RAPDs were inconclusive, showing either low or significant diversity depending on the technique used [17,32]. These molecular markers have a high mutation rate and can change from one generation to the next [33], or even in between bulbils produced asexually from the same mother plant [34]. ...
Traditional agave spirits such as mezcal or tequila are produced all over Mexico using different species of Agave. Amongst them, A. angustifolia is the most popular given its agricultural extension. A. angustifolia is a wild species extensively distributed from North to Central America, and previous studies show that it is highly related to the tequila agave A. tequilana. In different regions of Mexico, A. angustifolia is cultivated under different types and levels of management, and although traditional producers identify several landraces, for the non-trained eye there are no perceivable differences. After interviews with producers from different localities in Jalisco, Mexico, we sampled A. angustifolia plants classified as different landraces, measured several morphological traits, and characterized their genetic differentiation and diversity at the genome-wide level. We included additional samples identified as A. tequilana and A. rhodacantha to evaluate their relationship with A. angustifolia. In contrast with previous studies, our pool of ca 20K high quality unlinked SNPs provided more information and helped us to distinguish different genetic groups that are congruent with the ethnobotanical landraces. We found no evidence to genetically delimitate A. tequilana, A. rhodacantha and A. angustifolia. Our large genome level dataset allows a better understanding of the genetic identity of important A. angustifolia traditional and autochthonous landraces.
... The reproduction of agave is then based on clonal sprouts from a small pool of plants that are removed and resown. Unsurprisingly, this intensive technique of reproduction has been detrimental to genetic diversity (Gil Vega et al 2001, Vargas-Ponce et al 2009, Ruiz Mondragon et al 2022. The commercial farming of blue agave has an additional unintended negative side effect: harming bat populations. ...
Tequila has a designation of origin by which it can only be produced with the blue agave (Agave tequilana Weber blue variety) grown in certain regions of Mexico. For several decades, an intensive asexual technique of reproduction has prevailed in the tequila industry. This has not only been detrimental for the agave’s genetic diversity, but it has also harmed bat populations. The low levels of diversity have increased the agave’s exposure to pests and diseases and have limited the possibilities for artificial selection of beneficial characteristics that can improve production. Arguably, with the cooperation of producers and consumers, it is possible to have an environmentally friendly and resilient production system. We focus on the agave producers and analyze their willingness to let some of their plants flower in exchange of some monetary and non-monetary benefits. By analyzing data from a choice experiment, we show that farmers are willing to let some of their plants flower even when they only receive partial compensation for the value of the plants. Farmers also show a willingness to participate in a conservation program if they receive a non-monetary transfer or expect an increase in yield because of the program. Our results provide a relevant piece of information that can help policymakers, producers, NGOs, and other participants in the tequila industry, in the design of effective strategies that can lead to the genetic conservation of blue agave and the protection of magueyero bats.
... En el caso de muchas de las plantas cultivadas, se ha perdido la variación durante el proceso de domesticación y su posterior manejo. En el caso de los agaves, se detectó una reducción en la variación genética del 58.81% en la heterocigosis esperada en H-W en cada población, siendo el ejemplo más dramático el agave azul que se usa para producir el tequila, donde en las plantaciones comerciales se ha detectado nula variación genética (Gil-Vega et al., 2001) Para decidir mejor cuáles poblaciones conservar, desarrollamos una estrategia cuantitativa usando datos genéticos y ecológicos, y la aplicamos en el agave del desierto chihuahuense A. victoriaereginae, que se encuentra fuertemente amenazado por la destrucción de su hábitat y por la sobre-colecta para usarlo como ornato. Usamos información genética obtenida de isoenzimas, junto con datos ecológicos del tamaño poblacional para definir a las localidades prioritarias para su conservación. ...
... Furthermore, the genetic diversity of species such as A. tequilana var. Azul seems to be compromised (Gil-Vega et al., 2001;Vargas-Ponce et al., 2009;Trejo et al., 2018). Nevertheless, more recent studies based on single-nucleotide polymorphisms (SNPs) found heterozygosity excess and negative inbreeding index at plantations with clonally propagated agave plants (Ruiz-Mondragon et al., 2022;Cabrera-Toledo et al., 2022). ...
