Hoy-Taek Kim

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars ‘Orin’ and ‘Akane’ using F1 seedlings derived from a cross between these cultivars. The ‘Orin’ map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the ‘Akane’ map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by ‘Akane’ are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.

Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.).

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.

Molecular characterization of novel Ty1-copia-like retrotransposons in pear (Pyrus pyrifolia)

Retrotransposons are present in all plant genomes and play important roles in genome size, genome structure remodeling, gene function, and genome evolution. Eight novel long terminal repeat retrotransposons were identified from a bacterial artificial chromosome library of Japanese pear (Pyrus pyrifolia). On the basis of the order of gene arrangement within the gag and pol domains (protease, integrase, reverse transcriptase, and RNase H), these newly identified retrotransposons appear to be closely related to Ty1-copia retrotransposons. They were designated Ppcrt1–8 and classified into two groups based on the presence or absence of a 142-amino-acid deletion within the group-specific antigen DNA-binding domain. Ppcrt1–8 were grouped with the copia-like retrotransposons RIRE1 and BARE-1 by phylogenetic analysis based on the amino acid sequences encoded by the gag and pol domains. Fluorescence in situ hybridization analysis showed that sequences homologous to Ppcrt4 were dispersed throughout more than half of the pear chromosomes. Southern blot analysis suggested that many copies of Ppcrt retrotransposons exist in the pear genome. Sequence information from these eight retrotransposons should be useful for the development of retrotransposon-based molecular marker systems in Japanese pear.

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S2S3S4), ‘Winesap’ (S1S28), ‘York Imperial’ (S2S31), ‘Stark Earliblaze’ (S1S28), and ‘Burgundy’ (S20S32), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S31 and S32, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S31 showed high similarity to S20 (94%), S32 exhibited 58% (to S24) to 76% (to S25) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S31- and S32-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.

Genome-wide expression profiling of aquaporin genes confer responses to abiotic and biotic stresses in Brassica rapa

BackgroundPlants contain a range of aquaporin (AQP) proteins, which act as transporter of water and nutrient molecules through living membranes. AQPs also participate in water uptake through the roots and contribute to water homeostasis in leaves.ResultsIn this study, we identified 59 AQP genes in the B. rapa database and Br135K microarray dataset. Phylogenetic analysis revealed four distinct subfamilies of AQP genes: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). Microarray analysis showed that the majority of PIP subfamily genes had differential transcript abundance between two B. rapa inbred lines Chiifu and Kenshin that differ in their susceptibility to cold. In addition, all BrPIP genes showed organ-specific expression. Out of 22 genes, 12, 7 and 17 were up-regulated in response to cold, drought and salt stresses, respectively. In addition, 18 BrPIP genes were up-regulated under ABA treatment and 4 BrPIP genes were up-regulated upon F. oxysporum f. sp. conglutinans infection. Moreover, all BrPIP genes showed down-regulation under waterlogging stress, reflecting likely the inactivation of AQPs controlling symplastic water movement.ConclusionsThis study provides a comprehensive analysis of AQPs in B. rapa and details the expression of 22 members of the BrPIP subfamily. These results provide insight into stress-related biological functions of each PIP gene of the AQP family, which will promote B. rapa breeding programs.

Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, ‘SCNU1154’, ‘Edisto47’, ‘MR-1’, and ‘PMR5’. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon.

Expression Profiling of MLO Family Genes under Podosphaera xanthii Infection and Exogenous Application of Phytohormones in Cucumis melo L.

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phytohormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

Identification and characterization of S-RNase genes in apple rootstock and the diversity of S-RNases in Malus species

We isolated and confirmed two S-RNases, denoted as mpS1 and mpS2, from apple rootstock ``Marubakaido`` (Malus prunifolia Borkh. Var. ringo Asami). These S-RNases contained and conserved five cysteine residues and two histidine residues, which are essential for RNase activity. The mpS1 showed high similarity to S5 (99.1%) of Malus spectabilis, whereas the mpS2 showed 99.5% nucleotide sequence similarity to S26 of (Malus × domestica) and 99.6% to S35 of (Malus sieversii) when compared with reported S-RNases. In amino acid sequences, the mpS1-RNase was almost similar to the S5-RNase of Malus spectabilis, and the mpS2-RNase was similar to the S35 of Malus sieversii, with only one bp being different from the S26-RNase of Malus × domestica. The 57 S-RNases of Malus species were renamed and rearranged containing the new S-RNases, as mprpS35 (mpS2) and mprpS57 (mpS1), for determining S-genotypes and identifying new alleles from apple species (Malus spp.).

Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance

Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbita maxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin’s 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs.

Identification and Characterization of Anthocyanin Biosynthesis-Related Genes in Kohlrabi

Kohlrabi (Brassica oleracea var. gongylodes L.) is an important vegetable of the Brassicaceae family. The main edible part of kohlrabi is the swollen stem. The purple cultivars make anthocyanin mainly in the peel of the swollen stem, while in the leaf, it is limited to the midrib, but green cultivars do not. Anthocyanins are advantageous for both plants as well as humans. Two anthocyanin compounds were detected by high pressure liquid chromatography (HPLC) only in the peel of the purple kohlrabi cultivar. Three MYBs, three bHLHs, and one WD40 TF were identified as the candidate regulatory genes in kohlrabi. There was an abundance of transcript levels for the late biosynthetic genes more specifically for BoF3′H, BoDFR, BoLDOX, and BoGST in the purple peel while scarcely detectable in other tissues for both cultivars. The expression of BoPAP2 and BoTT8 was higher in the peel of the purple cultivar than the green cultivar. The expression of BoMYBL2.2 orthologue of Arabidopsis MYBL2, a negative regulator of anthocyanins, was dramatically decreased in the purple peel. The expression of BoACO1, a key gene for ethylene biosynthesis, and BoNCED3, an important gene of the ABA pathway, was down- and upregulated, respectively, in the peel of purple kohlrabi.