Genome-scale signatures of adaptive gene expression changes in an invasive seaweed Gracilaria vermiculophylla.

Invasive species can successfully and rapidly colonize new niches and expand ranges via founder effects and enhanced tolerance towards environmental stresses. However, the underpinning molecular mechanisms (i.e., gene expression changes) facilitating rapid adaptation to harsh environments are still poorly understood. The red seaweed Gracilaria vermiculophylla, which is native to the northwest Pacific but invaded North American and European coastal habitats over the last 100 years, provides an excellent model to examine whether enhanced tolerance at the level of gene expression contributed to its invasion success. We collected G. vermiculophylla from its native range in Japan and from two non-native regions along the Delmarva Peninsula (Eastern United States) and in Germany. Thalli were reared in a common garden for 4 months at which time we performed comparative transcriptome (mRNA) and microRNA (miRNA) sequencing. MRNA-expression profiling identified 59 genes that were differently expressed between native and non-native thalli. Of these genes, most were involved in metabolic pathways, including photosynthesis, abiotic stress, and biosynthesis of products and hormones in all four non-native sites. MiRNA-based target-gene correlation analysis in native/non-native pairs revealed that some target genes are positively or negatively regulated via epigenetic mechanisms. Importantly, these genes are mostly associated with metabolism and defence capability (e.g., metal transporter Nramp5, senescence-associated protein, cell wall-associated hydrolase, ycf68 protein and cytochrome P450-like TBP). Thus, our gene expression results indicate that resource reallocation to metabolic processes is most likely a predominant mechanism contributing to the range-wide persistence and adaptation of G. vermiculophylla in the invaded range. This study, therefore, provides molecular insight into the speed and nature of invasion-mediated rapid adaption.

Intraspecific genetic variation matters when predicting seagrass distribution under climate change.

Seagrasses play a vital role in structuring coastal marine ecosystems, but their distributional range and genetic diversity have declined rapidly in recent decades. To improve conservation of seagrass species, it is important to predict how climate change may impact their ranges. Such predictions are typically made with correlative species distribution models (SDMs), which can estimate a species' potential distribution under present and future climatic scenarios given species' presence data and climatic predictor variables. However, these models are typically constructed with species-level data, and thus ignore intraspecific genetic variability, which can give rise to populations with adaptations to heterogeneous climatic conditions. Here, we explore the link between intraspecific adaptation and niche differentiation in Thalassia hemprichii, a seagrass broadly distributed in the tropical Indo-Pacific Ocean and a crucial provider of habitat for numerous marine species. By retrieving and re-analysing microsatellite data from previous studies, we delimited two distinct phylogeographical lineages within the nominal species and found an intermediate level of differentiation in their multidimensional environmental niches, suggesting the possibility for local adaptation. We then compared projections of the species' habitat suitability under climate change scenarios using species-level and lineage-level SDMs. In the Central Tropical Indo-Pacific region, models for both levels predicted considerable range contraction in the future, but the lineage-level models predicted more severe habitat loss. Importantly, the two modelling approaches predicted opposite patterns of habitat change in the Western Tropical Indo-Pacific region. Our results highlight the necessity of conserving distinct populations and genetic pools to avoid regional extinction due to climate change and have important implications for guiding future management of seagrasses.

An underlying mechanism of qE deficiency in marine angiosperm Zostera marina.

Non-photochemical quenching (NPQ) of photosystem II (PSII) fluorescence is one of the most important protective mechanisms enabling the survival of phototropic organisms under high-light conditions. A low-efficiency NPQ, characterized by weak NPQ induction capacity and a low level of protective NPQ, was observed in the marine angiosperm Zostera marina, which inhabits the shallow water regions. Furthermore, chlorophyll fluorescence and Western blot analysis verified that the fast-inducted component of NPQ, i.e., the energy-dependent quenching (qE), was not present in this species. In contrast with the lack of PSII antenna quenching sites for qE induction in brown algae and the lack of functional XC in Ulvophyceae belonging to green algae, all the antenna proteins and the functional XC are present in Z. marina. A novel underlying mechanism was observed that the limited construction of the trans-thylakoid proton gradient (ΔpH) caused by photoinactivation of the oxygen evolving complex (OEC) did not induce protonation of PsbS, thus explaining the inability to form quenching sites for qE induction. Although the ΔpH established under light exposure activated violaxanthin (V) de-epoxidase enzyme to catalyze conversion of V via antheraxanthin (A) and then to zeaxanthin (Z), the quenching capacity of de-epoxidized pigment was weak in Z. marina. We suggest that the low-efficiency NPQ was conducive to efficiently utilize the limited electrons to perform photosynthesis, resisting the adverse effect of OEC photoinactivation on the photosynthetic rate.

Chlororespiration Serves as Photoprotection for the Photo-Inactivated Oxygen-Evolving Complex in Zostera marina, a Marine Angiosperm.

As an alternative electron sink, chlororespiration, comprising the NAD(P)H dehydrogenase complex and plastid terminal plastoquinone oxidase, may play a significant role in sustaining the redox equilibrium between stroma and thylakoid membrane. This study identified a distinct role for chlororespiration in the marine angiosperm Zostera marina, whose oxygen-evolving complex (OEC) is prone to photo-inactivation as a result of its inherent susceptibility to excess irradiation. The strong connectivity between OEC peripheral proteins and key chlororespiratory enzymes, as demonstrated in the interaction network of differentially expressed genes, suggested that the recovery of photo-inactivated OEC was connected with chlororespiration. Chlorophyll fluorescence, transcriptome and Western blot data verified a new physiological role for chlororespiration to function as photoprotection and generate a proton gradient across the thylakoid membrane for the recovery of photo-inactivated OEC. Chlororespiration was only activated in darkness following excess irradiation exposure, which might be related to electron deficiency in the electron transport chain because of the continuous impairment of the OEC. The activation of chlororespiration in Z. marina was prone to proactivity, which was also supported by the further activation of the oxidative pentose-phosphate pathway synthesizing NADPH to meet the demand of chlororespiration during darkness. This phenomenon is distinct from the common assumption that chlororespiration is prone to consuming redundant reducing power during the short transition phase from light to dark.

The highly efficient NDH-dependent photosystem I cyclic electron flow pathway in the marine angiosperm Zostera marina.

Zostera marina, a fully submerged marine angiosperm with a unique evolutionary history associated with its terrestrial origin, has distinct photochemical characteristics caused by its oxygen-evolving complex (OEC) being prone to deactivation in visible light. Based on the present phylogenetic analysis, the chloroplast NADPH dehydrogenase-like (NDH) complex was found to be completed in of Z. marina, unlike other marine plants, suggesting its crucial role. Thus, the responses of electron transport to irradiation were investigated through multiple chlorophyll fluorescence techniques and Western blot analysis. Moreover, the respective contribution of the two photosystem I cyclic electron flow (PSI-CEF) pathways to the generation of trans-thylakoid proton gradient (∆pH) was also examined using inhibitors. The contributions of the two PSI-CEF pathways to ∆pH were similar; furthermore, there was a trade-off between the two pathways under excess irradiation: the PGR5/L1-dependent PSI-CEF decreased gradually following its activation during the initial illumination, while NDH-dependent PSI-CEF was activated gradually with exposure duration. OEC inactivation was continuously under excess irradiation, which exhibits a positive linear correlation with the activation of NDH-dependent PSI-CEF. We suggest that PGR5/L1-dependent PSI-CEF was preferentially activated to handle the excess electron caused by the operation of OEC during the initial illumination. Subsequently, the increasing OEC inactivation with exposure duration resulted in a deficit of electrons. Limited electrons from PSI might preferentially synthesize NADPH, which could support the function of NDH-dependent PSI-CEF to generate ∆pH and ATP via reducing ferredoxin, thereby maintaining OEC stability.

Oncoprotein HBXIP induces PKM2 via transcription factor E2F1 to promote cell proliferation in ER-positive breast cancer.