Premise:
The central Oaxaca Basin has a century-long history of agave cultivation, and it is hypothesized to be the region of origin of other cultivated crops. Widely cultivated for mezcal production, the perennial crop known as "espadín" is putatively derived from wild Agave angustifolia. Nevertheless, little is known about its genetic relationship to the wild A. angustifolia or how the decades-long clonal propagation has affected its genetics.
Methods:
Using RADseq and over 8,000 SNPs, we studied aspects of the population genomics of wild and cultivated A. angustifolia in Puebla and Oaxaca, Mexico. We assessed patterns of genetic diversity, inbreeding, distribution of genetic variation, and differentiation among and within wild populations and plantations.
Results:
Genetic differentiation between wild and cultivated plants was strong, and both gene pools harbored multiple unique alleles. Nevertheless, we found several cultivated individuals with high genetic affinity with wild samples. Higher heterozygosity was observed in the cultivated individuals, while in total, they harbored considerably fewer alleles and presented higher linkage disequilibrium compared to the wild plants. Independently of geographic distance among sampled plantations, the genetic relatedness of the cultivated plants was high, suggesting a common origin and prevalent role of clonal propagation.
Conclusions:
The considerable heterozygosity found in "espadín" is contained within a network of highly related individuals, displaying high linkage disequilibrium generated by decades of clonal propagation and possibly by the accumulation of somatic mutations. Wild A. angustifolia, on the other hand, represents a significant genetic diversity reservoir that should be carefully studied and conserved. This article is protected by copyright. All rights reserved.
... Species such as A. tequilana for making tequila or A. fourcroydes to manufacture henequen, use clonal propagation, where suckers of the same genotype are grown and thus their genetic diversity is eroded generation after generation. The loss of genetic diversity carries a reduction of its adaptability to environmental and climate changes, pathogens and pests [2,23,[30][31][32][33]. ...
We compiled an updated database of all Agave species found in Mexico and analyzed it with specific criteria according to their biological parameters to evaluate the conservation and knowledge status of each species. Analyzing the present status of all Agave species not only provides crucial information for each species, but also helps determine which ones require special protection, especially those which are heavily used or cultivated for the production of distilled beverages. We conducted an extensive literature review search and compiled the conservation status of each species using mainstream criteria by IUCN. The information gaps in the database indicate a lack of knowledge and research regarding specific Agave species and it validates the need to conduct more studies on this genus. In total, 168 Agave species were included in our study, from which 89 are in the subgenus Agave and 79 in the subgenus Littaea. Agave lurida and A. nizandensis, in the subgenus Agave and Littaea, respectively, are severely endangered, due to their endemism, lack of knowledge about pollinators and floral visitors, and their endangered status according to the IUCN Red List. Some species are at risk due to the loss of genetic diversity resulting from production practices (i.e., Agave tequilana), and others because of excessive and unchecked overharvesting of wild plants, such as A. guadalajarana, A. victoriae-reginae, A. kristenii, and others. Given the huge economic and ecological importance of plants in the genus Agave, our review will be a milestone to ensure their future and continued provision of ecosystem services for humans, as well as encouraging further research in Agave species in an effort to enhance awareness of their conservation needs and sustainable use, and the implementation of eco-friendly practices in the species management.
... There are molecular researches with the purpose to determine the genetic 56 variability among agave phenotypes. Gil-Vega et al. (2001) reported studies among 40 field selections of A. tequilana cultivar blue, only 124 random amplified polymorphic DNA (RAPD) products (0.8%) were polymorphic and 39 of 40 were completely isogenic. This is one of the lowest levels of polymorphism detected to date for the analysis of a crop species. ...
