Kyong-Cheul Park

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.

Study of genetic diversity and relationships among accessions of foxtail millet [Setaria italica (L.) P. Beauv.] in Korea, China, and Pakistan using SSR markers

In this study, 28 simple sequence repeat (SSR) primer sets were used to analyze the genetic diversity, population structure, and genetic relationships among 37 accessions of foxtail millet from Korea, China and Pakistan. A total of 298 alleles were detected with an average allele number of 10.6 per locus among 37 foxtail millet accessions. The number of alleles per locus ranged from 2 (b226) to 20 (b236). Of the 298 alleles, 138 alleles (46.3%) were rare (frequency < 0.05), 152 alleles (51.0%) were detected at an intermediate frequency (range, 0.05–0.50), and eight alleles (2.7%) were abundant (frequency > 0.50), respectively. The average gene diversity values were 0.652, 0.692, and 0.491 and polymorphic information content values were 0.621, 0.653, and 0.438, for accessions from Korea, China, and Pakistan, respectively. The accessions from China showed higher SSR diversity than those from Korea and Pakistan. A phylogenetic tree constructed using the un-weighted pair group methods with arithmetic mean algorithm revealed three major groups of accessions that were not congruent with geographical distribution patterns with a few exceptions. The lack of correlation between the accession clusters and their geographic location indicates that the diffusion of foxtail millet from China to Korea might have occurred through multiple routes. Our results provide support for the origin and diffusion route of foxtail millet in East Asia. This SSR-based assessment of genetic diversity, genetic relationships, and population structure among genetic resources of foxtail millet landraces will be valuable to foxtail millet breeding and genetic conservation programs in Korea.

Intron loss mediated structural dynamics and functional differentiation of the polygalacturonase gene family in land plants

The polygalacturonase (PG) gene family is one of the largest gene families in plants. PGs are involved in various plant development steps. The evolutionary processes accounting for the functional divergence and the specialized functions of PGs in land plants are unclear. Whole sets of PG genes were retrieved from the genome web sites of model organisms in algae and land plants. The number of PG genes was expanded by lineage-specific manner with the biological complexity of the organism. Differentiation of PGs was related with phylogenetic hierarchy such as presence of rhamno-PGs from algae to plants, endo- and exo-PGs in land plants, exo-PGs in flowering plants. Gene structure analysis revealed that land plant PG genes resulted from differential intron gain and loss, with the latter event predominating. Differential intron losses partitioned the PGs into separate clades to be expressed differentially during plant development. Intron position and phase were not conserved between PGs of algae and land plants but conserved among PG genes of land plants from moss to vascular plants, indicating that the current introns in the PGs in land plants appeared after the split between unicellular algae and multicelluar land plants. The results demonstrate that the functional divergence and differentiation of PGs in land plants is attributable to intron losses.

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.

LTR-retrotransposons and inter-retrotransposon amplified polymorphism (IRAP) analysis in Lilium species

LTR-retrotransposons are ubiquitous and highly abundant in plant genomes. Moreover, LTR-retrotransposons can often cause genome obesity in plants. Although Lilium species have been known carrying large genomes among flowering plants, reports on the LTR-retrotransposons in Lilium species are rather limited. We isolated a novel Ty3/gypsy-like retrotransposon, LIRE-del, and two Ty1/copia-like retrotransposons, a LIRE-del and an unclassified, from a fosmid clone of Lilium longiflorum. Decayed internal ORF sequences indicated that they were non-autonomous elements. IRAP protocol was developed based on the LTR sequences of the isolated LTR-retrotransposons. Fourteen primer combinations showed clear distinctive PCR amplification bands that were highly informative in the analysis of species relationship among Lilium species. The phylogenetic relationship based on the IRAP profile revealed some discordant with phylogenetic studies based on the ITS sequences of 45S ribosomal gene and matK gene variations in a few species. Thus, the phylogenetic relationship among Lilium species may need to be re-evaluated with other tools such as cross compatibility and selectively neutral genetic markers.