We have reported that hepatitis B X-interacting protein (HBXIP, also termed LAMTOR5) can act as an oncogenic transcriptional co-activator to modulate gene expression, promoting breast cancer development. Pyruvate kinase muscle isozyme M2 (PKM2), encoded by PKM gene, has emerged as a key oncoprotein in breast cancer. Yet, the regulatory mechanism of PKM2 is still unexplored. Here, we report that HBXIP can upregulate PKM2 to accelerate proliferation of estrogen receptor positive (ER+) breast cancer. Immunohistochemistry analysis using breast cancer tissue microarray uncovered a positive association between the expression of HBXIP and PKM2. We also discovered that PKM2 expression was positively related with HBXIP expression in clinical breast cancer patients by real-time PCR assay. Interestingly, in ER+ breast cancer cells, HBXIP was capable of upregulating PKM2 expression at mRNA and protein levels in a dose-dependent manner, as well as increasing the activity of PKM promoter. Mechanistically, HBXIP could stimulate PKM promoter through binding to the -779/-579 promoter region involving co-activation of E2F transcription factor 1 (E2F1). In function, cell viability, EdU, colony formation, and xenograft tumor growth assays showed that HBXIP contributed to accelerating cell proliferation through PKM2 in ER+ breast cancer. Collectively, we conclude that HBXIP induces PKM2 through transcription factor E2F1 to facilitate ER+ breast cancer cell proliferation. We provide new evidence for the mechanism of transcription regulation of PKM2 in promotion of breast cancer progression.

Magnetic ring anastomosis of suprahepatic vena cava: novel technique for liver transplantation in rat.

To improve the technique of suprahepatic vena cava (SHVC) reconstruction in rat OLT, novel magnetic rings were designed and manufactured to facilitate reconstruction of SHVC and shorten the anhepatic time. One-hundred and twenty adult male Wistar rats were randomly divided into two groups: rings group (n = 30), using magnetic rings for SHVC reconstruction; suture group (n = 30), 7/0 prolene suture was used for SHVC running anastomosis as control. Cuff techniques were used for portal vein and infrahepatic vena cava reconstruction as Kamada and Calne described. The bile duct was reconnected with a stent. The hepatic re-arterialization was omitted. In the rings group, the SHVC reconstruction took 0.91 ± 0.24 (mean ± SD) min; the anhepatic phase and the recipient operation time were 5.63 ± 0.65 min and 36.02 ± 8.02 min, respectively. In suture group, the anastomotic time of SHVC was 10.40 ± 2.11 min; the anhepatic phase and the recipient operation time were 17.76 ± 2.51 and 49.38 ± 12.06 min, respectively, and there was statistically significant difference between the two groups. The ALT levels reached peak at 24 h post-OLT (186.2 ± 32.5 IU/l) and restored to normal level at 96 h gradually. In the rings group, 29 of 30 rats survived at day 7 and 28 of 30 rats survived at day 30. In contrast, only 25 of 30 recipients in suture group remained alive at day 7 and 22 of 30 remained alive at day 30 (P < 0.05). Better anastomotic healing was founded in rings group by pathology and scanning electron microscope. The magnetic rings technique provides a novel, simple method for SHVC reconstruction of OLT in rat. It significantly shortens anhepatic phase, while the success rate of the operation is satisfactory.

Antitumor effects of new glycoconjugated PtII agents dual-targeting GLUT1 and Pgp proteins.

A novel and highly efficient dual-targeting PtII system was designed to improve the drug delivery capacity and selectivity in cancer treatment. The dual-targeting monofunctional PtII complexes (1-8) having glycosylated pendants as tridentated ligand were achieved by introducing glycosylation modification in the thioaminocarbazone compounds with potential lysosomal targeting ability. The structures and stability of 1-8 were further established by various techniques. Molecular docking studies showed that 2 was efficiently docked into glucose transporters protein 1 (GLUT1) and P-glycoprotein (Pgp) proteins with the optimal CDocker-interaction-energy of -64.84 and -48.85 kcal mol-1. Complex 2 with higher protein binding capacity demonstrated significant and broad-spectrum antitumor efficacy in vitro, even exhibiting a half maximal inhibitory concentration (IC50) value (∼10 µM) than cisplatin (∼17 µM) against human lung adenocarcinoma cells (A549). The inhibitor experiment revealed GLUT-mediated uptake of 2, and the subcellular localization experiment in A549 also proved that 2 could be localized in the lysosome, thereby causing cell apoptosis. Moreover, cellular thermal shift assay (CETSA) confirmed the binding of 2 with the target proteins of GLUT1 and Pgp. The above results indicated that 2 represents a potential anticancer candidate with dual-targeting functions.

Photoinactivation of the oxygen-evolving complex regulates the photosynthetic strategy of the seagrass Zostera marina.

