Ju Kyong Lee

Rim 2/Hipa CACTA transposon display ; A new genetic marker technique in Oryza species

BackgroundTransposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa-TD) protocol for the study of genetic markers in map construction and the study of genetic diversity in rice.ResultsRim2/Hipa-TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa-TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa-TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa-TD. While Rim2/Hipa-TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC, EE, BBCC and CCDD, profiles demonstrated that most of the amplified fragments were larger than 400 bp, and that our methods were insufficient to clearly distinguish between these fragments. However, the overall amplification profiles between species in the Oryza genus were fully distinct. Phenetic relationships among the AA diploid Oryza species, as evidenced by the Rim2/Hipa-TD markers, were matched with their geographical distributions.ConclusionThe abundance of the Rim2/Hipa TIR sequences is very informative since the Rim2/Hipa-TD produced high polymorphic profiles with ample reproducibility within a species as well as between species in the Oryza genus. Therefore, Rim2/Hipa-TD markers can be useful in the development of high-density of genetic map around the centromeric regions. Rim2/Hipa-TD may also prove useful in evaluations of genetic variation and species relationships in the Oryza species.

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.

Comparison of seed characteristics between the cultivated and the weedy types of Perilla species

We evaluated the morphological characteristics of 54 Perilla accessions by examining two quantitative and four qualitative characteristics in order to understand better the relationships between the cultivated types and the weedy types of Perilla species. The size, germination rate, hardness, and color of seeds were examined. The cultivated type of Perillafrutescens var. frutescens showed variation in seed size (2.08 to 2.96 mm) and germination rate (54 to 99%). Most accessions had soft seeds, but the seed colors of white, gray, brown, and dark brown. The weedy type of var. frutescens also showed the variation in seed size (1.54 to 1.94 mm), germination rate (0 to 46%), and had only the hard seeds in dark brown. The cultivated type of var. crispa showed the seed size range of 1.6 to 1.72 mm, and the germination rate of 3 to 34%, and had only the hard seeds in dark brown. The weedy type of var. crispa showed the seed size of 1.45 to 1.96 mm, the germination rate of 0 to 27%, and also hard seeds with dark brown color. The accessions of cultivated type of var. frutescens exhibited the bigger variation in seed characteristics than the weedy type of var. frutescens and the cultivated and weedy types of var. crispa. Principal component analyses clearly discriminated between the cultivated and weedy types in var. frutescens, and between var. frutescens and var. crispa in the two cultivated types. However, the seed characteristics of the cultivated type and the weedy type in var. crispa were not clearly discriminated. In this study, the size, germination rate, and hardness of seed were the useful characters for discriminating var. frutescens and var. crispa in the cultivated types, and the cultivated type and the weedy type in var. frutescens. The cultivated type of var. frutescens might be regarded as a more domesticated type than the cultivated type of var. crispa.

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.

Gene set by de novo assembly of Perilla species and expression profiling between P. frutescens (L.) var. frutescens and var. crispa

Perilla frutescens (L.) Britt. is a self-pollinating annual species and is widely cultivated in China, Korea and Japan as an economic crop and a source of medicine and spices. In this study, we sequenced one cultivar variety (PF98095) of P. frutescens (L.) var. frutescens Britt., which was assembled as reference and other three varieties (PF11109, weedy of var. frutescens, PF06336 and PF06353, cultivars of varieties crispa) in order to carry out comparative expression profiling within cultivar and weedy in varieties frutescens and between varieties frutescens and varieties crispa of cultivar type in P. frutescens. Assembly of PF98095, annotation mapping, DEG (differentially expressed gene) profiling, and comparative analysis were performed. We found that more than 65% of the reads were mapped to the reference of P. frutescens gene set. Moreover, we detected 22,962 DEGs in the weedy variety compared to the cultivar, and also, 22,138 and 23,845 DEGs were identified in two cultivars according to the reference, respectively. The DEGs and functional classification were developed to analyze the differences between weedy and cultivar and between varieties frutescens and varieties crispa of Perilla. Furthermore, candidate genes for the different color and seed size of Perilla were identified that could be further investigated in future study. The herein results may play a significant role, and contribute in functional transcriptome studies of Perilla.

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.

QTL analysis for eating quality-related traits in an F2:3 population derived from waxy corn × sweet corn cross

In order to identify quantitative trait loci (QTL) for the eating quality of waxy corn and sweet corn (Zea mays L.), QTL analysis was conducted on an F2 population derived from a cross between a waxy corn inbred line and a sweet corn inbred line. Ten QTLs for pericarp thickness (PER), amylose content (AMY), dextrose content (DEX) and sucrose content (SUC) were found in the 158 F2 families. Among them, four QTLs, qAMY4 (10.43%), qAMY9 (19.33%), qDEX4 (21.31%) and qSUC4 (30.71%), may be considered as major QTLs. Three of these, qAMY4, qDEX4 and qSUC4, were found to be located within a region flanked by two adjacent SSR markers on chromosome 4 (umc1088 and bnlg1265), making this SSR marker pair a useful selection tool for screening the eating quality traits of AMY, DEX and SUC. The QTL for amylose content was found to be located between markers phi027 and umc1634, raising the possibility of its identity being the Wx1 gene, which encodes a granule-bound amylose synthase. The new QTLs identified by the present study could serve as useful molecular markers for selecting important eating quality traits in subsequent waxy corn breeding studies.

Molecular authentication of two medicinal plants Ligularia fischeri and Ligularia stenocephala using allele-specific PCR (AS-PCR) strategy

Ligularia fischeri (Gom-chi) and Ligularia stenocephala (Gon-dal-bi) are popular edible herbs in Korea. L. fischeri is used to treat jaundice, hepatitis, rheumatoid arthritis, and scarlet fever, while L. stenocephala is used to treat anxiety, weakness, and menstrual disorders. The herbal medicinal activities of these two herbs differ, but they are very difficult to distinguish based on their morphologies, especially in their dried forms. In an effort to distinguish these two plant species, we sequenced three barcoding genes in plastids, matK, rbcL, and trnH-psbA. From the analysis of sequence variations, we detected five single nucleotide polymorphisms (SNPs) between two the species. Allele specific (AS)-primers in the SNPs were employed in discrimination of the two species. Of the five AS-primer sets, one primer pair in matK gene showed reproducibly distinguishable PCR amplification between plants of L. fischeri and L. stenocephala. The method is reproducible and efficient, and is the first reported molecular method to discriminate between L. fischeri and L. stenocephala.

Genetic diversity and population structure in cultivated and weedy types of Perilla in East Asia and other countries as revealed by SSR markers

In this study, 17 simple sequence repeat (SSR) primer sets were used to analyze the genetic diversity, genetic relationships, and population structure among 81 accessions of two cultivated types of Perilla crop and their corresponding weedy types in East Asia and other countries. A total of 166 alleles were identified with an average of 9.8 alleles per locus. The average gene diversity (GD) value was 0.709. The average polymorphic information content (PIC) value was 0.679. The GD of each locus for accessions of cultivated var. frutescens, weedy var. frutescens, cultivated var. crispa, and weedy var. crispa were 0.557, 0.740, 0.429, and 0.509, respectively. Both weedy accessions exhibited higher GD and PIC values than their corresponding cultivated types. The cultivated and weedy types of the Perilla crop had much higher GD and PIC values in East Asia than in other countries. Particularly, in East Asia, the Perilla accessions from China showed higher SSR diversity than those from Korea, Japan, and other countries. Population structure analysis identified three groups, Group I, Group II, and an admixed group. Phylogenetic analysis produced four major clusters, but there was no clear geographic relationship between the two cultivated types of Perilla crop and their weedy types based on their regional distribution. This study demonstrated the utility of SSR analysis for performing genetic and population analysis of cultivated and weedy types of Perilla accessions in East Asia and other countries.

Genetic diversity and population structure among accessions of Perilla frutescens (L.) Britton in East Asia using new developed microsatellite markers

SSRs were successfully isolated from the Perilla crop in our current study, and used to analyze Perilla accessions from East Asia. Analyses of the clear genetic diversity and relationship for Perilla crop still remain insufficient. In this study, 40 new simple sequence repeat (SSR) primer sets were developed from RNA sequences using transcriptome analysis. These new SSR markers were applied to analyze the diversity, relationships, and population structure among 35 accessions of the two cultivated types of Perilla crop and their weedy types. A total of 220 alleles were identified at all loci, with an average of 5.5 alleles per locus and a range between 2 and 10 alleles per locus. The MAF (major allele frequency) per locus varied from 0.229 to 0.943, with an average of 0.466. The average polymorphic information content (PIC) value was 0.603, ranging from 0.102 to 0.837. The genetic diversity (GD) ranged from 0.108 to 0.854, with an average of 0.654. Based on population structure analysis, all accessions were divided into three groups: Group I, Group II and the admixed group. This study demonstrated the utility of new SSR analysis for the study of genetic diversity and population structure among 35 Perilla accessions. The GD of each locus for accessions of cultivated var. frutescens, weedy var. frutescens, cultivated var. crispa, and weedy var. crispa were 0.415, 0.606, 0.308, and 0.480, respectively. Both weedy accessions exhibited higher GD and PIC values than their cultivated types in East Asia. The new SSR primers of Perilla species reported in this study may provide potential genetic markers for population genetics to enhance our understanding of the genetic diversity, genetic relationship and population structure of the cultivated and weedy types of P. frutescens in East Asia. In addition, new Perilla SSR primers developed from RNA-seq can be used in the future for cultivar identification, conservation of Perilla germplasm resources, genome mapping and tagging of important genes/QTLs for Perilla breeding programs.