Species relationships among Allium species by ISSR analysis

The genus Allium includes several important condiment and ornamental vegetable species. The botanical status of many more than 600 species in the genus Allium has not been clearly classified. We used inter simple sequence repeat (ISSR) technique to analyze the phylogenetic relationships among 24 individual accessions and species that represent cultivated Allium species and their ancestral species. Nucleotide sequence analysis was used to rule out homoplasy, the possibility that unrelated ISSR-bands migrated to the same positions during gel electrophoresis. To avoid spurious results, PCR reactions were conducted in duplicate on independent thermocyclers and only reproducible bands were used for analysis. We detected 153 polymorphic bands from 20 ISSR primers. The ISSR polymorphism profiles were used to group most of the Allium species and accessions according to previous classification. However, A. chinense, previously included in the Rhizirideum section, was distinct from other species in the phylogenetic dendrogram. A. wakegi, the Korean endemic ‘Jjokpa’ and hybrid between A. cepa and A. fistulosum, was grouped in the Cepa section. A. monanthum, another Korean endemic ‘Dalae,’ was grouped in the Rhizirideum section. Because phylogenetic analysis of these three Korean endemic species has not been reported previously, the current report may allow these species to be included in future phylogenetic studies.

Identification of genetic variations of cultivated and weedy types of Perilla species in Korea and Japan using morphological and SSR markers

To better understand the genetic diversity and relationships of the two cultivated types of Perilla crop and their weedy types in Korea and Japan, we evaluated the genetic variations of 56 accessions by assessing five morphological characteristics and 18 SSR markers. The two cultivated types of var. frutescens and var. crispa were clearly distinguished by seed size, whereas most accessions of cultivated and weedy types of var. crispa cannot be distinguished strictly by seed characteristics. A total of 165 alleles with the SSR analysis were detected with an average number of 9.2 alleles per locus among the 56 Perilla accessions. The number of alleles per locus ranged from two for KWPE-56 and KWPE-39 to 21 for GBPFM-204. Additionally, the genetic diversity of each locus ranged from 0.497 at KWPE-56 and KWPE-39 to 0.959 at GBPFM-204, with an average of 0.692. The average genetic diversity values were 0.549, 0.685, 0.451 and 0.557 for cultivated and weedy types of var. frutescens and for cultivated and weedy types of var. crispa, respectively. The weedy type accessions of var. frutescens and var. crispa evidenced greater variation than the corresponding cultivated type accessions. The accessions of the cultivated and weedy types of var. frutescens and var. crispa from Korea exhibited greater SSR diversity than those of Japan. An UPGMA phylogenetic tree revealed three major groups, which was congruent with their morphological characteristics except for a few odd accessions. SSR markers clarified the genetic relationships between var. frutescens and var. crispa and helped improve our understanding of the genetic diversity of the two cultivated types of P. frutescens and their weedy types in Korea and Japan.

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.

CACTA transposon-derived Ti-SCARs for cultivar fingerprinting in rapeseed

Transposon display (TD) is a technique for analyzing polymorphic insertions and excisions of transposable elements. We used the CACTA transposon to develop cultivar-specific transposon insertion-sequence characterized amplified regions (Ti-SCARs) for rapeseed (Brassica napus). Using 16 combinations of TD primers, we detected 19 cultivar-specific fragments among six rapeseed cultivars. Of the 16 primer pairs, 12 successfully amplified targets in B. napus and six amplified novel, cultivar-specific markers. Cultivar-specific markers can be used for cultivar fingerprinting and marker-assisted selection in the rapeseed breeding program. Moreover, because Ti-SCARs are based on transposon insertions, Ti-SCAR markers may be used in reverse genetic techniques for isolating novel genes in plants.

A new active CACTA element and transposition activity in ecotype differentiation of Arabidopsis

The Arabidopsis genome was mined for whole copies of CACTA and CACTA-like elements and a new active element, AtCAC24024, was isolated. Like the previously identified CAC1 element, the genes encoding transposase (TPase) in AtCAC24024 were expressed in a methylation-defective mutant background, inducing its transposition. The AtCAC24024 element is structurally different from CAC1 in that it has a TPase_24 domain instead of the TPase_23 domain that exists in the TNP-A open reading frame of CAC1. The transposition activity of AtCAC24024 was low compared to CAC1. Phylogenetic analysis revealed that elements with both domains were present in both monocotyledonous and dicotyledonous plants, and originated independently and proliferated separately during plant differentiation. In a joint analysis of transposon display and amplified fragment length polymorphism polymerase chain reaction in various Arabidopsis ecotypes, the CAC1 element showed three transpositions that were followed by random loss during ecotype differentiation, leading to incremental increases in copy number in recent ecotypes. Copy numbers of AtCAC24024 increased explosively in the first differentiation period, followed by random loss during ecotype differentiation. Although transposition might not directly cause ecotype differentiation, the possibility of any direct or indirect involvement still exists in ecotype differentiation by insertion into or excision out of critically functioning genes.