Zostera marina, a widespread seagrass, evolved from a freshwater ancestor of terrestrial monocots and successfully transitioned into a completely submerged seagrass. We found that its oxygen-evolving complex (OEC) was partially inactivated in response to light exposure, as evidenced by both the increment of the relative variable fluorescence at the K-step and the downregulation of the OEC genes and proteins. This photosynthetic regulation was further addressed at both proteome and physiology levels by an in vivo study. The unchanged content of the ΔpH sensor PsbS protein and the non-photochemical quenching induction dynamics, described by a single exponential function, verified the absence of the fast qE component. Contents and activities of chlororespiration, Mehler reaction, malic acid synthesis, and photorespiration key enzymes were not upregulated, suggesting that alternative electron flows remained unactivated. Furthermore, neither significant production of singlet oxygen nor increment of total antioxidative capacity indicated that reactive oxygen species were not produced during light exposure. In summary, these low electron consumptions may allow Z. marina to efficiently use the limited electrons caused by partial OEC photoinactivation to maintain a normal carbon assimilation level.

Interaction of high seawater temperature and light intensity on photosynthetic electron transport of eelgrass (Zostera marina L.).

The interaction of widely recognized causes of eelgrass decline (high seawater temperature and limited light intensity) on photosynthetic electron transport was investigated via chlorophyll fluorescence technique. High seawater temperature combined light intensity significantly increasing the relative maximum electron transport rate (rETRmax); at critical temperature of 30 °C, the rETRmax increased with the enhancement of light intensity, indicating the elevation of overall photosynthetic performance. Based on the magnitude of effect size (η2), light intensity was the predominant factor affecting the performance index (PIABS), indicating that photosystem II (PSII) was sensitive to light intensity. Moreover, the donor side was severely damaged as evidenced by the higher decrease amplitude of fast component and its subsequent incomplete recovery. The reaction center exhibited limited flexibility due to the slight decrease amplitude in maximum photochemical quantum yield. In contrast with PSII, photosystem I (PSI) was more sensitive to high seawater temperature, based on the magnitude of η2 derived from the maximal decrease in slope. High seawater temperature significantly increased PSI activity, plastoquinol reoxidation capacity, and probability for electron transfer to final PSI electron acceptors. Moreover, it combined elevated light intensity significantly stimulated the activity of cyclic electron flow (CEF) around PSI. Higher activity of both PSI and CEF contributed to balancing the linear electron transport via alleviating the over-reduction of the plastoquinone pool, exhibiting flexible regulation of photosynthetic electron transport at critical temperature. Therefore, limited light intensity decreased the tolerance of eelgrass to critical temperature, which might be a factor contributing factor in the observed decline.

Cryopreservation of gametophytes of Laminaria japonica (phaeophyta) with two-step cooling: interactions between variables related to post-thaw survival.

Little attention has been given to the effect of interactions between different cryogenic parameters on the viability of cryopreserved algae. Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen by two-step cooling, and interactions were tested for optimizing the cooling protocol and improving freeze-tolerance. UNOVA of a general linear model suggested that interactions between both cooling rate and holding time and between holding temperature and holding time significantly affected the survival of thawed gametophytes. Based on the magnitude of effect, the importance order of factors was found to be: holding temperature, holding time, cooling rate, cooling rate x holding temperature, holding temperature x holding time. UNOVA also suggested significant main effects of pre-culture conditions and sex on survival of thawed gametophytes. Under the optimal procedure, post-thaw survivals obtained were 84 percent for male and 69 percent for female gametophytes. Male gametophytes were observed to be more tolerant to the whole procedure of cryopreservation than females. Following thawing viable gametophytes could grow asexually or give rise to morphologically normal sporophytes.

Seasonal dynamics of photosynthetic activity in the representive brown macroalgae Sagrassum thunbergii (Sargassaceae Phaeophyta).

