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Robert Griesbach* and Ronald Beck

The genetic distance for three Petunia species was determined based upon an intron in the chalcone synthase gene. The sequence of the intron was obtained for P. integriflolia ssp. integriflolia var. depauperata, P. integriflolia ssp. integriflolia Torres ecotype, P. altiplana and P. littoralis. These species are very closely related and believed by some taxonomists to be part of a large single species complex. In all the taxa, the intron contained multiple repeated and inverted sequences. The P. integriflolia ssp. integriflolia Torres ecotype intron differed from the P. integriflolia subsp. integriflolia var. depauperata intron in 3 of 930 nucleotides. While, the P. littoralis intron differed from the P. integrifolia subsp. integrifolia var. depauperata intron in 15 of 930 nucleotides. As compared to the P. integrifolia subsp. integriflolia var. depauperata intron, the intron in P. altiplana intron was longer (1125 bp), had a section of 338 nucleotides with a completely different sequence, and differed by 27 of 787 nucleotides in the common sequence.

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R.J. Griesbach, R.M. Beck, and J.R. Stehmann

A method was developed to characterize the genetic heterogeneity of the chalcone synthase gene intron within the Petunia integrifolia (Hook.) Schinz & Thell. species complex. The DNA from wild species collected from known locations was used to amplify the chalcone synthase gene intron through the polymerase chain reaction (PCR). The resulting PCR product was then characterized by Rsa 1 restriction, revealing a degree of heterogeneity that could be used to characterize the species genetically. Of the four different species that were characterized, two could be placed in the same genetic grouping. This study shows that the variation in the intron of the Chs A gene may be species-specific.

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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

perennial ( P. domestica ) for the development of virus resistance ( Hily et al., 2005 ; Scorza et al., 2001 ). In this case, resistance was not produced by an intron-hairpin-RNA (ihpRNA) vector, but rather PTGS developed as a result of peculiarities of the

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Li Yingzhi, Cheng Yunjiang, Tao Nengguo, and Deng Xiuxin

solve many of the longstanding problems in the classification and breeding of Citrus ( Moore, 2001 ). DNA sequences of the chloroplast genome are widely used in the field of molecular plant systematics. The trn L intron and trn L- trn F intergenic

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Jiahua Xie, Todd C. Wehner, Kurt Wollenberg, Michael D. Purugganan, and Mark A. Conkling

Aquaporin proteins are part of an ancient family that functions as water transporting facilitators in all organisms. Phylogenetic and physiological analyses have revealed that plant aquaporins consist of two groups: the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs). Using the conserved asparagineproline-alanine (NPA) to NPA motif regions, we studied the evolution of 35 plant aquaporins that included nine of our newly cloned cucumber aquaporins and 26 from the GenBank database. Results indicated that NPA repeated regions were effective for phylogenetically characterizing the plant aquaporin family, and to accurately localize the introns. Phylogenetic analysis showed that 35 plant aquaporins fell into two distinct groups (except for the Arabidopsis gene AtMip)—PIPs and TIPs. The nine cucumber aquaporins belong to the PIP group that were localized further into two different sub-groups. The intron analysis showed that introns of plant aquaporins mainly consist of two types. Eighteen PIPs shared identical intron positions localized in connecting loop C between amino acids 95 and 96. Nine TIPs shared the other identical intron positions localized in connecting loop D between amino acids 44 and 45. Cucumber aquaporins CRB9 and CRB10 (with no intron in the repeated NPA regions) may be the result of intron loss events, while intronless rice (Orzya sativa) Os-TIP1 and Os-TIP2 may have resulted from other intron loss events. PIP11 and Os-PIP do not have the same amino acid number as major PIP members, but combined phylogenetic analysis results along with intron positions and phases showed that they belong to the PIP group. The phylogenetic tree and intron position information suggest that AtMip was mis-annotated as a member of aquaporin, and is a homologue of the glycerol facilitator-like protein. Introns share identical positions and phases within the PIP group (except PIP13) or the TIP group, but differ between the plasma and the tonoplast membrane aquaporins matching the phylogenetic analysis results. Intron positions of the repeated NPA regions of plant aquaporins that have stable inheritance can act as molecular markers for phylogenetic studies.