p> Background: Blue agave ( Agave tequilana Weber var. azul) , it is commonly known as “Agave azul or agave tequilero”. The Agave crop is fundamental part of the productive chain Agave-Tequila, since is the distinctive source of reducing sugars for the elaboration of the liqueur known as Tequila, according with the Appellation of Origen Tequila to fulfill the National Normativity (NOM-). Besides, Tequila is the most spirit drink exported from Mexico worldwide. According with the latest evaluation over this crop, the susceptibility to pathogens generate in cases total lost of the agave fields. Objective. to achieve somatic embryos that could be resistant, generated from plantlets irradiated with Co<sup>60</sup>gamma rays; which were obtained from axillary buds. Methodology: The arisen of embryo was reached in the modified Murashige and Skoog (MS) medium supplemented with of 2,4-D combined with BA or KIN. The plantlets were radiated 12 weeks after the somatic embryo induction with Co<sup>60</sup> gamma rays. The doses were 0 (test control), 10, 20 and 30 Gy. Results: There was a significant difference in the induction of embryonic callus, development and mature somatic embryos, which were reduced as the doses of radiation increases. Since only somatic embryos were achieved in tissues treated with 10 and 20 Gy doses, in higher doses induction of embryonic callus was inhibited. The regression confirmed the negative results with the increase of radiation. Implications: There were made protocols to achieve somatic embryos and plants from tissues irradiated with 10 and 20 Gy. Conclusions : The radiation effect over somatic tissue was crucial as the doses affect the conversion into plantlets, nevertheless such radiation allowed the conversion of the embryos to complete agave plantlets</p
... For this reason, it has been vegetatively propagated for centuries for commercial purposes, using mainly the rhizome offshoots produced around the agave plants from the second year of this crop cycle. It has therefore been presumed that agave crops had lost genetic diversity with a higher vulnerability to pathogens (Gil-Vega et al. 2001;Abraham-Juárez et al. 2009;Valenzuela 2011). However, additional reports have shown a signi cant level of genetic diversity using ampli ed fragment length polymorphism (AFLP) and inverse sequence-tagged repeat DNA markers (Gil-Vega et al. 2006;Torres-Morán et al. 2013). ...
Agave (Agave tequilana Weber var. azul) is considered a crop with low genetic diversity because it has been propagated vegetatively for centuries for commercial purposes, and consequently, it could be equally susceptible to pests and diseases. However, the present study employs plant material derived from field grown plants exhibiting phenotypic variability in susceptibility to agave wilt. The offshoots from rhizomes of these plants were reproduced in vitro and classified as potentially resistant or susceptible. Amplified fragment length polymorphism (AFLP) analysis confirmed wide genetic differences among individuals, but these differences were not correlated with the observed phenotypic variability in resistance. Propagated plantlets were inoculated with Fusarium solani in two time-lapse confrontations for 72 h and 30 days. The early biochemical response showed statistically superior levels in the accumulation of shikimic acid, phenolic compounds, and chitinase activity in potentially resistant plantlets. There was an inverse correlation of these early biochemical responses and salicylic acid and the incidence of diseased root cells in isogenic plantlets in the 30-day confrontation with F. solani, suggesting that these activities and accumulation of molecules were primordial in the defence against this pathogen.
Hellenia speciosa (J. Koenig) S. R. Dutta (Syn: Costus speciosus (J. Koenig) Sm.) is well known for its anti-diabetic properties and is in high demand for its steroidal sapogenin, diosgenin, which is used in the pharmaceutical industry for manufacturing steroidal drugs. The increasing demand for this plant has led to the depletion of its wild population. This study was conducted to reveal the genetic diversity of H. speciosa collected from various agro-ecological regions of India for use in devising conservational strategies, and cultivation and breeding programs. Twenty inter-simple sequence repeat markers yielded 304 products with 100% polymorphism demonstrating high genetic diversity. Nei′s gene diversity and Shannon information indexes were estimated among the populations as 0.19 ± 0.23 and 0.28 ± 0.25, respectively. High genetic differentiation (GST = 0.40) and low gene flow (Nm = 0.74) were observed, which corroborated with the analysis of molecular variance revealing maximum (86.75%) variance within populations. The unweighted pair group method with arithmetic mean analysis grouped all accessions into three major clusters. Principal component analysis showed that the first three components accounted for 30.66%, 4.54%, and 3.98% variation, respectively, and Bayesian clustering through structure showed the presence of two genetic population (K = 2). This study revealed high genetic diversity among the populations collected from high altitude compared to those from the plain. A positive but low correlation among genetic and geographic distances was obtained (r = 0.282, p < 0.0001). Identification of genetic diversity and intra population variation of a species are prime factors to take defined conservation strategies.