The present study evaluates the seasonal photosynthetic performances of Sargassum thunbergii via chlorophyll fluorescence technique. During summer and early winter, no significant change was observed in maximum photochemical efficiency (Fv/Fm), and performance index (PIabs). During late winter and early spring, Fv/Fm, and PIabs decreased significantly, implying that S. thunbergii photosystem II (PSII) suffered apparent photoinhibition. Subsequently, PSII gradually recovered during late spring and summer, as evidenced by an increase of both parameters. Throughout the year, the maximum decrease in the slope of MR/MR0 maintained low values indicated that photosystem I (PSI) was incative, the initial rate of P700+ re-reduction maintained low value indicated that cyclic electron transport (CET) were inactive; nevertheless, a seasonal down-regulation of both PSI and CET during late winter and early spring could be detected. The weak performance of PSI and CET can potentially limit the flexibility in response to winter stress and result in a delayed recovery of PSII. In conclusion, the seasonal variability of S. thunbergii photosynthetic activity was characterized by three periods: active state, down-regulation and restoration. The rapid growth during early spring was accompanied by weak photosynthetic performance, indicating that the carbohydrates consumed during this period were derived from previously stored starch.

Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.).

Responses of electron transport to three levels of irradiation (20, 200, and 1200 µmol photons m-2 s-1 PAR; exposures called LL, ML and HL, respectively) were investigated in eelgrass (Zostera marina L.) utilizing the chlorophyll a fluorescence technique. Exposure to ML and HL reduced the maximum quantum yield of photosystem II (PSII) (Fv/Fm) and the maximum slope decrease of MR/MRO (VPSI), indicating the occurrence of photoinhibition of both PSII and photosystem I (PSI). A comparatively slow recovery rate of Fv/Fm due to longer half-life recovery time of PSII and 40% lower descending amplitude compared to other higher plants implied the poor resilience of the PSII. Comparatively, PSI demonstrated high resilience and cyclic electron transport (CEF) around PSI maintained high activity. With sustained exposure, the amplitudes of the kinetic components (L1 and L2), the probability of electron transfer from PSII to plastoquinone pool (ψET2o), and the connectivity among PSII units decreased, accompanied by an enhancement of energy dissipation. Principle component analysis revealed that both VPSI and Fv/Fm contributed to the same component, which was consistent with high connectivity between PSII and PSI, suggesting close coordination between both photosystems. Such coordination was likely beneficial for the adaption of high light. Exposure to LL significantly increased the activity of both PSI and CEF, which could lead to increased light harvesting. Moreover, smooth electron transport as indicated by the enhancement of L1, L2, ψET2o and the probability of electron transport to the final PSI acceptor sides, could contribute to an increase in light utilization efficiency.

The alternation between PSII and PSI in ivy (Hedera nepalensis) demonstrated by in vivo chlorophyll a fluorescence and modulated 820 nm reflection.

To examine the coordination between photosystem II (PSII) and photosystem I (PSI) in response to varying environmental conditions, both diurnal fluctuations and seasonal variability of photosynthetic electron transport activity in ivy (Hedera nepalensis, Araliaceae) were investigated: by measuring prompt fluorescence, delayed fluorescence (DF) and modulated reflection of 820 nm light (MR). During diurnal fluctuations, the PSII electron donor side was damaged, as evidenced by decreases of the fast amplitude of DF decay kinetics at I1, although there was no significant change in relative variable fluorescence at K-step to amplitude of FJ - Fo. Decreases in the maximum photochemical efficiency (i.e., PSII photoinactivation) were accompanied by an increased maximum decrease in the slope of MR/MRo (i.e., PSI photoactivation). Subsequently, PSII recovery and PSI relaxation occurred in the afternoon. Throughout the season, alternations between PSII and PSI were also suggested by the down-regulation of PSII and the up-regulation of PSI from summer to winter. Significant negative linear correlations between the activity of PSII and PSI across both diurnal fluctuations and seasonal variability were verified by correlation analyses. In addition, PSI was active throughout the year, suggesting PSI is independent from high temperatures. High PSI activity may maintain the functional integrity of the photosynthetic apparatus in overwintering ivy. The alternation between PSII and PSI activity may regulate the distribution of excitation energy between the two photosystems and balance the redox state of the electron transport change, thereby enabling ivy to respond to varying environmental conditions.

[Photosynthetic activity of Gloiopeltis furcata (intertidal red macroalga) in response to desiccation].

