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Citrus unshiu Marc. cv. Miyagawa-wase is the most widely cultivated citrus variety in Korea. To determine whether the C. unshiu genome used in this study shows genetic variation compared to the published reference genome (C. unshiu Marc. Miyagawa-wase), we conducted genome re-sequencing of two C. unshiu cultivars (Miyagawa-1 and Miyagawa-2) cultivated on Jeju in Korea. Compared with the reference genome, 1,198,650 and 1,207,084 single-nucleotide polymorphisms (SNPs) and 172,259 and 172,391 insertion/deletion polymorphisms (InDels) were detected in the Miyagawa-1 and -2 genomes, respectively. In SNP and InDel classifications by genome annotation, 367,591 and 369,068 SNPs and 45,362 and 45,464 InDels were located in the genic regions of Miyagawa-1 and -2, respectively. Among the SNPs of Miyagawa-1 and -2, transitions were more frequent than transversions. The majority of InDels was distributed in 1-bp InDels in both cultivars. The comparative number of total SNPs between Miyagawa-1 and -2 was smaller than the number of SNPs between the reference genome and Miyagawa-1 or -2. Gene ontology (GO) analysis showed that 23,164 and 23,049 genes with SNPs and 16,830 and 16,774 genes with InDels were annotated in the GO database. Taken together, Miyagawa-1 and -2 show genome-wide variation, including SNPs and InDels, compared to the published C. unshiu Marc. cv. Miyagawa-wase genome. This study suggests it would be more accurate to use the Miyagawa-1 and -2 genome sequences as a reference when conducting research using C. unshiu cultivated in Korea.

알로에는 화장품 및 약용 식물로 널리 사용되고 있는 식물로서 알로에 젤 추출물에서 항산화, 항암, 항 당뇨, 면역, 미백 등 여러 효과가 보고되었지만, 알로에 가공에서 발생하는 부산물을 이용한 연구는 많이 수행되지 않았다. 본 연구에서는 알로에 가공 과정에서 나온 부산물을 이용한 추출물이 기능성 바이오 소재로 활용될 수 있는지를 조사하였다. 먼저 믹서 추출물과 착즙 추출물 모두에서 세포 독성은 보이지 않았다. 3T3-L1 지방세포 분화의 억제는 믹서 추출물에서만 확인되었다. 믹서 추출물의 처리 농도에 따라 히알루론산 축적 및 Tyrosinase억제가 증가하는 것을 확인하였다. 믹서 추출물의 항균 활성은 Porphyromonas gingivalis 균주에서 관찰되었지만, Streptococcus mutans 균주에서는 관찰되지 않았다. DPPH 및 SOD 분석을 통한 항산화 활성은 믹서 추출물의 농도에 따라 증가하였다. 본 연구를 통해 알로에 부산물의 믹서 추출물은 지방 세포 분화억제와 보습, 미백, 항산화 효과가 있음을 확인하였으며, 건강 음료나 미용 마스크 등의 기능성 바이오 소재로 활용이 가능한 것을 시사하고 있다.

Satsuma mandarin (

Aloe is widely used as a cosmetic and medicinal plant. Numerous studies have reported that aloe gel extract has antioxidant, anticancer, antidiabetic, immunity, and skin antiaging properties. However, few studies have investigated the properties of fermentation products of aloe processing byproducts. Aloe stalks and leaves remain as byproducts after the aloe beverage manufacturing process. This study evaluated whether fermentation products of blender and press extracts of aloe processing byproducts (BF and PF, respectively) that remain after beverage manufacturing were useful as functional biomaterial by investigating their effects on adipocyte differentiation, hyaluronic acid (HA) production, tyrosinase activity, and antioxidant activity. Co-fermentation of G. xylinus and S. cerevisiae was conducted for fermentation of aloe processing byproducts. The BF and PF products did not induce observable cytotoxicity effects. However, BF and PF products did inhibit a 3T3-L1 adipocyte differentiation compared with control, with the BF product displaying greater inhibition of 3T3-L1 adipocyte differentiation than the PF product. HA production increased in HaCaT cell cultures as the concentration of the MF product increased, as compared with the untreated control. The levels of tyrosinase inhibition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and superoxide dismutase (SOD)-like activity also depended on the MF product concentration. This study indicates that the fermented products of aloe processing byproducts have biological potential for applications in the manufacturing of cosmetics, pharmaceuticals, and beverages. These laboratory bench results provide the foundation for future studies of scaling and practical applications at the industrial level.

Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.