Sexual propagation of Agave plants is an incipient cultivation method, these plants withstand drought and adverse growing conditions; therefore, research on Agave’s diversity, seed processing, and storage could support its cultivation on marginal lands. The aim of this work was to evaluate seed morphology, germination, and seedling genetic diversity of six seed origins (species × provenance) of Agave plants collected in five provenances from Mexico. Seed longevity was evaluated in two seed origins after a 10-year storage period. Seed morphology and seedling genetic variation results demonstrated intra- and interspecific variation within Agave salmiana and with the other seed origins, respectively. After a 10-year storage period seed germination of two A. salmiana seed origins remained relatively stable, storage conditions, and seed variables of this work can serve as reference parameters for future analyses. To the best authors’ knowledge, this is the first report of Agave’s seed longevity evaluation after a 10-year storage period.
RAPD analysis was employed to assess genetic variation in named cultivars of garlic (Allium sativum L.), and to examine the relationships between cultivated garlic varieties and the wild progenitor Allium longicuspis. Twenty-seven accessions were subjected to RAPD analysis using 26 oligonucleotide primers. Of a total of 292 bands, 63 (21%) were polymorphic. Cluster analysis revealed groupings that in part reflected patterns of morphological variation. All bolting forms (including wild and cultivated) grouped separately from the nonbolting cultivars. A. longicuspis and var. ophioscorodon grouped together, indicating close taxonomic affinity. Based upon relative levels of variation within different groups, we suggest potential relationships within the A. sativum/A. longicuspis complex.
The use of the RAPD technique was investigated on a set of 30 grapevine (Vitis ssp.) rootstock cultivars. The DNA was extracted directly from woody canes using a CDAB technique. The screening of 21 decamer oligonucleotides allowed the selection of six primers used for the analysis. They revealed a high level of polymorphism among the rootstocks. Twenty bands, intense and easy to score, were chosen as markers. Identification of 23 out of the 30 rootstocks was obtained with only two primers (opP17 and opA09). The addition of one primer (opA20) allowed the identification of the complete set of rootstocks. Based on simple matching coefficient calculated from RAPD data, a tree was generated. It clearly reflected the genetic relationships between the rootstocks. The stability of the markers was tested in very diverse conditions. The main factor of variability was the origin of the Taq polymerase. Given a preliminary screening step, RAPD markers proved to be very stable and useful for identification purpose since they are quick and easy to use.
A chloroplast DNA restriction site analysis of 40 taxa in the Agavaceae s.l. was undertaken to test the accuracy of several current systems of classification. In Cronquist's system, the Agavaceae contain 18 genera of fibrous-leaved, more or less woody plants such as Yucca, Hesperaloe, Agave, Manfreda, Polianthes, Dracaena, Sansevieria, Nolina, Beaucarnea, Dasylirion, and Cordyline. In Dahlgren's system, the Agavaceae are restricted to Yucca, Hesperaloe, Agave, Manfreda, Polianthes, Beschorneria, and Furcraea, while the other genera are placed in different families. These contrasting phylogenetic schemes were investigated by a chloroplast DNA restriction site analysis of these genera plus Xanthorrhoea, Hosta, Hypoxis, Hemerocallis, Asparagus, Liriope, Aspidistra, Maianthemum, and Convallaria. Phylogenetic analysis of 110 restriction site mutations, 90 of which were phylogenetically informative, strongly supports the conclusion that there are two major lineages in this group of taxa. One well supported clade contains, sensu Dahlgren, the Nolinaceae, Dracaenaceae, and Convallariaceae. It appears that Nolina and Dasylirion are more closely related to genera such as Liriope, Maianthemum, and Dracaena than to Yucca and Agave. The other major lineage contains Yucca, Hesperaloe, Agave, Manfreda, Polianthes, Beschorneria, and Furcraea, with Hosta and perhaps Xanthorrhoea at the base. Asparagus appears to be intermediate between the two lineages. The cpDNA suggests that Cordyline, Hypoxis, and Hemcrocallis are not closely related to the Agavaceae.
