Pachanoor S. Devanand

Genetic Analysis of Egyptian Date (Phoenix dactylifera L.) Accessions Using AFLP Markers

Forty-seven samples of date palm (Phoenix dactylifera L.) collected from eight locations in Egypt were studied using four sets of amplified fragment length polymorphism (AFLP) markers with near infrared fluorescence labeled primers. These samples belonged to 21 named accessions and 9 of unknown pedigrees. A total of 350 bands were scored and 233 (66.6%) were polymorphic. Twenty-seven Egyptian accessions and ‘Medjool’and ‘Deglet Noor’accessions from California could beclassified into the major cluster. This major cluster may represent a major group of date palm germplasm in North Africa. There were four other clusters, each containing one or two accessions. The variety ‘Halawy’and one accession of unknown provenance were most likely from hybridization between two clusters. Six groups of accessions of which had the same names, revealed similar but not identical AFLP profiles suggesting these accessions might derive from seedlings rather thanthrough clonal offshoot propagation.

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants.

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, ‘Pink Allusion’ was selected from ‘White Butterfly’. Tissue culture of ‘Pink Allusion’ through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of ‘Pink Allusion’ obtained a cultivar, ‘Regina Red Allusion’, and tissue culture propagation of ‘Regina Red Allusion’ led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within ‘White Butterfly’ and ‘Regina Red Allusion’ groups, however, were only 1.2% and 0.4%, respectively. Jaccards similarity coefficients among somaclonal cultivars derived from ‘White Butterfly’ and ‘Regina Red Allusion’, on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the ‘White Butterfly’ group and three out of six from the ‘Regina Red Allusion’ group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the ‘White Butterfly’ group and three from the ‘Regina Red Allusion’ group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.

Genetic variation within ‘Medjool’ and ‘Deglet Noor’ date (Phoenix dactylifera L.) cultivars in California detected by fluorescent-AFLP markers

Summary Date palm (Phoenix dactylifera L.) is one of the most important fruit crops of the arid climate region in countries of North Africa and Middle East and there is limited production in California. All date palms in California have always been propagated by offshoots, a vegetative propagation. Throughout the world and in California, two of the most important, widely grown date cultivars are ‘Medjool’ and ‘Deglet Noor’. In the past, it has been difficult to identify date palm cultivars based on morphological characteristics, and even more difficult to identify genetic strains of commercial cultivars. To identify the date cultivars in California, we developed a PCR based amplified fragment length polymorphism (AFLP) analysis with near infrared fluorescence labelled primers. The AFLP markers generate large number of polymorphic bands among different date cultivars and facilitate easy identification of each cultivar. Using four AFLP primer sets (IRD700 E1TA/M1CAG, IRD800 E1AC/M1CAG, IRD700 E1TG/M1CAT, and IRD800 E1AG/ M1CAT), we evaluated the genetic variation within twenty-three ‘Medjool’ and thirty-three ‘Deglet Noor’ date accessions in California. We were able to identify large intra-varietal variation within ‘Medjool’ and almost none in ‘Deglet Noor’ using AFLP markers. These accessions of ‘Medjool’ date in California are representatives of the genetic constituents of ‘Medjool’ date from its origin. ‘Medjool’ dates most likely exist as a landrace variety at its location of origin, the Tafilalt region of Morocco. Another possibility is that ‘Medjool’ date may have a very high mutation rate. The identification of genetic variation of date cultivars using fluorescent-AFLP markers will change the direction of future germplasm collection and preservation effort of dates. The information also will be useful in future date breeding and improvement. This study demonstrated the utility of fluorescent-AFLP markers in detecting polymorphism and estimating genetic diversity among date cultivars and accessions.

Practical strategy for identification of single nucleotide polymorphisms in fruiting mei (Prunus mume Sieb. et zucc.) from amplified fragment length polymorphism fragments

Single nucleotide polymorphism (SNP) is the most abundant form of genetic variation among individuals within a species. SNPs can be used as markers for gene discovery and for assessment of diversity. We established a practical strategy for identification of SNPs in fruiting mei (Prunus mume Sieb. et Zucc.) from amplified fragment length polymorphism (AFLP) fragments. The main modification of this procedure was optimization of the reamplification of bands excised from an AFLP gel by using a single enzyme (EcoRI) in digestion reaction to generate large AFLP fragments and to lower the number of bands on gels, using lower-concentration polyacrylamide gels (4%) and loading each sample into 4 continuous lanes, using a newly modified protocol for purification of AFLP bands from the gel, and using additional cycles for reamplification of AFLP bands. In this study, 15 groups of bands with identical migration distances from 10 fruiting mei cultivars were selected for purification. Eighty-one of the 150 chosen bands were successfully reamplified, and 67 of these reamplified polymerase chain reaction products yielded reliable sequences belonging to 13 groups. The alignment of 13 group sequences yielded 95 SNPs, for a total of 5252 bp. Among these SNPs, 73 were heterozygous in the loci of some individual cultivars. The SNP distribution was 58% transition, 40% transversion, and 2% indels. There was also 1 dinucleotide polymorphism and 1 tetranucleotide deletion.

Interspecific relationships of Alocasia revealed by AFLP analysis

Summary This study analysed genetic relationships of 23 Alocasia cultivars across 17 species using amplified fragment length polymorphism (AFLP) markers. Six primer sets, selected from an initial screening of 48, generated a total of 578 scorable AFLP fragments of which 334 (58.4%) were polymorphic. All cultivars were clearly detected by their AFLP fingerprints. A dendrogram was constructed using the unweighted pair-group method of arithmetic averages (UPGMA). Principal coordinated analysis (PCOA) was carried out to show multiple dimensional distributions of cultivars. Both UPGMA and PCOA analyses separated the 23 cultivars into three clusters. Cluster I comprises 16 cultivars, mainly derived from A. crassifolia, A. cuprea, A. longiloba, A. grandis, A. guttata, A. plumbea, A. macrorrhiza, A. micholitziana, and A. villeneuvei or hybrids of A. lowii A. sanderiana and A. cuprea A. veitchii. Jaccard’s similarity coefficients for these species ranged from 0.43 to 0.77. Cluster II contains six cultivars, which include A. cadieri, A. cucullata, A. gageana, A. odora, and A. portei. Jaccard’s similarity coefficients varied from 0.52 to 0.83. There is only one cultivar, ‘Hilo Beauty’ in the cluster III, whose low similarity (0.21) with the rest of the Alocasia species may suggest that it could actually belong to another genus of Araceae. Based on documented interspecific hybrids, it appears that hybrids were developed from species exclusively within the identified clusters. This may suggest that Alocasia species sharing high Jaccard’s similarity coefficients are more likely to be intercrossable. The interspecific relationships detected by the AFLP analysis could provide the genetic basis for selecting parents for future hybrid development.