Citrus fruits are rich sources of different phytochemicals for human health due to their high anti-oxidant capacity. However, the anti-aging effect of citrus fruits has not been well understood. In this study, methanol extracts taken at various developmental stages from tissues of Citrus unshiu was used to investigate its anti-aging effect by an elastase/collagenase inhibition assay, and a gas chromatography-mass spectrometry (GC-MS) analysis was carried out to identify the potential anti-aging compositions. The elastase/collagenase inhibitory activity was greatest in the flesh of immature green fruit (i.e., early July flesh (EJF)), and four candidate compounds were selected by GC-MS and evaluated by a collagenase inhibition assay. Three of the four candidate compounds (heptadecanoic acid, D-allose, and 5-hydroxymethyl-2-furaldehyde (HMF)) showed anti-aging activity, and the activity was highest in heptadecanoic acid, followed by D-allose and HMF. The total phenolic content (TPC), total flavonoid content (TFC), and anti-oxidant activity (DPPH and ferric reducing anti-oxidant power (FRAP) assay) were also investigated. Interestingly, the patterns of the total phenolic/flavonoid content and the anti-oxidant activity were different from that of the elastase/collagenase inhibitory activity. Flowers had the most anti-oxidant activity followed by immature fruit, and the fruit peels had more anti-oxidant activity than its flesh at all stages of development. This study demonstrated that the flesh of immature fruit and flowers of C. unshiu could be sources of anti-aging and anti-oxidant agents for human health, respectively.

A novel disposable all-printed electronic biosensor is proposed for a fast detection and classification of bacteria. This biosensor is applied to classify three types of popular pathogens: Salmonella typhimurium, and the Escherichia coli strains JM109 and DH5-α. The proposed sensor consists of inter-digital silver electrodes fabricated through an inkjet material printer and silver nanowires uniformly decorated on the electrodes through the electrohydrodynamic technique on a polyamide based polyethylene terephthalate substrate. The best sensitivity of the proposed sensor is achieved at 200 µm teeth spaces of the inter-digital electrodes along the density of the silver nanowires at 30 × 103/mm2. The biosensor operates on ±2.5 V and gives the impedance value against each bacteria type in 8 min after sample injection. The sample data are measured through an impedance analyzer and analyzed through pattern recognition methods such as linear discriminate analysis, maximum likelihood, and back propagation artificial neural network to classify each type of bacteria. A perfect classification and cross-validation is achieved by using the unique fingerprints extracted from the proposed biosensor through all the applied classifiers. The overall experimental results demonstrate that the proposed disposable all-printed biosensor is applicable for the rapid detection and classification of pathogens.

Chrysanthemum is one of the most popular and economically important ornamental plants due to its huge diversity in growing habits, wide range of colors, and different patterns of flower. In the present study, we conducted the karyotype analysis in four naturally occurring genotypes of Chrysanthemum boreale. Karyotype studies based on the fluorescence in situ hybridization (FISH) using 5S and 45S rDNAs. Four chrysanthemum genoteyps showed an aneuploid chromosome number of 2n=18+2 (111016 and 111021) or a diploid of 2n=18 (121001 and 121002). All the genoteyps had the same karyotype formula of 14 metacentrics and 4 submetacentrics. In 111016, the chromosome length during somatic metaphase ranged from 3.11 ± 0.26 ㎛ (shortest) to 3.94 ± 0.20 ㎛ (longest), with a total length of 32.94 ㎛. The chromosome length at the mitotic metaphase ranged from 3.11 to 6.46 μm, with a total length of 32.94 ㎛ in 111016 and 51.05, 32.81, and 46.00 ㎛ in 111021, 121001, and 121002, respectively. The 5S rDNA and 45S rDNA signals recorded different in all four wildly grown genotypes of C. boreale. This information can be useful in cultivar improvement, as well as in elucidation of the evolution of the chrysanthemum.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identification of aDart in pyl-v was first performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an efficient transposon-tagging system in rice are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00438-008-0410-x) contains supplementary material, which is available to authorized users.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

In contrast to angiosperms, some gymnosperms form well-development suspensors during somatic embryogenesis. This feature is highly useful to studies of suspensor biology. In cell cultures of Japanese larch ( Larix leptolepis Gordon), somatic embryos rarely formed when the initial cell density was lower than 0.1 ml packed cell volume (PCV) l −1 . However, in the presence of phytosulfokine (PSK), a peptidyl plant growth factor, mitotic activity during somatic embryogenesis was stimulated, even when the cell density was lower than 0.1 ml PCV l −1 , particularly, the development of the suspensor. Manual separation of somatic embryos into the embryo proper and the suspensor arrested further development, but when embryos proper lacking a suspensor were treated with PSK, the suspensor regenerated and development proceeded to maturity. Because no division of the suspensor cells was observed, the cells of the regenerated suspensor probably originated from basal cells of the embryo proper. In contrast, PSK treatment of suspensors lacking the embryo proper did not stimulate further development. These results indicate that PSK stimulates not only cell division of embryo proper but also development of the suspensor.

In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.