Several systems of classification have been proposed for the family Agavaceae. A distinctive bimodal karyotype and similarities of fruits and seeds strongly support close relationship among Yucca, Hesperaloe, Beschorneria, Furcraea, Agave, Manfreda, Polianthes, Prochnyanthes, and perhaps Hosta. However, Dasylirion, Beaucarnea, Nolina, Calibanus, Dracaena, and Sansevieria differ in so many cytological and morphological features that many have been concluded they should he excluded from Agavaceae and separated into two families. Nolinaceae and Dracaenaceae, Chloroplast DNA restriction site data support these separations and indicate that Nolinaceae and Dracaenaceae are very close to Convallariaceae (Maianthemum. Convallaria, Aspidistra, Liriope, etc.). In this paper we report the results of an ITS rDNA sequencing study of 40 taxa in Agavaceae sensu lato and related groups in the order Asparagales. Sequence alignments were optimized using the Consistency Index. Retention index, and rescaled Consistency Index to find the alignment that least amount of homoplasy. The results of our study are congruent with the conclusions drawn from cytological, immunological, cpDNA and rbcL studies, which support a narrow interpretation of Agavaceae and a close relationship among Convallariaceae, Dracaenaceae, and Nolinaceae. In addition, the ITS sequence data provides evidence for some interesting relationships within these families.
Yucca filamentosa and its species-specific pollinator, the yucca moth, Tegeticula yuccasella (Lepidoptera: Prodoxidae), form a relationship that is often cited as a classic example of a coevolved plant-pollinator mutualism. Observations of the moth's behavior have led to predictions that moth dispersal is relatively limited and that, as a consequence, the self-compatible Y. filamentosa should experience relatively high rates of self-fertilization, In contrast, analyses of its mating system indicated that Y. filamentosa was predominantly outcrossed. To better understand effective breeding patterns in Y. filamentosa populations, 10 polymorphic allozyme loci were investigated to analyze the breeding structure of a natural Y. filamentosa population. Analyses revealed that Y. filamentosa is predominantly outcrossed, has multiply sired fruits, and that each fruit was sired by a different set of pollen donors. The effective number of pollen donors per fruit ranged from 1.56 to 3.13, indicating that some correlated mating exists within fruits. Paternity analyses revealed that pollen moved from 6 m to 293 m (mean = 118 m) within the study population and that a minimum of 10% of the progeny were sired by pollen originating outside of the population. These results are discussed in the context of the yucca-yucca moth mutualism.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Limonium cavanillesii is an extremely endangered plant species endemic to the east Mediterranean region of Spain. Regarded as extinct for several years, the recent discovery of a small population (only 29 individuals) has prompted the adoption of measures for its conservation by official agencies. As part of this effort, we have analysed genetic variation in this population by means of random amplified polymorphic DNA (RAPDs). The analysis of 29 individuals with 11 different primers produced 131 monomorphic bands. To our knowledge, this is the lowest level of genetic variation detected in plants using RAPD markers. This result could be explained both by the apomictic reproductive system of this species and by the passage through a severe bottleneck in recent times, after which there has been no chance for mutation to restore detectable genetic variation.
Molecular diversity in Saccharum complex was studied using 195 RAPD markers generated by 12 random primers. Among the Saccharum species, S. officinarum showed a low level of genetic diversity while S. sinense was found to be more diverse. Six taxonomical groups were clearly resolved in the cluster analysis. S. officinarum, S. robustum, S. spontaneum and Erianthus spp. formed discrete groups. S. barberi and S. sinense formed a single cluster, so also Narenga and Sclerostachya. S. officinarum was found to be closer to S. robustum and distant from S. spontaneum. Among the related genera, Sclerostachya was closer to Saccharum while Erianthus was found to be highly divergent from all the Saccharum species. Six of the primers used generated RAPD fragments unique to Erianthus. It is suggested that the Erianthus spp. can contribute substantially towards sugarcane varietal improvement in view of its greater divergence with Saccharum.
Forty-one of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the United States and Canada were examined
for RAPD (randomly amplified polymorphic DNA) marker polymorphisms using 10mer primers (>50% GC content). A set of 10 primers
produced 15 polymorphic fragments ranging in size between 450 and 1200 bp, which were more than sufficient to distinguish
among all tested cultivars. Ten of the markers derived from seven primers were absolutely required for distinguishing the
cultivars. A DNA fingerprinting table was constructed based on these results. In addition, similarity coefficients were calculated
based on RAPD marker data and a dendogram was constructed using the unweighted pair group method of arithmetic averages (UPGMA).
These results were compared with known pedigree data for the cultivars. Our results demonstrate that RAPD markers can be used
effectively for strawberry cultivar identification.