Soo-Jin Kwon

Accumulation of trehalose within transgenic chloroplasts confers drought tolerance

Yeast trehalose phosphate synthase(TPS1) gene was introduced into the tobacco chloroplast ornuclear genomes to study resultant phenotypes. PCR and Southern blots confirmedstable integration of TPS1 into the chloroplast genomes ofT1, T2 and T3 transgenic plants. Northern blotanalysis of transgenic plants showed that the chloroplast transformantexpressed169-fold more TPS1 transcript than the best survivingnuclear transgenic plant. Although both the chloroplast and nuclear transgenicplants showed significant TPS1 enzyme activity, no significant trehaloseaccumulation was observed in T0/T1 nuclear transgenicplants whereas chloroplast transgenic plants showed 15–25 fold higheraccumulation of trehalose than the best surviving nuclear transgenic plants.Nuclear transgenic plants (T0) that showed even small amounts oftrehalose accumulation showed stunted phenotype, sterility and otherpleiotropiceffects whereas chloroplast transgenic plants (T1, T2,T3) showed normal growth and no pleiotropic effects. Transgenicchloroplast thylakoid membranes showed high integrity under osmotic stress asevidenced by retention of chlorophyll even when grown in 6% PEG whereaschloroplasts in untransformed plants were bleached. After 7 hrdrying, chloroplast transgenic seedlings (T1, T3)successfully rehydrated while control plants died. There was no differencebetween control and transgenic plants in water loss during dehydration butdehydrated leaves from transgenic plants (not watered for 24 days) recoveredupon rehydration turning green while control leaves dried out. Theseobservations suggest that trehalose functions by protecting biologicalmembranesrather than regulating water potential. In order to prevent escape of droughttolerance trait to weeds and associated pleiotropic traits to related crops, itmay be desirable to engineer crop plants for drought tolerance via thechloroplast genome instead of the nuclear genome.

Sequence-Level Analysis of the Diploidization Process in the Triplicated FLOWERING LOCUS C Region of Brassica rapa

Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred ∼0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.

Comparative Analysis between Homoeologous Genome Segments of Brassica napus and Its Progenitor Species Reveals Extensive Sequence-Level Divergence

Homoeologous regions of Brassica genomes were analyzed at the sequence level. These represent segments of the Brassica A genome as found in Brassica rapa and Brassica napus and the corresponding segments of the Brassica C genome as found in Brassica oleracea and B. napus. Analysis of synonymous base substitution rates within modeled genes revealed a relatively broad range of times (0.12 to 1.37 million years ago) since the divergence of orthologous genome segments as represented in B. napus and the diploid species. Similar, and consistent, ranges were also identified for single nucleotide polymorphism and insertion-deletion variation. Genes conserved across the Brassica genomes and the homoeologous segments of the genome of Arabidopsis thaliana showed almost perfect collinearity. Numerous examples of apparent transduplication of gene fragments, as previously reported in B. oleracea, were observed in B. rapa and B. napus, indicating that this phenomenon is widespread in Brassica species. In the majority of the regions studied, the C genome segments were expanded in size relative to their A genome counterparts. The considerable variation that we observed, even between the different versions of the same Brassica genome, for gene fragments and annotated putative genes suggest that the concept of the pan-genome might be particularly appropriate when considering Brassica genomes.

Mapping quantitative trait loci for yield components and morphological traits in an advanced backcross population between Oryza grandiglumis and the O. sativa japonica cultivar Hwaseongbyeo