Phytosulfokine-α (PSK-α), a peptidyl plant growth factor that was originally isolated from conditioned medium derived from asparagus mesophyll cell culture, stimulates cell proliferation in some plants in suspension culture. However, the physiological relationship between phytohormones and this growth factor in plants is not well understood. Using carrot ( Daucus carota L.) non-embryogenic cell culture, we investigated the stimulatory effects of PSK-α on the cell proliferation induced by different combinations and concentrations of auxin [2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), or naphthaleneacetic acid (NAA)] and PSK-α. Cell proliferation was not stimulated by PSK-α at high auxin concentrations, but was promoted at low auxin concentrations. There was no proliferation in the absence of auxin. These results indicate that PSK-α stimulates cell proliferation when cell division activity is low, and that auxin is required for this process. We also evaluated PSK-α production using medium derived from non-embryogenic cell culture with high or low concentrations of auxin (2,4-D, IAA or NAA) in a competition ELISA system. More PSK-α was produced at high auxin concentrations than at low auxin concentrations. These results suggest that auxin participates in PSK-α production.

Expression of carrot embryogenic cell specific protein genes (DcECPs) that are late-embryogenesis abundant proteins is ABA-inducible and embryo-specific. The expression of some DcECPs is controlled by an embryo-specific transcription factor C-ABI3, a carrot homologue of Arabidopsis ABI3. In order to understand the molecular mechanisms regulating ABA-inducible and embryo-specific expression of DcECP genes, we carried out a detailed analysis of DcECP31 promoter with deletion analysis, base-substitution mutagenesis and electrophoretic mobility shift assay (EMSA). We identified two important elements in the promoter of DcECP31, motif X (CACACGTGGG) and motif Y (CACACGTATC), which are sufficient for the embryo-specific and ABA-inducible promoter activity. By precise EMSA analysis, it was demonstrated that a nuclear protein which has sequence specific-binding ability binds to the flanking ACGT core motifs.

Expression of carrot ECP (DcECP) genes encoding late-embryogenesis abundant proteins is embryo-specific and ABA-inducible. The expression is regulated by C-ABI3, a carrot homologue of Arabidopsis ABI3. To understand the molecular mechanisms controlling ABA-inducible and embryo-specific gene expression, we compared and analyzed the promoter regions of some ECP genes. The accumulation of DcECPs mRNA in response to ABA was not inhibited by an inhibitor of protein synthesis, cycloheximide. These results indicate that the ABA-inducible expression of the DcECPs did not require de novo protein synthesis. Sequence comparison among some ECP gene promoters revealed that the promoters contained several conserved motifs including ABRE (ABA responsive element)-like ACGT core motifs, and an Sph box (CATGCATG), which has been identified as a motif mediating gene activation of maize antocyanin regulatory gene C1. To investigate the promoter activity of DcECP31 promoter region, we carried out a deletion analysis with a transient assay system using protoplasts of embryogenic cells or with a transformation system using embryogenic cells of carrot. We found that the -250bp upstream region of the DcECP31 promoter is sufficient for embryo-specific and ABA-inducible promoter activity. Following deletion analysis of DcECP40 promoter, we clarified that the distal (-670∼-390) and proximal regions (-140∼-50), are essential for the ABA-inducible expression.

Cells in culture with a high cell density can proliferate in medium that contains phytohormones, but cells at low Phytosulphokine-a (PSK-a) is the first chemically char- cell density cannot. However, cells at a low cell density acterized peptide that acts as a plant growth factor. can grow in the presence of conditioned medium (Stuart It stimulates the proliferation of asparagus and rice and Street, 1969; Somers et al., 1987; Birnberg et al., cells, but no information is yet available on its effects 1988; Bellincampi and Morpurgo, 1987, 1989; Huang on plant morphogenesis. The effects of PSK-a on et al., 1990; Bellincampi et al., 1993). None of the somatic embryogenesis in carrot (Daucus carota L.) phytohormones characterized to date is able to substitute were examined. PSK-a, when added to the induction for the conditioned medium. Thus, it was postulated medium for somatic embryogenesis, increased the that certain factors accumulate in conditioned medium number of somatic embryos. The chemical analogues and promote the division of cells cultured at low cell [2‐5]PSK-a and tyrosine sulphate ester (Tyr-SO 3 H), density. Matsubayashi and Sakagami (1996) isolated which have been used as negative controls in other phytosulphokine-a (PSK-a) as such a factor from the systems, had no effect. Moreover, the proliferation conditioned medium of a culture of mesophyll cells of of cells during somatic embryogenesis was also Asparagus oYcinalis L. PSK-a was determined to be a enhanced by PSK-a. These results indicate that PSK- sulphated pentapeptide: H-Tyr(SO

Haptoglobin, the product of the gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, and , and 1 minor allele, . Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing—phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring . The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases.