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Jinggui Fang and ChihCheng T. Chao

DNA methylation plays an important role in the regulation of gene expression in eukaryotes. The extent and patterns of DNA methylation were assessed in the date palm (Phoenix dactylifera L.) mother plants and their offshoots using the methylation sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I bands were present in both EcoR I + Hpa II and EcoR I + Msp I lanes; type II bands were present in EcoR I + Hpa II lanes, but not in EcoR I + Msp I lanes; and type III bands were present in EcoR I + Msp I lanes, but not in EcoR I + Hpa II lanes. The total numbers of these three types of bands were 782, 55, and 34. Among these three types of bands, the polymorphic bands were 34, 10, and 0, respectively. The distribution of polymorphic bands among mother-plants and offshoots could suggest the methylation variation occurred to the mother plants and offshoots. The methylation variation during offshoot growth of date palm was characterized as a process involving mainly of demethylation. Hypomethylation of DNA in offshoots compared with mother plants reflects the marked expression of this molecular feature, which may related to gene expression during development of offshoots. The methylation or demethylation status of specific loci in the mother plants and their offshoots might not relate their lineage but occurred randomly.

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Jinggui Fang and Chih Cheng T. Chao

MicroRNAs (miRNA) are endogenous tiny RNAs (about 22 nucleotides in length) that can play important regulatory roles in plants and animals by targeting mRNAs for cleavage or involved in translational suppression. Based on the sequence conservation of many miRNA genes in different plant genomes, it is possible to identify miRNAs in citrus. Identification of miRNA is the prerequisite for understanding the miRNA function in citrus. Citrus is an important fruit crop in the world and the publicly available citrus EST databases are increasing. Thirty known miRNAs from Arabidopsis were used to search the citrus EST databases for miRNA precursors. Nine possible citrus miRNA sequences were predicted to have fold-back structures. The Northern results indicated most of the 26 Arabidopsis miRNAs are expressed ubiquitously in the leaf, young shoot, flower, and root tissues of Nules Clementine mandarin (Citrus clementina Hort. Ex Tan.) and Trifoliate orange (Poncirus trifoliata [L.] Raf.). Some miRNAs accumulated preferentially in different tissues.

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Chih-Cheng T. Chao, Jinggui Fang and Pachanoor S. Devanand

Production of seedless mandarins such as `Nules' clementine mandarin (Citrus clementina Hort. Ex Tan.) and `Afourer' mandarin [C. sinensis (L.) Osbeck × C. reticulata Blanco] is increasing in California as consumers' interest in seedless, easy peeling, and good tasting mandarins increases. The fruit would produce seeds if cross-pollination with compatible pollen source occurred. It is almost impossible to prevent cross-pollination between compatible mandarin cultivars by honeybees (Apis mellifera L.) within the multi-faceted agricultural environment in California. To produce seedless mandarin, growers either plant a single cultivar in a large solid block or try to use pollen-sterile navel oranges (C. sinensis) or satsuma mandarins (C. unshiu Marco.) as buffers to prevent cross-pollination. The question of how many rows of buffer trees or spacing can effectively prevent cross-pollination by honeybees between compatible mandarins is unclear. We initiated a study using fluorescent-labeled AFLP markers to determine the pollen parentages of `Nules' clementine seedlings and `Afourer' mandarin seedlings from two orchards in California. The longest distance of pollen flow at an orchard near Madera was 521 m. The pollen of `Minneola' tangelo (C. reticulata × C. paradisi Macf.) was able to disperse across a minimum of 92 rows of `Lane Late' navel oranges plus two rows of `Afourer' mandarins to pollinate `Afourer' mandarins. We also found that all the seedlings of `Nules' clementine mandarin at an orchard near Bakersfield had been pollinated by `Afourer' mandarin pollen. The pollen of `Afourer' mandarin was able to disperse up to distances between 837 and 960 m to pollinate `Nules' clementine. The pollen dispersal distance found in this study was at least 16 times longer than previously reported in a citrus orchard. Growers need to consider a much larger space or buffer rows to prevent cross-pollination and produce seedless mandarins in California.