Introgression has been achieved from wild species Oryza grandiglumis (2n=48, CCDD, Acc. No. 101154) into O. sativa subsp. japonica cv. Hwaseongbyeo as a recurrent parent. An advanced introgression (backcross) line, HG101, produced from a single plant from BC5F3 families resembled Hwaseongbyeo, but it showed differences from Hwaseongbyeo in several traits, including days to heading and culm length. To detect the introgressions, 450 microsatellite markers of known chromosomal position were used for the parental survey. Of the 450 markers, 51 (11.3%) detected O. grandiglumis segments in HG101. To characterize the effects of alien genes introgressed into HG101, an F2:3 population (150 families) from the cross Hwaseongbyeo/HG101 was developed and evaluated for 13 agronomic traits. Several lines outperformed Hwaseongbyeo in several traits, including days to heading. Genotypes were determined for 150 F2 plants using simple sequence repeat markers. Qualitative trait locus (QTL) analysis was carried out to determine the relationship between marker genotype and the traits evaluated. A total of 39 QTL and 1 gene conferring resistance to blast isolate were identified using single-point analysis. Phenotypic variation associated with each QTL ranged from 4.2 to 30.5%. For 18 (46.2%) of the QTL identified in this study, the O. grandiglumis-derived alleles contributed a desirable agronomic effect despite the overall undesirable characteristics of the wild phenotype. Favorable wild alleles were detected for days to heading, spikelets per panicle, and grain shape traits. Grain shape QTL for grain weight, thickness, and width identified in the F2:3 lines were further confirmed based on the F4 progeny test. The confirmed locus, tgw2 for grain weight is of particular interest because of its independence from undesirable height and maturity. Several QTL controlling amylose content and grain traits have not been detected in the previous QTL studies between Oryza cultivars, indicating potentially novel alleles from O. grandiglumis. The QTL detected in this study could be a rich source of natural genetic variation underlying the evolution and breeding of rice.

Genome-wide identification of glucosinolate synthesis genes in Brassica rapa.

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate‐derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa, we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B. rapa. Glucosinolate genes in B. rapa share 72–94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers. The exon/intron split pattern of B. rapa is almost identical to that of Arabidopsis, although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B. rapa, whereas only three were found in Arabidopsis. The expression of those paralogs was not tissue‐specific but varied greatly depending on B. rapa tissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation in B. rapa appears to be more complex than that of Arabidopsis. With the isolation and further characterization of the endogenous genes, health‐beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.

The Korea Brassica Genome Project: A glimpse of the Brassica genome based on comparative genome analysis with Arabidopsis

A complete genome sequence provides unlimited information in the sequenced organism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequencing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa. We have selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. Among them, 30 BAC clones have been sequenced and 18 are on the way. Comparative genome analyses of the EST sequences and sequenced BAC clones from Brassica chromosome 1 revealed their homeologous partner regions on the Arabidopsis genome and a syntenic comparative map between Brassica chromosome 1 and Arabidopsis chromosomes. In silico chromosome walking and clone validation have been successfully applied to extending sequence contigs based on the comparative map and BAC end sequences. In addition, we have defined the (peri)centromeric heterochromatin blocks with centromeric tandem repeats, rDNA and centromeric retrotransposons. In-depth sequence analyses of five homeologous BAC clones and an Arabidopsis chromosomal region reveal overall co-linearity, with 82% sequence similarity. The data indicate that the Brassica genome has undergone triplication and subsequent gene losses after the divergence of Arabidopsis and Brassica. Based on in-depth comparative genome analyses, we propose a comparative genomics approach for conquering the Brassica genome. In 2005 we intend to construct an integrated physical map, including sequence information from 500 BAC clones and integration of fingerprinting data and end sequence data of more than 100 000 BAC clones. The sequences have been submitted to GenBank with accession numbers: 10 204 BAC ends of the KBrH library (CW978640–CW988843); KBrH138P04, AC155338; KBrH117N09, AC155337; KBrH097M21, AC155348; KBrH093K03, AC155347; KBrH081N08, AC155346; KBrH080L24, AC155345; KBrH077A05, AC155343; KBrH020D15, AC155340; KBrH015H17, AC155339; KBrH001H24, AC155335; KBrH080A08, AC155344; KBrH004D11, AC155341; KBrH117M18, AC146875; KBrH052O08, AC155342.

Complexity of genome evolution by segmental rearrangement in Brassica rapa revealed by sequence-level analysis

BackgroundThe Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level.ResultsWe developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events.ConclusionOur results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions of genes in B. rapa based on the locations of orthologues in A. thaliana can be misleading. Our results will be of relevance to a wide range of plants that have polyploid genomes, many of which are being considered according to a paradigm of comprising conserved synteny blocks with respect to sequenced, related genomes.

Progress in Understanding and Sequencing the Genome of Brassica rapa

Brassica rapa, which is closely related to Arabidopsis thaliana, is an important crop and a model plant for studying genome evolution via polyploidization. We report the current understanding of the genome structure of B. rapa and efforts for the whole-genome sequencing of the species. The tribe Brassicaceae, which comprises ca. 240 species, descended from a common hexaploid ancestor with a basic genome similar to that of Arabidopsis. Chromosome rearrangements, including fusions and/or fissions, resulted in the present-day “diploid” Brassica species with variation in chromosome number and phenotype. Triplicated genomic segments of B. rapa are collinear to those of A. thaliana with InDels. The genome triplication has led to an approximately 1.7-fold increase in the B. rapa gene number compared to that of A. thaliana. Repetitive DNA of B. rapa has also been extensively amplified and has diverged from that of A. thaliana. For its whole-genome sequencing, the Brassica rapa Genome Sequencing Project (BrGSP) consortium has developed suitable genomic resources and constructed genetic and physical maps. Ten chromosomes of B. rapa are being allocated to BrGSP consortium participants, and each chromosome will be sequenced by a BAC-by-BAC approach. Genome sequencing of B. rapa will offer a new perspective for plant biology and evolution in the context of polyploidization.

Major repeat components covering one-third of the ginseng (Panax ginseng C.A. Meyer) genome and evidence for allotetraploidy

Ginseng (Panax ginseng) is a famous medicinal herb, but the composition and structure of its genome are largely unknown. Here we characterized the major repeat components and inspected their distribution in the ginseng genome. By analyzing three repeat-rich bacterial artificial chromosome (BAC) sequences from ginseng, we identified complex insertion patterns of 34 long terminal repeat retrotransposons (LTR-RTs) and 11 LTR-RT derivatives accounting for more than 80% of the BAC sequences. The LTR-RTs were classified into three Ty3/gypsy (PgDel, PgTat and PgAthila) and two Ty1/Copia (PgTork and PgOryco) families. Mapping of 30-Gbp Illumina whole-genome shotgun reads to the BAC sequences revealed that these five LTR-RT families occupy at least 34% of the ginseng genome. The Ty3/Gypsy families were predominant, comprising 74 and 33% of the BAC sequences and the genome, respectively. In particular, the PgDel family accounted for 29% of the genome and presumably played major roles in enlargement of the size of the ginseng genome. Fluorescence in situ hybridization (FISH) revealed that the PgDel1 elements are distributed throughout the chromosomes along dispersed heterochromatic regions except for ribosomal DNA blocks. The intensity of the PgDel2 FISH signals was biased toward 24 out of 48 chromosomes. Unique gene probes showed two pairs of signals with different locations, one pair in subtelomeric regions on PgDel2-rich chromosomes and the other in interstitial regions on PgDel2-poor chromosomes, demonstrating allotetraploidy in ginseng. Our findings promote understanding of the evolution of the ginseng genome and of that of related species in the Araliaceae.

Sequence-level comparative analysis of the Brassica napus genome around two stearoyl-ACP desaturase loci.

We conducted a sequence-level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl-ACP desaturase-encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (approximately 56%) is lower than has been reported previously between A. thaliana and Brassica (approximately 66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 +/- 3.9% and 85.8 +/- 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 +/- 3.1% between the B. napus A genome and B. rapa, and 93.1 +/- 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.