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R.J. Griesbach and R.M. Beck

The sequence of the intron within the chalcone synthase A gene (ChsA) was used to characterize Petunia integrifolia subsp. integrifolia var. depauperata (Fries) Smith et Downs, P. altiplana Ando et Hashimoto, P. littoralis Smith et Downs, and an unknown taxon from the town of Torres in Brazil. Based upon the intron, the Torres taxon most closely resembled P. integrifolia. The unrooted phylogenetic tree suggested that P. integrifolia was more closely related to P. littoralis than P. altiplana.

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R.J. Griesbach and R. Beck

A new method was developed to analyze genetic diversity among Petunia species. The first intron of the chalcone synthase A gene was cloned through the polymerase chain reaction (PCR) and partially sequenced. This sequence was used to dissect the intron into two halves (3' and 5' halves). The PCR primers for the 5' half amplified a single fragment that was the same length for all of the species that were studied. Restriction fragment length polymorphism (RFLP) analysis of the 5' half resulted in the same number and length of fragments for all the species that were evaluated. The PCR primers for the 3' half amplified a number of fragments that were characteristic for each species. This research provides a new tool for measuring genetic diversity. Genetic diversity measured with this tool should be closely related to evolutionary distance.

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Santiago Vilanova, Carlos Romero, Gerardo Llácer, María Luisa Badenes, and Lorenzo Burgos

This report shows the PCR-based identification of the eight known self-(in)compatibility alleles (S 1 to S 7 and S c) of apricot (Prunus armeniaca L.). Two sets of consensus primers, designed from P. armeniaca S-RNase genomic sequences and sweet cherry (P. avium L.) S-RNase-cDNAs, were used to amplify fragments containing the first and the second S-RNase intron, respectively. When the results obtained from the two PCRs were combined, all S-alleles could be distinguished. The identity of the amplified S-alleles was verified by sequencing the first intron and 135 base pairs (bp) of the second exon. The deduced amino acid sequences of these fragments showed the presence of the C1 and C2 Prunus L. S-RNase conserved regions. These results allowed us to confirm S-genotypes previously assigned by stylar ribonuclease analyses and to propose one self-(in)compatibility group (I) and one universal donor group (O) containing unique S-genotypes and self-compatible cultivars (SC). This PCR-based typing system also facilitates the identification of the S c-allele and might be a very useful tool for predicting self-compatibility in apricot breeding progenies.

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Hui Wei, Yan Fu, and Rajeev Arora

Intron length polymorphisms were used to investigate relationships among eight Rhododendron L. species (R. catawbiense Michaux., R. minus Michaux., R. ponticum L., R. keiskei Miquel., R. arboreum Sm., R. dichroanthum Diels., ssp. scyphocalyx Cowan., R. maximum L. and R. dauricum L.) and two hybrid cultivars [i.e., R. `PJM' (R. minus var. minus × R. dauricum) and R. `Chionoides' (R. ponticum × unknown)]. A total of 27 of these markers were used to estimate phylogenetic relationships among the species and draw inferences about the parentage of the cultivars, which is partially unknown. In general the expressed sequence tag-polymerase chain reaction (EST-PCR) marker-based phylogenetic map of the eight species is congruent with the currently accepted morphology-based classification of these species at the subgenus as well as the section level. However, the constructed phylogenetic tree revealed that, at the subsection level, two species, R. arboreum (subsection Arborea Sleum.) and R. dichroanthum (subsection Neriiflora Sleum.), are grouped under the same “clade” (80% bootstrap score), suggesting that these species are more closely related than indicated in the current classification system that places them in separate subsections/clades. Moreover, our phylogenetic analysis of the three species belonging to section Ponticum G. Don. demonstrated a closer phylogenetic relationship between R. ponticum and R. maximum (bootstrap score of 74%) than between these species and R. catawbiense; such observation is consistent with a recent phylogenetic analysis of section Ponticum by Milne (2004) using the sequences of a chloroplast gene. Parentage analysis for the two cultivars confirmed the interspecific lineage of R. `PJM' and provided genetic support for the speculated R. ponticum and R. maximum parentage of R. `Chionoides'. Our results indicate that, in addition to their use in mapping studies, intron-flanking EST-based PCR markers are valuable tools for conducting phylogenetic and parentage analyses and/or gene flow studies.

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Júlia Halász, Attila Hegedűs, Zoltán Szabó, József Nyéki, and Andrzej Pedryc

two introns at the same sites as those of other Prunus species ( Igic and Kohn, 2001 ). Both introns varied in size in an S -haplotype-specific manner. Sapir et al. (2004) cloned five additional S -alleles from three commercially important