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Xiucai Fan, Renzong Zhao, Qianqian Wang, Chonghuai Liu and Jinggui Fang

In this study, we measured the anthocyanin composition and content in the ‘Kyoho’ grape cultivar and its derivatives via ultra-performance liquid chromatography–mass spectrometry and characterized the MybA-related genes at the color locus via capillary electrophoresis and quantitative real-time polymerase chain reaction. A total of 30 anthocyanins (15 monoglucoside and 15 diglucoside) were detected. Peonidin-3-O-(t-6''-O-coumaroyl)-glucoside-5-O-glucoside was the most abundant component, and the content of malvidin-3-O-(c-6''-O-coumaroyl)-glucoside-5-O-glucoside was low in all cultivars. All 49 cultivars contained VvmybA1, VvmybA2, and VvmybA3, whereas only the black-skinned cultivars contained VlmybA2. The anthocyanin content in the cultivars that contained VlmybA2 was significantly higher than other cultivars. These results could provide information for future color breeding programs in grapes.

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Jinggui Fang, Pachanoor S. Devanand and ChihCheng T. Chao

Single-nucleotide-polymorphism (SNP) is the most abundant genetic variation among individuals within a species. SNPs can be used as markers for gene discovery and for assessment of biodiversity. We established a practical strategy for discovering candidate SNPs in fruiting-mei (Prunusmume Sieb. et Zucc.), a non-model tree fruit, from amplified-fragment-length-polymorphism (AFLP) fragments. Eighty-one of the 150 chosen bands from 10 cultivars of fruiting-mei were successfully re-amplified and 67 of these re-amplified PCR products yielded 13 groups of reliable sequences. The sequencing results from both directions of 23 randomly selected PCR products using the corresponding selective primers showed that all the purified fragments from the gels were EcoR I-EcoR I fragments. The sequence alignment of 13 groups of sequence yielded 95 SNPs from a total of 5252 bp, averaging one SNP every 55 bp. Among these SNPs, 73 were heterozygous in the loci of some individual cultivars. The SNPs distribution were: 58% transition, 40% transversion, and 2% InDels. There were also one di-nucleotide polymorphism and one tetra-nucleotide deletion. The procedure of SNP isolation from AFLP fragments can be useful for transferring AFLP markers into sequence-tagged-site markers.

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Jinggui Fang, Chih Cheng Chao, Richard J. Henny and Jianjun Chen

Plant tissue culture can induce a variety of genetic and epigenetic changes in regenerated plantlets, a phenomenon known as somaclonal variation. Such variation has been widely used in the ornamental foliage plant industry as a source for selection of new cultivars. In ornamental aroids alone, at least 63 somaclonal-derived cultivars have been released. In addition to morphological differences, many somaclonal aroid cultivars can be distinguished by amplified fragment length polymorphism (AFLP) analysis. However, a few cultivars have no detectable polymorphisms with their parents or close relatives by AFLP fingerprints. It is postulated that DNA methylation may be involved in the morphological changes of these cultivars. In this study, methylation-sensitive amplification polymorphism (MSAP) technique was used to study DNA methylation in selected somaclonal cultivars of Alocasia, Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium. Results showed that polymorphisms were detected in the somaclonal cultivars, suggesting that DNA methylation polymorphisms may associate with tissue culture-induced mutation in ornamental aroids. This is the first study of methylation variation in somaclonal variants of ornamental foliage plants. The results clearly demonstrate that the MSAP technique is highly efficient in detecting DNA methylation events in somaclonal-derived cultivars.

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Jinggui Fang, Tal Twito, Zhen Zhang and Chih Cheng T. Chao

The genetic relationship among 50 fruiting-mei (Prunus mume Sieb. et Zucc.) cultivars from China and Japan was investigated using 767 amplified fragment length polymorphism (AFLP) and 103 single nucleotide polymorphism (SNP) markers. The polymorphism among the cultivars was 69.77% based on EcoR I + Mse I AFLP primer pairs. The sequence alignment of 11 group sequences derived from 50 samples yielded 103 SNPs with a total length of 3683-bp genomic sequences. Among these SNPs, 73 were heterozygous in the loci of different cultivars. The SNP distribution were: 58% transition, 40% transversion, and 2% InDels. There was also one tri-nucleotide deletion. Both AFLP and SNP allowed the evaluation of genetic diversity of these 50 fruiting-mei cultivars; however, the two derived cladograms have some differences: 1) all the cultivars formed two sub-clusters (1A and 1B) within cladogram based on AFLP polymorphisms, and there were three sub-clusters (2A, 2B and 2C) formed in the cladogram based on SNP polymorphisms; and 2) most cultivars from G-F, Y-H-S regions and Japan are grouped in cluster 1A and 18 (78.26%) out of 23 cultivars from J-Z origin are grouped in cluster 1B in the cladogram generated based on AFLP polymorphisms. The results show cultivars from Japan are clustered within cultivars from China and supports the hypothesis that fruitingmei in Japan was introduced from China in the past. Cultivars from J-Z region of China have higher genetic similarities. Cultivars from G-F and Y-S-H regions have lower genetic similarities and suggest more germplasm exchanges in the past.