In this study, the diurnal change of photosynthesis activity in response to various tidal patterns, the relationship between photosynthetic activity and tissue water content, and the interactive effect of desiccation and irradiance on photosynthetic activity in Gloiopeltis furcata were investigated by using portable pulse amplitude modulated (PAM) fluorometer. Results showed that Fv/Fm decreased more rapidly during the noon low tide than during the morning- or evening low tide. F/Fm decreased slowly at the beginning of desiccation during the morning low tide, but decreased rapidly throughout the evening low tide. Fv/Fm recovered to the initial values on the same day no matter when the low tide occurred, suggesting the occurrence of dynamic photoinhibition. These features endowed G. furcata with an ability to adapt to the periodic desiccation on high intertidal rocks. The maximum (Fv/Fm) and effective (Phi(PSII)) quantum yield declined with the decrease of tissue water content (TWC). However, photosynthetic activity could recover completely when TWC exceeded 6%, showing a strong ability of G. furcata to tolerate desiccation. The relationships between TWC and Fv/Fm and Phi (PS II) as were as follows: F/Fm = 0.68 + (0.44-0.68)/[1 +(TWC/ 66.96)]5 , R2 = 0.99; Phi(PSII) = 0.585 + (0.004-0.585)/[1+(TWC/73)10], R2 = 0.99. ANOVA result further showed that the interactive effect of irradiance and desiccation on photosynthetic activity was significant, and that the photoinhibition degree increased with elevation of irradiation and duration of desiccation. The extreme condition (6 h desiccation at 1000 micromol photons x m(-2) x s(-1)) resulted in a serious photoinhibition, with the longest period of complete recovery for photosynthesis activity.

[Spatiotemporal variation and related affecting factors of Gloiopeltis furcata biomass and length around Xiaoheishan Island, Shandong of China].

Gloiopeltis furcata, an important economic red macroalga, has been severely depleted due to human disturbances. Taking the natural G. furcata populations around the Xiaoheishan Island of Shandong, China as test objects, this paper studied the spatiotemporal variation pattern of their biomass and length and related affecting factors. The two-way ANCOVA showed that at island scale, both the subbottom and the Baardseth index had significant effects on the G. furcata biomass and length. The steady subbottom and the high Baardseth index were beneficial to the G. furcata growth. At micerohabitat scale, wave action and tidal level had significant effects on the G. furcata biomass and length. Wave wash and moderate tidal level promoted the G. furcata growth. The one-way repeated measurements ANOVA showed there existed seasonal variation of G. furcat biomass and length. The optimal growth period of G. furcata was from March to April, with the peak values of biomass and length appeared in late spring and early summer.

[Sexual reproductive allocation of Sargassum thunbergii at Taiping Cape of Yellow Sea].

This paper studied the dynamics of reproductive allocation (RA) of Sargassum thunbergii during its sexual reproductive season and the related environmental factors at the Taiping Cape of Yellow Sea. The sexual reproduction of S. thunbergii initiated in early June, peaked in mid July when the sea water temperature was about 22 degrees C (the mean proportion of biomass allocated to reproductive organs on July 19 was 76.7%), and ended in late August. The RA had a significant linear correlation with the average length of thallus branches (r = 0.855, P < 0.01). The thalli with a length less than 10 cm showed a lower RA in the whole sexual reproductive season, while the thalli longer than 10 cm had a RA up to averagely 70.0% at the peak maturing stage. UNIANOVA analysis showed that both tidal level and wave strength had significant effects on the RA of S. thunbergii (tidal level: F = 175.62, P < 0.01; wave strength: F = 95.35, P < 0.01), and there was a significant interaction between tidal level and wave strength (F = 9.14, P < 0.05). The sizes of the effects were in the order of tidal level > wave strength > tidal level x wave strength.

[Methodological study for detecting gene mutation of family with genotyping of compound heterogenicity of SEA alpha-thalassemia 1 and HbCS].

This study was aimed to establish a method of PCR combination with PCR-RFLP for detecting the South-East Asian (SEA) deletion type alpha-thalassemia 1 and non-deletion mutation of Hb Constant Spring (CS), and to investigate the application value of this method. For the members of the families with alpha-thalassemia, SEA deletion mutation was detected by PCR, then the HbCS point mutation was screened by PCR-RFLP. The results indicated that 15 carriers with alpha-thalassemia (--(SEA)/) were found in 19 members from 7 families, and 2 families with genotype of --(SEA)/alpha(CS)alpha were screened out successfully. It is concluded that the PCR combination with PCR-RFLP is a simple, rapid, and reliable method for screening HbH disease with genotype of --(SEA)/alpha(CS)alpha.