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Mohammed Aziz Elhoumaizi, Panchanoor S. Devanand, Jinggui Fang and Chih-Cheng T. Chao

We studied 66 `Medjool' date palm (Phoenix dactylifera L.) accessions from Morocco, six varieties of dates from Egypt, and four `Medjool' accessions and one `Deglet Noor' accession from California to investigate the hypothesis that `Medjool' is a landrace variety in Morocco. We used four sets of fluorescent-labeled amplified fragment length polymorphism (AFLP) markers to examine these accessions. A total of 402 bands were generated, of which 217 were polymorphic (54.0%). The 66 `Medjool' accessions from Morocco shared a minimum of 79% genetic similarity. These results support the hypothesis that `Medjool' is a landrace variety in Morocco and it is not genetically uniform. `Medjool' is the first confirmed landrace variety of date palm in the world. This finding raises the possibility that other date palms may be landrace varieties in different growing areas. Confirmation of a landrace variety in date palm has significant implication for future date palm germplasm collection and preservation. The mechanism(s) generating the genetic variation within `Medjool' accessions remains unknown. One possibility is that spontaneous genetic changes could occur frequently within vegetative tissues like offshoots under high temperature and drought stress.

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Jinggui Fang, Jianjun Chen, Richard J. Henny and Chih-Cheng T. Chao

Ornamental Ficus L. is a group of lactiferous trees, shrubs, and woody root-climbing vines that are cultivated either as landscape plants in the tropics and subtropics or as foliage plants used worldwide for interiorscaping. With the recent rapid expansion of the ornamental plant industry, more new Ficus species and cultivars have been introduced. However, no study has thus far addressed the genetic relationships of cultivated ornamental Ficus. Using amplified fragment length polymorphism (AFLP) markers with near-infrared fluorescence-labeled primers, this study analyzed the genetic relatedness of 56 commercial cultivars across 12 species. Forty-eight EcoRI + 2/MseI + 3 primer set combinations were initially screened, from which six primer sets were selected and used in this investigation. Most cultivars were differentiated by their AFLP fingerprints, and their relationships were determined using the unweighted pair-group method of arithmetic average cluster analysis. The 56 cultivars were divided into 12 clusters that correspond to 12 species, indicating that no interspecific hybrids of ornamental Ficus are in commercial production. The 12 species are genetically diverse, with Jaccard's similarity coefficients ranging from 0.21 to 0.43. However, cultivars within three species—Ficus benjamina L., Ficus elastica Roxb. Ex Hornem., and Ficus pumila L.—are genetically close. Twenty-seven of the 29 cultivars of F. benjamina and five cultivars of F. pumila had Jaccard's similarity coefficients of 0.98 or higher respectively. Nine cultivars of F. elastica shared Jaccard's coefficients higher than 0.96. These results indicate potential genetic vulnerability of these cultivars within the three species. Because there are increasing reports of invasive pests in the ornamental plant industry, strategies for conserving genetic resources and broadening genetic diversity of cultivated Ficus are discussed.

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Jinggui Fang, Panchanoor S. Devanand, Chih Cheng T. Chao, Philip A. Roberts and Jeff D. Ehlers

Cowpea (2n=2x=22) is a high protein, short-cycle, and essential legume food crop of the tropics, especially in the low input agricultural areas of sub-Saharan Africa, Asia, and South America. Lack of genetic diversity within breeding programs can limit long-term gains from selection. The cowpea gene pool is thought to be narrow and the genetic diversity within breeding programs could be even less diverse. Genetic relationships among 87 cowpea accessions, including 60 advanced breeding lines from six breeding programs in Africa and the United States, and 27 accessions from Africa, Asia, and South America were examined using amplified fragment length polymorphism (AFLP) markers with six near-infrared fluorescence labeled EcoR I + 3/Mse I + 3 primer sets. A total of 382 bands were scored among the accessions with 207 polymorphic bands (54.2%). Overall, the 87 cowpea accessions have narrow genetic basis and they shared minimum 86% genetic similarities. The data also show that the advanced breeding lines of different programs have higher genetic affinities with lines from the same program but not with lines from other programs. The results suggest that there is a need to incorporate additional germplasm of different genetic background into these breeding lines and to ensure the long-term genetic gains of the programs.